HK1238175A1 - Compositions, systems, and methods for generating inner ear hair cells for treatment of hearing loss - Google Patents

Abstract

Method and compositions for inducing the self-renewal of stem/progenitor supporting cells comprised by a cochlear cell population, including inducing the stem/progenitor cells to proliferate while maintaining, in the daughter cells, the capacity to differentiate into hair cells.

Description

Compositions, systems and methods for generating inner ear hair cells to treat hearing loss

RELATED APPLICATIONS

This application claims the benefit of united states provisional application No. 62/045,506 filed on 9/3/2014 and united states provisional application No. 62/051,003 filed on 9/16/2014. The entire teachings of the above application are incorporated into this application by reference.

Government support

The completion of the present invention was supported by government support granted by the national institutes of health under grant R01DE 013023. The completion of the present invention was supported by government support awarded by the national institutes of health, No. HL 095722. The government has certain rights in the invention.

Background

In a large proportion of the population, permanent damage to the inner ear hair cells leads to sensorineural hearing loss, which makes communication difficult. Hair cells are the sensory cells that transduce auditory stimuli. Regeneration of damaged hair cells provides a way to treat conditions that currently have no other treatment than a prosthetic organ device. Although hair cells do not regenerate in the mammalian cochlea, new hair cells are produced in lower vertebrates by epithelial cells surrounding the hair cells, which are called supporting cells.

The development of hearing loss following cochlear injury in mammals is believed to be due to the lack of spontaneous regeneration of hair cells and/or neurons that are the main components for sensing sound (Wong and Ryan, 2015). Humans have 15,000 inner ear hair cells at birth, and hair cells do not regenerate after birth. In neonatal mice after ototoxic injury, supporting cells surrounding hair cells in the normal cochlear epithelium have the potential to differentiate into hair cells (Bramhall et al, 2014). Using a lineage tracing approach, new hair cells, mainly outer hair cells, have been shown to be produced by Lgr 5-expressing inner column cells and third defiters cells, and it has been shown that gradual increase in regeneration of new hair cells can be achieved by pharmacological inhibition of Notch (Bramhall et al, 2014, Mizutari et al, 2014). It has been proposed that neonatal mammalian cochlea has some capacity for hair cell regeneration after only injury (Cox et al, 2014), while Lgr5 is positive (Lgr 5)⁺) The cells act as hair cell progenitors in the cochlea (Chai et al, 2011, Shi et al, 2012).

Auditory dysfunction in humans is a continuing problem in the medical fields of otology and audiology. Auditory dysfunction typically results from acute and chronic exposure to loud sounds, ototoxic chemicals, and aging. Hearing loss can be caused by sounds in excess of 85 decibels, which are produced by sources such as firearm shots, explosive bombs, jet engines, power tools, and concerts. Other common everyday activities and products also produce high levels of noise, such as the use of hair dryers, MP3 players, weed killers and blenders. Military personnel are especially at risk of noise-induced hearing loss due to common military noise exposure. Side effects of noise-induced hearing loss include tinnitus (ringing in the ear), reduced speech intelligibility, hyperacusis, heavy vibrations, and impaired auditory processing of various types. Exposure to commonly used drugs may also induce auditory dysfunction. For example, patients treated with anti-cancer therapies, antibiotics, and other drugs often develop hearing loss as a side effect. In addition, exposure to industrial chemicals and gases can induce hearing damage. In western countries, auditory dysfunction is a common consequence of aging. Hearing impairment can be attributed to a variety of causes, including infection (e.g., otitis media), genetic predisposition, mechanical injury, tumors, loud sounds, or long-term exposure to noise, aging, and chemical-induced ototoxicity (e.g., antibiotics or platins drugs) that destroy neurons and/or hair cells of the peripheral auditory system. It may be caused by acute noise or may develop over time.

Currently, few cases of hearing loss can be practically cured. Audiological devices (e.g., hearing aids) have limitations including: speech intelligibility cannot be improved. Of those affected by hearing impairment, less than 20% are currently using hearing instruments. For age-related auditory dysfunction or noise or drug-induced auditory dysfunction, the only effective method currently used to "treat" or reduce the severity of the condition is often prophylactic: avoiding excessive noise and the use of ear protectors, implementing a healthy lifestyle, and avoiding as much as possible exposure to ototoxic drugs and substances.

Once hearing loss has developed, people can use hearing aids to correct the hearing loss. However, despite the advances in performance of these prostheses, they still have major limitations. For example, hearing aids primarily amplify sound and are unable to correct suprathreshold or post-cochlear impairments, such as impaired speech intelligibility, speech deficits in noise, tinnitus, hyperacusis, loudness repetition, and various types of central auditory processing disorders. Hearing aids are basically amplifying sounds that stimulate intact cells, but there is no treatment to aid in the recovery of damaged cells or to maximize the function of existing intact cells.

For total deafness or deep deafness, cochlear implants may be used. Such devices deliver electrical stimulation through electrodes surgically implanted in the cochlea. Cochlear implants are particularly helpful for deaf children if implanted at the age of two or three, which is the fastest growing stage of language skills. However, cochlear implants involve invasive surgery and are relatively expensive. Furthermore, cochlear implants require living neurons to achieve beneficial effects.

About 17% of americans have hearing loss, half of which are less than 65 years old. It is predicted that by 2030, the number of americans with hearing loss will exceed seventy million.

Currently, about 3 hundred million people worldwide suffer from moderate to severe hearing loss, and this figure is expected to increase to 7 hundred million by 2015. Most of these people suffer from noise-induced hearing loss, and 1/4's americans develop permanent hearing loss due to occupational exposure to noise hazards. VA costs more than 10 billion dollars in hearing loss compensation as reported by the U.S. department of defense and the advanced technology commercialization center for VA. Navy, navy army and air force (in total) record 22,000 new hearing loss reimbursements, with hearing losses costing more than $ 560 million in economic investment per year.

Thus, there is a long felt need to meet: protect auditory cells prior to injury and retain/promote the function of existing cells after injury. As disclosed below, in certain embodiments, the present invention provides compositions, systems and methods for preventing and treating auditory dysfunction.

The use of new therapies to prevent or treat hearing loss can help many patient populations, for example, patients with vertigo, tinnitus, or patients in need of cochlear implants, patients with hearing loss but not fit for cochlear implants, and patients with chronic low/moderate or severe hearing loss.

Previous work has shown that direct inhibitors of cell cycle activation (e.g. p27kip1, Rb1, p19ink4d, p21cip1) cause proliferation of many cells, including hair cells. Hair cells that re-enter the Cell cycle then die and hearing deteriorates (salivi r.j. hair Cell Regeneration Repair and Protection, Sage et al, 2005,2006). After proliferation of the supporting cells by manipulation of cell cycle genes (e.g. p27Kip1 or Rb), no differentiation into hair cells was seen (Yu et al, 2010, Liu et al, 2012).

Stem cells exhibit a very strong ability to produce multiple cell types in vivo. In addition to embryonic stem cells, tissue-specific stem cells play a key role during development and in adult homeostasis and injury repair. Stem cells self-renew by proliferation and produce tissue-specific cell types by differentiation. The characteristics of different stem cells differ in different tissues and are determined by their intrinsic and epigenetic states. However, the balance between self-renewal and differentiation is tightly controlled among different stem cells. Uncontrolled self-renewal can lead to overgrowth of stem cells and possibly to tumor formation, while uncontrolled differentiation can deplete the stem cell pool, leading to an impaired ability to maintain tissue homeostasis. Thus, stem cells continuously sense their surroundings and respond appropriately by proliferation, differentiation or apoptosis. It would be desirable to drive regeneration by controlling the timing and extent of stem cell proliferation and differentiation. The control of proliferation with small molecules that are eliminated over time will allow control of the timing and extent of stem cell proliferation and differentiation. Significantly, tissue stem cells from different tissues share limited several signaling pathways that regulate their self-renewal and differentiation, but in a manner that is very dependent on the environment. One of these pathways is the Notch pathway.

The Notch pathway is an evolutionarily conserved signaling pathway with a simple but unique mode of action. The core Notch pathway contains only a few components. The classical Notch pathway is activated by the binding of Notch ligands on the signaling cell surface to Notch receptors on adjacent signaling cells. This event initiates a proteolytic cleavage cascade of the Notch receptor, including gamma-secretase-mediated release of the Notch intracellular domain (NICD). The NICD fragment then enters the nucleus, inducing transcription of the target gene. In most cases, the classical Notch pathway requires physical contact between adjacent cells, and thus it links the fate of one cell to that of an intermediate cell, providing a mature method of controlling self-renewal and differentiation of stem cells. The Notch pathway has been shown to regulate many types of stem cells, including embryonic stem cells, neural stem cells, hematopoietic stem cells, and Lgr5 epithelial stem cells (Koch et al, 2013; VanDussen et al, 2012).

Lgr5 is expressed in a variety of tissues and has been identified as a biomarker of adult stem cells in certain tissues, such as the intestinal epithelium (Barker et al, 2007), kidney, hair follicle and stomach (Barker et al, 2010; Haegebarth)&Clevers, 2009). 2011 discloses for the first time that mammalian inner ear hair cells are derived from LGR5⁺Cells (Chai et al, 2011, Shi et al, 2012.) Lgr5 is a known component of the Wnt/β -catenin pathway that has been shown to play a major role in differentiation, proliferation, and induction of stem cell characteristics (Barker et al, 2007).

Previous work has been focused on the transformation of supporting cell differentiation into hair cells by activation or forced expression of genes that lead to hair cell formation, and of particular interest is the mechanism that enhances expression of Atoh1 (Bermingham et al, 1999; Zheng and Gao, 2000; Izumikawa et al, 2005; Mizutari et al, 2013). Interestingly, cells transduced with the Atoh1 vector have been shown to acquire the vestibular phenotype (Kawamoto et al, 2003; Huang et al, 2009; Yang et al, 2012,2013) and lack complete development. As described, Atoh1, which has been shown to be upregulated by gene insertion, produces non-cochlear cell types that behave in a manner that does not occur in the native cochlea. In addition, these methods increase hair cell number, but decrease the number of supporting cells. Since supporting cells are known to have specialized functions (amirez-Camancho 2006, Dale and Jagger2010), loss of these cells can be problematic for normal cochlear function.

Disclosure of Invention

Thus, in aspects of the present disclosure, a method of activating the Wnt pathway in a population of cochlear support cells to increase the self-renewal capacity (i.e., the ability to repeatedly produce progeny cells with equivalent proliferative potential and "cell fate designation" potential) and differentiation capacity (i.e., the ability to produce progeny cells designated for differentiation) of the population may be noted. Preferably, the Wnt pathway is activated upstream of the c-myc gene of a member of the population without any genetic modification to the population. Alternatively, it is preferred to activate the Wnt pathway with small molecules that transiently induce such activity. Furthermore, the support cell population preferably comprises LGR5 homologous to the organ of Corti⁺Supporting the cell.

Another aspect of the disclosure is a method of inducing self-renewal of stem/progenitor support cells contained in a cochlear cell population. That is, the stem/progenitor support cells are induced to proliferate (i.e., divide and form daughter cells) while maintaining the ability to differentiate into hair cells in the daughter cells. In contrast, if only stem/progenitor support cell proliferation is induced (without maintaining pluripotency), the progeny cells will lack the ability to divide into hair cells. Furthermore, merely enhancing the differentiation of a pre-existing stem/progenitor cell population may deplete the stem cell pool. Accordingly, the present disclosure provides a method of: wherein pre-existing cochlear support cells are induced to proliferate prior to differentiation, and subsequently allow the expanded population (or in some embodiments even induce it) to differentiate into hair cells. Preferably, proliferation is induced by activation of the Wnt pathway upstream of the c-myc gene of a member of the population, without any genetic modification to the population. Alternatively, it is preferred to activate proliferation with small molecules that transiently induce such activity. Furthermore, in certain embodiments, the population of support cells preferably comprises LGR5 homologous to Corti apparatus⁺Supporting the cell.

Thus, in certain embodiments, the present disclosure provides compositions capable of inducing self-renewal of a supporting cell population. These compositions are capable of activating pathways and mechanisms known to be involved in inducing stem cell properties, such as those used to generate "induced pluripotent stem cells" (in combination with Wnt stimulation, HDAC inhibition, TGF- β inhibition, RAR activation, DKK1 inhibition). Preferably, these pathways are activated with small molecules. For example, when administered in vitro to a support cell population, preferred compositions induce the population to proliferate to a high degree and purity in a stem cell proliferation assay, and allow the population to differentiate into a high purity hair cell population in a stem cell differentiation assay. In one such embodiment, the composition induces and maintains stem cell properties by proliferating produced stem cells that are capable of dividing through many generations and maintaining a high proportion of the capacity of the produced cells to differentiate into hair cells. In addition, stem cell markers expressed by proliferating stem cells may include one or more of Lgr5, Sox2, operml, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3, Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt3l, Utf1, Tcl1, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3, Smad1, Smad2, Smad2/3, Smad 39 4, Smad5, and Smad 7.

In certain embodiments, the present disclosure provides a method of expanding a cochlear cell population in a cochlear tissue comprising a parental cell population. In this embodiment, the method comprises contacting cochlear tissue with a stem cell proliferating agent to form an expanded cell population in cochlear tissue, wherein

The stem cell proliferating agent is capable of (i) forming a proliferation assay final cell population from a proliferation assay initial cell population after undergoing a proliferation assay time period in a stem cell proliferation assay, and (ii) forming a differentiation assay final cell population from a differentiation assay initial cell population after undergoing a differentiation assay time period in a stem cell differentiation assay, wherein:

(a) the proliferation assay starting cell population has: (i) proliferation assay initial total cell count, (ii) proliferation assayFixed initial Lgr5⁺Cell number, (iii) proliferation assay initial hair cell number, (iv) proliferation assay initial Lgr5⁺Cell fraction equal to the initial Lgr5 of proliferation assay⁺(iv) the ratio of the number of cells to the total number of cells from which the proliferation assay was initiated, and (v) the proliferation assay initiation hair cell ratio, which is equal to the ratio of the proliferation assay initiation hair cell number to the total number of cells from which the proliferation assay was initiated;

(b) the proliferation assay final cell population has: (i) proliferation assay final total cell count, (ii) proliferation assay final Lgr5⁺Cell number, (iii) proliferation assay final hair cell number, (iv) proliferation assay final Lgr5⁺Cell fraction equal to the final Lgr5 of the proliferation assay⁺(iv) the ratio of the number of cells to the final number of cells for the proliferation assay, and (v) the proliferation assay final hair cell ratio, which is equal to the ratio of the proliferation assay final hair cell number to the proliferation assay final cell number;

(c) the differentiation assay initial cell population has: (i) differentiation assay initial total number of cells, (ii) differentiation assay initial Lgr5⁺Cell number, (iii) differentiation assay initial hair cell number, (iv) differentiation assay initial Lgr5⁺Cell proportion equal to the differentiation assay initial Lgr5⁺(iv) the ratio of the number of cells to the total number of cells from which differentiation is determined, and (v) the proportion of hair cells from which differentiation is determined, which is equal to the ratio of the number of hair cells from which differentiation is determined to the total number of cells from which differentiation is determined;

(d) the final cell population of the differentiation assay has: (i) differentiation assay final total cell count, (ii) differentiation assay final Lgr5⁺Cell number, (iii) differentiation assay final hair cell number, (iv) differentiation assay final Lgr5⁺Cell proportion, which is equal to the final Lgr5 of the differentiation assay⁺(iv) the ratio of the number of cells to the total number of cells at the end of the differentiation assay, and (v) the differentiation assay final hair cell ratio, which is equal to the ratio of the number of cells at the end of the differentiation assay to the total number of cells at the end of the differentiation assay;

(e) proliferation assay Final Lgr5⁺Cell number is the initial Lgr5 of proliferation assay⁺At least 10 times the number of cells; and

(f) differentiation assay the final hair cell number was not 0.

In certain embodiments, the stem cell proliferator comprises a sternness driver. In certain embodiments, the stem cell proliferating agent comprises a differentiation inhibitor. In certain embodiments, the stem cell proliferator comprises a sternness driver and a differentiation inhibitor.

In certain embodiments, the present disclosure provides a method of increasing cell density of a support cell in a cochlear cell population. The method comprises the following steps: activating pathways and mechanisms in the support cells that induce stem cell properties, allowing the activated support cells to proliferate (while maintaining their pluripotent properties in the newly formed daughter cells), and subsequently allowing (or even inducing) the expanded population to differentiate into hair cells, thereby forming an expanded cochlear cell population, wherein the cell density of the hair cells in the expanded cochlear cell population is greater than the cell density of the hair cells in the original (unexpanded) cochlear cell population. In some embodiments, the support cell population is an in vitro support cell population. In other embodiments, the support cell population is an in vivo support cell population. Furthermore, the proliferation phase is preferably controlled to substantially maintain the natural architecture of the cochlear structure. Preferably, proliferation is induced by small molecules that transiently induce this activity, rather than by induction of c-myc, and without any genetic modification to the population. Furthermore, in certain embodiments, the population of support cells preferably comprises LGR5 homologous to Corti apparatus⁺Supporting the cell.

In certain embodiments, the present disclosure provides a method of increasing Lgr5⁺Methods of supporting cell density of cells in a cochlear cell population. The method comprises the following steps: activating Lgr5⁺Pathways and mechanisms supporting induction or maintenance of stem cell properties in cells, activated Lgr5⁺Supporting cell proliferation (while maintaining such stem cell properties), and subsequently differentiating (or even inducing) the expanded population into hair cells, thereby forming an expanded cochlear cell population, wherein the cell density of the hair cells in the expanded cochlear cell population is greater than that of the original (unexpanded) cochlear cellCell density of hair cells in the cell population. In some embodiments, Lgr5⁺The supporting cell population is Lgr5 in vitro⁺A population of stem cells. In other embodiments, Lgr5⁺The support cell population is an in vivo support cell population. Furthermore, in certain embodiments, the proliferation phase is preferably controlled to substantially maintain the natural architecture of the cochlear structure.

In certain embodiments, a composition comprising a sternness driver and a differentiation inhibitor is administered to a population of cochlear cells to induce stem cell proliferation and inhibit stem cell differentiation until a desired degree of expansion of the stem cell population is achieved. Subsequently, the amplified population is allowed (or optionally even induced) to differentiate into hair cells. Furthermore, the proliferation phase is preferably controlled to substantially maintain the natural architecture of the cochlear structure. In some embodiments, the sternness driver and the differentiation inhibitor are small molecules. In some embodiments, the stem cell population is an in vivo stem cell population. In other embodiments, the stem cell population is an in vitro stem cell population. In some embodiments, the stem cell population is in vivo Lgr5⁺A population of stem cells. In other embodiments, the stem cell population is in vitro Lgr5⁺A population of stem cells.

In certain embodiments, the present disclosure provides a method of increasing the cell density of hair cells in an initial population of cochlear cells (which may be an in vivo or in vitro population) comprising hair cells, Lgr^－Supporting cells and Lgr5⁺Supporting the cell. The method comprises the following steps: applying to the initial population a composition comprising a sternness driver and a differentiation inhibitor, wherein the composition is capable of inducing Lgr5 in the population in a stem cell proliferation assay⁺Supporting the number of cells to expand and allow Lgr5 in the population to differentiate in a stem cell assay⁺Supporting differentiation of the cells into a hair cell population.

In certain embodiments, the method produces expression of the stem cell marker Lgr5 in a stem cell proliferation assay⁺The stem cell of (1). In certain embodiments, if Lgr5 is used⁺And non-Lgr 5⁺The mixed population of stem cells is placed in a stem cell proliferation assay,the method increases Lgr5 in the population⁺The proportion of cells.

Expansion of the supporting cell population to the point of disrupting the natural architecture of the cochlear structure may inhibit cochlear function. Driving existing supporting cell proliferation with small molecule signals can make hair cell regeneration more controlled than using gene delivery, which cannot target specific cell types and can permanently alter the genetic information of the cell. In the desired near normal cochlear configuration, the support cells are between the columns of hair cells, and the hair cells do not contact other hair cells. In addition, it is desirable to avoid the use of genetic modifications to drive proliferation to produce large aggregates of cells in the cochlea that disrupt organ anatomy. In certain embodiments, it may be preferred to use non-carcinogenic compositions. In certain embodiments, it may be preferred to use compositions that proliferate stem cells independently of manipulation of the c-myc pathway, for example, using mechanisms effective in c-myc knockouts, or in cases where c-myc is inhibited or silenced.

In certain embodiments, the present disclosure provides a composition comprising a dry propellant, which can be used to drive the selective expansion of cochlear support cells. In some cases, a sternness driver can also induce differentiation of the supporting cells into hair cells if an effective differentiation-inhibiting concentration of a differentiation-inhibiting agent is not present. Examples of sternness drivers that can drive proliferation and differentiation include GSK3 β inhibitors and Wnt agonists. In certain embodiments, stem cell proliferation may be enhanced by the addition of a modulator of a cell cycle regulatory factor (e.g., a modulator of p27 or the Tgf β pathway). In certain such embodiments, the composition comprises the sternness driver and the differentiation inhibitor in a formulation that releases the sternness driver and the differentiation inhibitor at different rates in a release assay. Thus, for example, in one such embodiment, the formulation may provide a constant, sustained, prolonged, delayed or pulsed rate of release of the active agent into the inner ear environment, and thus avoid any variability in drug exposure.

In some embodimentsIn the formula, dry drivers may be used to drive Lgr5⁺Proliferation of stem cells. In some cases, a sternness driver may also induce Lgr5 if an effective differentiation-inhibiting concentration of a differentiation-inhibiting agent is not present⁺In certain such embodiments, the compositions comprise a dryly-driving agent and a differentiation inhibitor in a formulation that releases the dryly-driving agent and the differentiation inhibitor at different rates in a release assay.

In certain embodiments, the present disclosure provides a method of increasing cell density of hair cells in an initial population of cochlear cells comprising hair cells and support cells. The method comprises the following steps: selectively expanding the number of support cells in the initial population to form an intermediate cochlear cell population, wherein a ratio of the number of support cells to hair cells in the intermediate cochlear cell population is greater than a ratio of the number of support cells to hair cells in the initial cochlear cell population. The method further comprises the following steps: generating hair cells in the intermediate cochlear cell population to form an expanded cochlear cell population, wherein a ratio of number of hair cells to number of support cells in the expanded cochlear cell population is greater than a ratio of number of hair cells to number of support cells in the intermediate cochlear cell population.

In certain embodiments, the present disclosure provides a method of increasing Lgr5 in an initial population of cochlear cells⁺A method of supporting the number of cells or the activity of Lgr5, said initial population comprising hair cells and supporting cells. For example, in one such method, an intermediate population is formed in which Lgr5⁺The number of supporting cells is expanded relative to the initial population. Alternatively, in one such method, an intermediate population is formed in which the activity of Lgr5 of the supporting cells is increased relative to the initial population. Alternatively, there is a method of: by having a structure which is normally devoid of Lgr5 or hasActivation of Lgr5 in cell types with very low levels of Lgr5⁺Expressing in Lgr5⁺The number of cells is increased relative to the initial population of cells. As another example, an intermediate population is formed in which Lgr5 is relative to the initial population of cochlear cells⁺The number of supporting cells was expanded and Lgr5 activity was increased. Hair cells may then be generated in the intermediate cochlear cell population to form an expanded cochlear cell population, wherein a ratio of hair cells to support cells in the expanded cochlear cell population is greater than a ratio of hair cells to support cells in the intermediate cochlear cell population.

In some embodiments, the method, when applied to an adult mammal, produces adult mammal Lgr5 in S phase⁺A population of cells.

In each of the above embodiments of the disclosure, the differentiation inhibitor may be a Notch agonist or an HDAC inhibitor. In some such embodiments, there may be a prior proliferation phase of a differentiation inhibitor having an effective sternness driver concentration and an effective differentiation inhibitory concentration, followed by a differentiation phase of a differentiation inhibitor having an effective sternness driver concentration but no effective differentiation inhibitory concentration. In each of these embodiments, the sternness driver and the differentiation inhibitor are provided to the cochlear cell in formulations that release the sternness driver and the differentiation inhibitor at different rates. For example, the formulation may provide a constant, sustained, prolonged, delayed or pulsed rate of dry driver and differentiation inhibitor release into the inner ear environment. Importantly, however, the formulation releases the sternness driver and the differentiation inhibitor in a manner that provides a proproliferation phase with an effective sternness driver concentration and an effective differentiation inhibiting concentration, and a subsequent differentiation phase with an effective sternness driver concentration but no effective differentiation inhibiting concentration of the differentiation inhibitor.

In certain embodiments, the methods further comprise high throughput screening of inner ear progenitor/stem cells to identify sternness drivers and differentiation inhibitors, as used in stem cell proliferation assays and stem cell differentiation assays.

In each of the above embodiments of the disclosure, there may be a prior proliferative phase of a Wnt agonist or a GSK3 β inhibitor having an effective dryly driver concentration and a Notch agonist or a HDAC inhibitor having an effective differentiation inhibitory concentration, followed by a differentiation phase of a Wnt agonist or a GSK3 β inhibitor having an effective dryly driver concentration but no Notch agonist or HDAC inhibitor having an effective differentiation inhibitory concentration. Thus, in each of these embodiments, the formulation may provide a constant, sustained, prolonged, delayed or pulsed rate of release of the Wnt agonist or GSK3 β inhibitor and release of the Notch agonist or HDAC inhibitor into the inner ear environment. Importantly, however, the formulations release the Wnt agonist or GSK3 β inhibitor and the Notch agonist or HDAC inhibitor in a manner that provides for a pro-proliferative phase having an effective drydriver concentration of the Wnt agonist or GSK3 β inhibitor and an effective differentiation inhibitory concentration of the Notch agonist or HDAC inhibitor followed by a differentiation phase having an effective drydriver concentration of the Wnt agonist or GSK3 β inhibitor but no effective differentiation inhibitory concentration of the Notch agonist or HDAC inhibitor.

In some embodiments, the differentiation inhibitor is also a sternness driver. In some embodiments, the differentiation inhibitor is a Notch agonist and is also a sternness driver. In some embodiments, the differentiation inhibitor is valproic acid, which may be a sternness driver. If the differentiation inhibitor is also a sternness driver, the concentration of the differentiation inhibitor during the differentiation phase should be lower than the effective differentiation inhibiting concentration.

In each of the above embodiments, the differentiation inhibitor and the dryness driving agent may be contained in a sustained-release polymer gel. In some embodiments, the gel may be injected through a needle, but becomes solid in the middle ear space. In certain embodiments, the gel is comprised of a thermoreversible polymer, such as poloxamer 407.

The Notch pathway is known to be a key regulator supporting the differentiation process of cells into hair cells (Lanford et al, 1999). In some embodiments, the desiccating driver and differentiation inhibitor are applied such that the level of Notch activity in the support cells remains 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the level of Notch activity in the native state of the support cells.

In certain embodiments, the present disclosure provides a method and composition for producing hair cells, the method comprising: administering or causing the composition comprising (i) and (ii) to be administered to a population of stem cells (e.g., a population of stem cells of a sample/subject in vitro, ex vivo, or in vivo): (i) a GSK3 β inhibitor (or derivative or pharmaceutically acceptable salt thereof) and/or a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof), and (ii) a Notch agonist (or derivative or pharmaceutically acceptable salt thereof) and/or a HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof), thereby proliferating stem cells in the stem cell population and obtaining an expanded stem cell population; and exposing the expanded stem cell population to a GSK3 β inhibitor (or derivative or pharmaceutically acceptable salt thereof) and/or a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) and optionally a notch inhibitor (or derivative or pharmaceutically acceptable salt thereof), thereby promoting production of inner ear hair cells from the expanded stem cell population.

In certain embodiments, the present disclosure provides compositions, systems, and methods for preventing and treating auditory dysfunction. For example, in certain embodiments, the present disclosure provides a method of preventing or treating hearing impairment in a subject, the method comprising administering to the subject an effective amount of a composition comprising: (a) (ii) an HDAC inhibitor and/or Notch activator and (ii) a GSK3 β inhibitor, a derivative thereof (e.g., a derivative of an HDAC inhibitor, a derivative of a Notch activator, and/or a derivative of a GSK3 β inhibitor), a pharmaceutically acceptable salt thereof (e.g., a pharmaceutically acceptable salt of an HDAC inhibitor, a pharmaceutically acceptable salt of a Notch activator, and/or a pharmaceutically acceptable salt of a GSK3 β inhibitor), or a combination thereof; and (b) a pharmaceutically acceptable carrier or excipient. Thus, for example, the composition may comprise (a) an HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and a GSK3 β inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and (b) a pharmaceutically acceptable carrier or excipient. As another example, the composition may comprise (a) a Notch activator (or a derivative or pharmaceutically acceptable salt thereof) and a GSK3 β inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and (b) a pharmaceutically acceptable carrier or excipient. For another example, the composition may comprise (a) an HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof), a Notch activator (or a derivative or pharmaceutically acceptable salt thereof), and a GSK3 β inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and (b) a pharmaceutically acceptable carrier or excipient.

In certain embodiments, the disclosure also relates to ex vivo uses of the cells described herein. For example, the methods described herein can be used for hi and development purposes. For example, certain embodiments of the present disclosure can be used to identify agents that proliferate hair cell progenitors and/or increase hair cell numbers, as well as agents that protect (e.g., support the survival of) supporting cells and/or hair cells, and can also be used to identify agents that are toxic or non-toxic to supporting cells or to differentiated progeny, including hair cells.

In certain embodiments, the present disclosure provides a method of preventing or treating hearing impairment in a subject in need of treatment, the method comprising administering to the subject an effective amount of a composition comprising: an HDAC inhibitor and/or Notch activator and a GSK3 β inhibitor or derivative or pharmaceutically acceptable salt thereof, and an acceptable carrier or excipient.

In certain embodiments, the present disclosure provides a method of inhibiting loss or death of auditory system cells in a subject, comprising administering to the subject an effective amount of a composition described herein or a derivative or pharmaceutically acceptable salt thereof, and an acceptable carrier or excipient, thereby inhibiting loss or death of auditory system cells in the subject.

In certain embodiments, the present disclosure provides a method of maintaining or promoting growth of cells of the auditory system of a subject, the method comprising administering to the subject an effective amount of a composition comprising an agent described herein or a derivative or pharmaceutically acceptable salt thereof, and an acceptable carrier or excipient, to amplify or initiate endogenous repair, thereby maintaining or promoting growth of the cells of the auditory system of the subject.

The methods and compositions of the present disclosure allow for greater, and therefore more effective, dosing with these ototoxicity-inducing drugs while preventing or reducing the ototoxic effects caused by these drugs. The methods of the present disclosure provide a safe, effective, and long-term means for the prophylactic or curative treatment of hearing impairment associated with inner ear tissue damage, loss, or degeneration, particularly sound or age-and ototoxin-induced hearing impairment, especially involving inner ear hair cells. In certain embodiments, the present disclosure provides compositions and methods to achieve one or more of these or other goals.

The present disclosure relates generally to compositions, systems, and methods for inducing, promoting, or enhancing the growth, proliferation, or regeneration of inner ear tissue (e.g., inner ear support cells and/or inner ear hair cells).

Also described herein is a method of expanding a population of cochlear cells in cochlear tissue comprising a parent population of cells comprising a support cell and a plurality of Lgr5⁺A cell, the method comprising: contacting cochlear tissue with a stem cell proliferating agent to form an expanded cell population in cochlear tissue, wherein the stem cell proliferating agent is capable of (i) determining Lgr5 in a stem cell proliferation assay⁺The number of cells increased at least 10-fold, and (ii) in a stem cell differentiation assay from cells comprising Lgr5⁺The cell population of cells forms hair cells.

Also described herein is a method of expanding a population of cochlear cells in cochlear tissue comprising a population of parent cells, the parent population comprisingA support-containing cell, the method comprising: contacting the cochlear tissue with a stem cell proliferating agent to form an expanded cell population in the cochlear tissue. The stem cell proliferating agent is capable of (i) forming a proliferation assay final cell population from a proliferation assay initial cell population after undergoing a proliferation assay time period in a stem cell proliferation assay, and (ii) forming a differentiation assay final cell population from a differentiation assay initial cell population after undergoing a differentiation assay time period in a stem cell differentiation assay, wherein: (a) the proliferation assay starting cell population has: (i) proliferation assay initial total number of cells, (ii) proliferation assay initial Lgr5⁺Cell number, (iii) proliferation assay initial hair cell number, (iv) proliferation assay initial Lgr5⁺Cell fraction equal to the initial Lgr5 of proliferation assay⁺(iv) the ratio of the number of cells to the total number of cells from which the proliferation assay was initiated, and (v) the proliferation assay initiation hair cell ratio, which is equal to the ratio of the proliferation assay initiation hair cell number to the total number of cells from which the proliferation assay was initiated; (b) the proliferation assay final cell population has: (i) proliferation assay final total cell count, (ii) proliferation assay final Lgr5⁺Cell number, (iii) proliferation assay final hair cell number, (iv) proliferation assay final Lgr5⁺Cell fraction equal to the final Lgr5 of the proliferation assay⁺(iv) the ratio of the number of cells to the final number of cells for the proliferation assay, and (v) the proliferation assay final hair cell ratio, which is equal to the ratio of the proliferation assay final hair cell number to the proliferation assay final cell number; (c) the differentiation assay initial cell population has: (i) differentiation assay initial total number of cells, (ii) differentiation assay initial Lgr5⁺Cell number, (iii) differentiation assay initial hair cell number, (iv) differentiation assay initial Lgr5⁺Cell proportion equal to the differentiation assay initial Lgr5⁺(iv) the ratio of the number of cells to the total number of cells from which differentiation is determined, and (v) the proportion of hair cells from which differentiation is determined, which is equal to the ratio of the number of hair cells from which differentiation is determined to the total number of cells from which differentiation is determined; (d) the final cell population of the differentiation assay has: (i) differentiation assay final total cell count, (ii) differentiation assay final Lgr5⁺Cell number, (iii) differentiation assay final hair cell number, (iv) differentiation assay final Lgr5⁺Cell proportion, which is equal to the final Lgr5 of the differentiation assay⁺Ratio of cell number to the final total number of cells for differentiation assayAnd (v) a differentiation assay final hair cell ratio equal to the ratio of the differentiation assay final hair cell number to the differentiation assay final cell total number; (e) proliferation assay Final Lgr5⁺Cell number is the initial Lgr5 of proliferation assay⁺At least 10 times the number of cells; and (f) differentiation assay final hair cell number is not 0.

Proliferation assay Final Lgr5⁺The number of cells may be determined as the initial Lgr5 of the proliferation assay⁺At least 50 times or at least 100 times the number of cells. The expanded cell population in cochlear tissue may contain more hair cells than the parent population. Proliferation assay Final Lgr5⁺The cellular proportion may be the initial Lgr5 of a differentiation assay⁺At least 2 times the proportion of cells. The differentiation assay final hair cell ratio can be at least 2-fold greater than the proliferation assay initial hair cell ratio. The proliferation assay final hair cell fraction can be at least 25% lower than the proliferation assay initial hair cell fraction. Proliferation assay Final Lgr5⁺Cell proportion the initial Lgr5 can be determined as the specific proliferation⁺The proportion of cells is at least 10% higher. One or more morphological features of the cochlear tissue may be preserved. Can maintain natural morphology. The at least one stem cell proliferator may be dispersed in a biocompatible matrix, which may be a biocompatible gel or foam. The composition may be a controlled release formulation. The cochlear tissue may be in vivo cochlear tissue or ex vivo cochlear tissue. The method can produce Lgr5 in S phase⁺A population of cells. The at least one stem cell proliferator may include a sternness driver and a differentiation inhibitor. The contact may provide for cochlear tissue: at an initial stage, an effective proliferation concentration of at least a sternness driver and an effective differentiation inhibiting concentration of at least a differentiation inhibitor; at a later stage, at least an effective proliferation concentration of a desiccating driver and a differentiation inhibitor below an effective differentiation-inhibiting concentration. The cochlear tissue may be in a subject, and the step of contacting the cochlear tissue with the composition may be effected by administering the composition trans-tympanic to the subject. Contacting cochlear tissue with the composition may improve the hearing function of the subject.

Also described herein is a composition comprisingA composition of a biocompatible matrix and at least one stem cell proliferator, wherein the at least one stem cell proliferator is capable of expanding an initial test Lgr5 in a stem cell proliferation assay⁺A cell population to produce an expanded test population, and wherein the Lgr5 of the expanded test population⁺Lgr5 in which the cells are the initial test population⁺At least 10-fold of the cells.

Also described herein is a composition comprising a biocompatible matrix and at least one stem cell proliferator, wherein the at least one stem cell proliferator is capable of expanding a composition comprising Lgr5 in a stem cell proliferation assay⁺An initial population of cells to produce a final population of cells, and wherein the final population of cells is Lgr5⁺Lgr5 in which the cells are the primary cell population⁺At least 10-fold of the cells. Further, (a) the initial population of cells has: (i) initial total number of cells, (ii) initial Lgr5⁺Cell number, (iii) initial hair cell number, (iv) initial Lgr5⁺Cell fraction equal to the initial Lgr5 of proliferation assay⁺(iv) the ratio of the number of cells to the total number of initial cells of the proliferation assay, and (v) the ratio of initial hair cells, which is equal to the ratio of the number of initial hair cells to the total number of initial cells; and (b) the final population of cells has: (i) final total number of cells, (ii) final Lgr5⁺Cell number, (iii) final hair cell number, (iv) final Lgr5⁺Cell fraction equal to final Lgr5⁺(iv) the ratio of the number of cells to the final total number of cells, and (v) the final hair cell ratio, which is equal to the ratio of the final number of hair cells to the final total number of cells.

Final Lgr5⁺The number of cells may be the initial Lgr5⁺At least 50 times or at least 100 times the number of cells.

The at least one stem cell proliferator may be dispersed in a biocompatible matrix, which may be a biocompatible gel or foam. Proliferation assay Final Lgr5⁺Cell proportion the initial Lgr5 can be determined as the specific proliferation⁺The proportion of cells is at least 10% higher. The at least one stem cell proliferator may include at least one of a sternness driver and a differentiation inhibitor. The at least one stem cell proliferating agent may includeBoth desiccation drivers and differentiation inhibitors. The stem cell proliferator may comprise a sternness driver at a concentration 100 times the effective proliferation concentration of the sternness driver and a differentiation inhibitor at a concentration at least 100 times the effective differentiation inhibition concentration of the differentiation inhibitor. The composition may be a controlled release formulation. The controlled release formulation provides immediate release, delayed release, sustained release, extended release, variable release, pulsed release, or bimodal release of the stem cell proliferator when administered to a subject via the tympanic membrane. The controlled release formulation, when administered to a subject, can provide: (a) at an initial stage, an effective proliferation concentration of at least a sternness driver and an effective differentiation inhibiting concentration of at least a differentiation inhibitor; and (b) at a later stage, at least an effective proliferation concentration of a sternness driver and a less than effective differentiation inhibitory concentration of a differentiation inhibitor.

In the methods and compositions, the dry driver may be a GSK3 β inhibitor, a GSK3 β inhibitor derivative, a wnt agonist derivative, or a pharmaceutically acceptable salt of any of them. In the methods and compositions, the differentiation inhibitor may be a notch agonist, a notch agonist derivative, an hdac inhibitor derivative, or a pharmaceutically acceptable salt of any of them. In the methods and compositions, the dry drivers may be selected from the group consisting of CHIR99021, LY2090314, lithium, a1070722, BML-284, and SKL 2001.

In the methods and compositions, the differentiation inhibitor may be a Notch agonist or an HDAC inhibitor selected from the group consisting of valproic acid, SAHA, and Tubastatin a.

Also described herein is a method of treating a subject having hearing loss or at risk of developing hearing loss. The methods can include administering a composition comprising at least one stem cell proliferating agent trans-tympanic to cochlear tissue of a subject. The at least one stem cell proliferator may include at least one of a sternness driver and a differentiation inhibitor. The at least one stem cell proliferator may include both a sternness driver and a differentiation inhibitor.

The dry driver may be CHIR 99021. The differentiation inhibitor may be valproic acid. In some methods, the cochlear tissue may be further contacted with epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor 1, pVc, and 616452.

Also described herein is a method of producing a Myo7a + cochlear cell. The method may include: make Lgr5⁺Contacting a cochlear cell with a composition comprising a sternness driver and a differentiation inhibitor to produce expanded Lgr5⁺A population of cells; and amplifying the amplified Lgr5⁺The cell population is contacted with a notch inhibitor and a sternness driver to produce Myo7a + cochlear cells. The dry driver may be CHIR 99021. The differentiation inhibitor may be valproic acid. The notch inhibitor may be DAPT.

In some aspects, the invention includes compounds comprising one or more of the compounds described herein (e.g., stem cell proliferators). In some aspects, the invention encompasses kits, e.g., kits comprising a kinase inhibitor. In some aspects, the kit comprises instructions for use.

Other objects and features will be in part apparent and in part pointed out hereinafter.

Drawings

This patent or application document contains at least one color picture. Copies of this patent or patent application with color drawings will be provided by the office upon request for the necessary fee.

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the invention.

FIG. 1: lgr5-GFP inner ear support cells were expanded under a variety of conditions. (A) Brightfield and GFP fluorescence images of Lgr5-GFP inner ear progenitor cells cultured for 10 days in medium containing EGF, bFGF and IGF-1. (B) Lgr5-GFP cells were cultured in the presence of CHIR and VPA. Scale bar: 100 μm.

FIG. 2: small molecules (CHIR99021 and VPA) facilitate the expansion of inner ear progenitor cells. (A) FACS histograms of Lgr5-GFP cells under various conditions. (B) Quantification of cell proliferation and GFP expression of the cells shown in (A). (C) Corresponding cell quantification.

FIG. 3: addition of pVc increased cell proliferation of Lgr5 inner ear progenitor cells. Scale bar: 400 μm.

FIG. 4: increasing bFGF concentration promoted proliferation of Lgr5 inner ear progenitor cells. Scale bar: 400 μm.

FIG. 5: the inner ear progenitors were further screened for support factors for Lgr 5. TTNPB increased cell proliferation but did not increase GFP expression. In the presence of pVc, CHIR is essential for cell proliferation and GFP expression, and EGF, bFGF are important for cell proliferation but less important for GFP expression. VPA is important for GFP expression. IGF showed minimal beneficial effects in promoting cell proliferation and GFP expression. (A) The number of cells. (B) Percentage of GFP +.

FIG. 6: the inner ear progenitors were further screened for support factors for Lgr 5. Additional screening with modulators of the major signaling pathways indicated that manipulation of these signaling pathways did not promote expression of Lgr 5-GFP. Small molecules used in screening include: PD0325901(MEK inhibitor), VX745(p38 inhibitor), JNK-IN8(JNK inhibitor), Tofacitinib (JAK inhibitor), PH797804(p38 inhibitor), rapamycin (mTor inhibitor), LY294002(PI3K inhibitor), SC79(AKT activator), PKC412(PKC inhibitor), FR180209 (insulin receptor inhibitor), LDN193189(BMP inhibitor), BMS536924 (insulin and insulin-like growth factor-1 receptor inhibitor), 5IT (increasing beta cell proliferation), and 616452(Tgfb, ALK5 inhibitor).

FIG. 7: addition of 616452 to EFICVP increased the intensity of Lgr5-GFP and the size of colonies. Scale bar: 400 μm.

FIG. 8 depicts characterization of culture conditions that promote proliferation of inner ear progenitor cells. Fig. 8(a) shows GFP fluorescence and bright field images of single inner ear epithelial cells in the presence of EGF, bFGF, IGF1(EFI), EFI and CHIR99021, VPA, pVc, 616452 (eficgp 6) for 10 days. FIG. 8(b) is a graph showing Lgr5-GFP expression, cell proliferation (number of viable cells) and GFP in inner ear epithelial cells cultured for 10 days under various conditions⁺Quantification of cell number. The cell colonies were dispersed into single cells using trypsin. The total number of cells was counted using a hemocytometer. The cells were subsequently stained with Propidium Iodide (PI) and analyzed for Lgr5-GFP expression using a flow cytometer. Multiplication of GFP by the total number of cells⁺Percentage of cells calculation of GFP⁺The number of cells. EFICVP6 indicated culture conditions containing all factors (including EGF, bFGF, IGF1, CHIR, VPA, pVc and 616452) that showed the best results in supporting cell proliferation and GFP expression. Each individual factor was then removed from the medium and tested. Removal of bFGF or CHIR from the medium had a large effect on cell proliferation, while removal of CHIR also affected GFP expression to a large extent. Removal of EGF, 616452 showed greater effect on cell proliferation, while removal of VPA or pVc showed greater effect on GFP retention. Removal of IGF-1 showed minimal effects on cell proliferation or GFP maintenance. These results indicate that bFGF and CHIR are important in promoting cell proliferation and GFP expression, while several other factors are also important. FIG. 8(c) shows GFP fluorescence and bright field images of the cultures shown in (b). Scale bar: 200 μm (a) and 400 μm (c).

Figure 9 demonstrates how small molecules promote the retention of inner ear progenitor cells. FIG. 9(a) shows GFP expression in inner ear epithelial cells cultured under various conditions. W: wnt3 a. R: R-Spondin 1. FIG. 9(b) shows a histogram of GFP expression of cells cultured under various conditions. FIG. 9(c) shows GFP fluorescence and bright field images of Lgr5-GFP inner ear epithelial cell cultures subjected to 7 days under the indicated conditions. FIG. 9(d) shows GFP fluorescence and bright field images of Atoh1-GFP inner ear epithelial cells subjected to 7 days under the indicated conditions. GFP + cells are differentiated cells. The results show that VPA inhibits differentiation of inner ear progenitor cells into Atoh1 positive hair cells. FIG. 9(e) shows the selection of support factors for inner ear progenitor cells. GFP expression is shown for Lgr5-GFP inner ear progenitor cells cultured under various conditions. Small molecules were added based on control (EFICV) conditions. Laminin 511 was added to the matrigel. Two batches of cells were used for screening as indicated by Exp 1 and Exp 2. The results show that pVc promotes GFP expression in Lgr5-GFP inner ear progenitor cells. FIG. 9(f) shows fluorescence and bright field images of Lgr5-GFP cells cultured under the indicated conditions. Cells from passage 2 day 10 are shown. 616452 allow passage of cultured inner ear progenitor cells. Scale bar: 400 μm.

FIG. 10 shows the expansion of single sorted Lgr5-GFP cells. Left panel: sorted high GFP cells grew into large colonies that uniformly expressed high levels of GFP. Right panel: low GFP cells grew into colonies including high GFP (large arrow), low GFP (triangular arrow) and GFP-negative (small arrow) colonies.

FIG. 11 depicts the differentiation of expanded inner ear progenitor cells under various conditions. qPCR was performed to measure Myo7a expression 6 days after differentiation. Conditions without growth factors (EGF, bFGF, IGF) or small molecules (616452, pVC and VPA) resulted in the highest Myo7a expression.

FIG. 12 demonstrates that cultured inner ear progenitor cells produce hair cells in vitro. Fig. 12(a) shows that combination with a Gsk3 β inhibitor (e.g., CHIR) and a Notch inhibitor (e.g., DAPT) induces Myo7a positive and Prestin positive outer hair cells (top panel) and Myo7a positive and Prestin negative inner hair cells (bottom panel). FIG. 12(b) shows that in the presence of Wnt inhibitor IWP-2, little hair cell production from cultured inner ear progenitor cells was observed. Scale bar: 100 μm.

FIG. 13 depicts in vitro cultures of Lgr5-GFP inner ear progenitor cells from adult mice. (a) Cochlea isolated from 9-week-old mice was cultured for 9 days in the presence of eficgp 6. Shows the outgrowth of Lgr5-GFP cells. (b)) Same culture at day 13. show expansion of Lgr5-GFP cells passage 2 of same culture 5 days after passage (day 19. all scales: 100 μm. in particular, cochlea was isolated from 9-week-old adult mice, mixed directly with Matrigel (Matrigel), and plated in the center of the well of a 24-well plate after Matrigel gelation, medium was added in the presence of EGF, bFGF, IGF-1, CHIR99021, VPA, L-ascorbic acid 2-phosphate (pVc), and TGF- β RI kinase inhibitor II (616452). after Matrigel gelation, tissue was cultured for 2 weeks until Lgr5-GFP cells were expanded and formed to outgrow.2 weeks after (day 14), tissue was dispersed using trypsin, replated, and further cultured.A comparison of the results at day 9 and day 13 shows that our culture system can be used to support adult Lgr cell expansion from the epithelium of the inner ear and growth using 5-GFP, also from adult cells cultured with 5, and further showing growth conditions from Lgr culture using GFP⁺Cells were passable (fig. 13C).

FIG. 14 expansion of inner ear cells of Lgr5-GFP from adult mice. Under conditions of EFICVP6, the cells grew slowly. Addition of TTNPB increased cell proliferation and GFP + colony formation.

FIG. 15 amplification of Lgr5-GFP + inner ear cells from adult mice. The cells were cultured under conditions comprising EGF, bFGF, IGF1, CHIR, VPA, pVc, 616452, and TTNPB. Images were taken for the same field of view on days 2 and 5 in culture. Bright field and GFP fluorescence images are shown.

FIG. 16: (A) CHIR and (B) VPA were released cumulatively from the percentage of poloxamer 407 based hydrogel formulation in the dialysis bag.

FIG. 17: (A) CHIR and (B) cumulative release of VPA from poloxamer 407 based hydrogel formulation in a dialysis bag. The initial loading of CHIR and VPA was 41.7. mu.g/30. mu.l and 2.63 mg/30. mu.l, respectively.

FIG. 18: hearing recovery in CBA/CaJ mice. Animals treated with VPA/CHIR (n-7) showed significant recovery at all frequencies tested. Animals treated with LY411575(n ═ 8) showed significant recovery at 10kHz, 28.3kHz and 40 kHz. In animals treated with the control vehicle injection (n-8), no recovery occurred. [. p <0.05]

Corresponding reference characters indicate corresponding parts throughout the drawings.

Definition of

In this application, the use of "or" means "and/or" unless stated otherwise. As used herein, the term "comprising" and variations thereof (e.g., "comprises" and "comprising") are not intended to exclude other additives, components, integers or steps. As used herein, the terms "about" and "approximately" are used equivalently. Any number used in this application, whether about/about, is intended to cover any normal fluctuations as understood by one of ordinary skill in the art. In certain embodiments, the term "about" or "approximately" refers to a range of values that, in either direction (greater or less) fall within the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lower of the referenced value, unless otherwise stated or clearly evident from the context (except where the number would exceed 100% of the possible values).

"administering" refers to introducing a substance into a subject. In some embodiments, the administration is otic, intracochlear, vestibular, or trans-tympanic, for example by injection. In some embodiments, administration is directly to the inner ear, e.g., by round window, ear capsule, or vestibular canal injection. In some embodiments, administration is directly into the inner ear via a cochlear implant delivery system. In some embodiments, the substance is injected into the middle ear via the tympanic membrane. In certain embodiments, "causing … … to be administered" refers to administering the second component after the first component has been administered (e.g., at a different time and/or by a different actor).

"antibody" refers to an immunoglobulin polypeptide or fragment thereof having immunogen-binding ability.

As used herein, "agonist" refers to an agent that increases the expression or activity of a target gene, protein, or pathway, respectively. Thus, an agonist is able to bind to and activate its cognate receptor in a manner that exerts a physiological influence, either directly or indirectly, on a target gene or protein. Agonists are also capable of increasing the activity of a pathway by modulating the activity of a component of the pathway (e.g., by inhibiting the activity of a negative regulator of the pathway). Thus, a "Wnt agonist" may be defined as an agent that increases Wnt pathway activity, which may be measured by increased TCF/LEF mediated transcription in a cell. Thus, a "Wnt agonist" may be a true Wnt agonist that binds to and activates a member of the frizzled receptor family, including any and all members of the Wnt family of proteins, inhibitors of intracellular β -catenin degradation, and activators of TCF/LFE. A "Notch agonist" can be defined as an agent that increases Notch pathway activity, which can be determined by measuring the transcriptional activity of Notch.

An "antagonist" refers to an agent that binds to a receptor and thereby reduces or eliminates binding to other molecules.

"antisense" refers to a nucleic acid sequence, regardless of length, that is complementary to the coding strand or mRNA of the nucleic acid sequence. The antisense RNA can be introduced into cells, tissues or organoids of an individual. Antisense nucleic acids can comprise a modified backbone, for example, a phosphorothioate, phosphorodithioate, or other modified backbone known in the art, or can comprise non-natural internucleoside linkages.

As used herein, a "complementary nucleic acid sequence" is a nucleic acid sequence consisting of complementary nucleotide base pairs that is capable of hybridizing to another nucleic acid sequence. "hybridization" means pairing between complementary nucleotide bases under suitably stringent conditions to form a double-stranded molecule (e.g., in DNA adenine (a) forms a base pair with thymine (T), as does guanine (G) with cytosine (C)). (see, e.g., Wahl, G.M., and S.L.Berger (1987) Methods enzymol.152: 399; Kimmel, A.R, (1987) Methods enzymol.152: 507).

By "otic (otic) administration" is meant administration of the composition to the inner ear of a subject across the tympanic membrane using a catheter or stylet device. To facilitate insertion of the stylet or catheter, a syringe or suction tube of appropriate size may be used to perforate the tympanic membrane. These devices may also be inserted using other methods known to those skilled in the art, for example, surgical implantation of the devices. In certain embodiments, the stylet or catheter device may be a stand alone device, meaning that it is inserted into the ear of a subject and subsequently releases the composition in a controlled manner to the inner ear. In other particular embodiments, the stylet or catheter device may be connected or coupled to a pump or other device that allows for the administration of additional compositions. The pump may be automatically programmed to deliver a dosage unit, or may be controlled by the subject or a medical professional.

As used herein, a "biocompatible matrix" is a polymeric carrier that is acceptable for administration to a human for the release of a therapeutic agent. The biocompatible matrix may be a biocompatible gel or foam.

As used herein, "cell aggregates" refer to entities of cells in the organ of Corti that have proliferated to form clusters of a given cell type greater than 40 microns in diameter and/or to produce a morphology in which more than 3 cell layers reside perpendicular to the basement membrane. "cell aggregates" may also refer to the following processes: cell division creates an entity of cells that break one or more cell types through the otter board or the boundary between the endolymph and the perilymph.

As used herein, the "cell density" of a particular cell type is the average number of that cell type per unit area in a representative microscopic sample. The cell type may include, but is not limited to, Lgr5⁺Cells, hair cells or support cells. Cell density may be assessed with a given cell type in a given organ or tissue, including but not limited to cochlea or Corti organ. For example Lgr5 in Corti device⁺Cell density is measured across the Corti apparatus as Lgr5⁺Cell density of the cells. Typically, the support cells and Lgr5 will be counted by cutting a cross section of the Corti apparatus⁺A cell. Hair cells are typically counted by looking down on the surface of a Corti machine depicted in a representative microscopy sample, although cross sections may be used in some cases. Generally, Lgr5⁺The cell density of the cells will be measured by the following method: bulk preparations of the Corti apparatus were analyzed and the number of Lgr5 cells counted at a given distance along the epithelial surface as depicted in representative microscopy samples. Hair cells can be identified by their morphological characteristics, such as bundle or hair cell specific staining (e.g., myosin VIIa, Prestin, vgout 3, Pou4f3, Espin, conjugated phalloidin, PMCA2, Ribeye, Atoh1, etc.). Lgr5⁺Cells can be identified by specific staining or antibodies (e.g., Lgr5-GFP transgene reporter, anti-Lgr 5 antibodies, etc.).

"CHIR 99021" is of formula C₂₂H₁₈Cl₂N₈And chemical compositions of the following alias names: CT 99021; 6- [ [2- [ [4- (2, 4-dichlorophenyl) -5- (5-methyl-1H-imidazol-2-yl) -2-pyrimidinyl]Amino group]Ethyl radical]Amino group]-3-pyridinecarbonitrile. The chemical structure is as follows:

as used herein, "cochlear concentration" is the concentration of a given agent measured by sampling cochlear fluid. Unless otherwise indicated, the sample should contain a substantial enough fraction of cochlear fluid so that it is approximately representative of the average concentration of the agent in the cochlea. For example, a sample may be drawn from the vestibular canal and is a series of bodily fluid samples that are sampled sequentially such that each sample consists of the cochlear fluid of a particular portion of the cochlea.

"complementary nucleic acid sequence" refers to a nucleic acid sequence consisting of complementary nucleotide base pairs that is capable of hybridizing to another nucleic acid sequence.

As used herein, the "cross-sectional cell density" of a particular cell type is the transection of the tissue in a representative microscopic sampleAverage number of this cell type per unit area of the face. The cross section of the Corti instrument can also be used to determine the number of cells in a given plane. In general, hair cell cross-sectional cell density will be measured by the following method: whole preparations of the Corti machine were analyzed and the number of hair cells counted at a given distance in a cross section cut along the epithelial section as depicted in representative microscopy samples. Generally, Lgr5⁺The cross-sectional cell density of the cells will be measured by the following method: whole preparation of Corti apparatus was analyzed and Lgr5 was counted at given distances in cross sections cut along epithelial sections⁺The number of cells, as depicted in representative microscopy samples. Hair cells can be identified by their morphological characteristics, such as bundle or hair cell specific staining (suitable stains include, for example, myosin VIIa, Prestin, vGlut3, Pou4f3, conjugated phalloidin, PMCA2, Atoh1, and the like). Lgr5⁺Cells can be identified by specific staining or antibodies (suitable stains and antibodies include fluorescent in situ hybridization of Lgr5mRNA, Lgr5-GFP transgenic reporter system, anti-Lgr 5 antibodies, and the like).

By "reduce" is meant a reduction of at least 5%, e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, e.g., as compared to a reference level.

"reduce" also means a reduction of at least 1 fold, e.g., 1,2,3,4, 5,6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 fold or more, e.g., as compared to a reference level.

As used herein, a "differentiation inhibitor" is an agent that inhibits differentiation of inner ear stem cells into inner ear hair cells. Some differentiation inhibitors maintain expression of postnatal stem cell markers. Some differentiation inhibitors include, but are not limited to, Notch agonists and HDAC inhibitors.

As used herein, the "differentiation period" is the period during which an effective sternness driver concentration is present without an effective differentiation inhibitory concentration.

The "effective concentration" may be an effective dry driver concentration for a dry driver, or an effective diffusion-inhibiting concentration for a diffusion-inhibiting agent.

The "effective differentiation-inhibiting concentration" is the lowest concentration of the differentiation inhibitor that does not allow the proportion of hair cells in the total number of cells to increase by more than 50% at the end of the stem cell proliferation assay compared to the beginning of the stem cell proliferation assay. In measuring effective differentiation inhibitory concentrations, hair cell staining of cells can be used with flow cytometry to quantify hair cells of mouse strains other than Atoh1-GFP mice. Alternatively, the Atoh1-GFP mouse strain can also be used.

As used herein, the "effective release rate" (mass/time) is the effective concentration (mass/volume) 30 μ L/1 hour.

The "effective dry driver concentration" is the lowest concentration of dry driver, as compared to Lgr5 in a stem cell proliferation assay performed without dry driver and with equal concentrations of all other components⁺This concentration induces Lgr5 in a stem cell proliferation assay, as compared to cell number⁺The number of cells increased at least 1.5-fold.

By "eliminated" is meant that the level is reduced to an undetectable level.

"engraftment" or "engraftment" refers to the process of incorporating stem or progenitor cells into a tissue of interest in vivo by contact with existing cells of the tissue. By "epithelial progenitor cell" is meant a pluripotent cell that has the potential to become restricted to a cell lineage that eventually becomes an epithelial cell.

By "epithelial stem cell" is meant a pluripotent cell having the potential to become a specialized multiple cell lineage, including the cell lineage that eventually becomes an epithelial cell.

"fragment" refers to a portion of a polypeptide or nucleic acid molecule. This portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides or amino acids.

"GSK 3 beta", "GSK 3 beta" and "GSK 3B" are used interchangeably herein to short for glycogen synthase kinase 3 beta.

A "GSK 3 β inhibitor" is a composition that inhibits the activity of GSK3 β.

As used herein, "HDAC" is a shorthand for histone deacetylase.

An "HDAC inhibitor" is a composition that inhibits HDAC activity.

"hybridization" refers to the pairing between complementary nucleotide bases under suitably stringent conditions to form a double-stranded molecule (e.g., adenine (A) forms a base pair with thymine (T) and guanine (G) forms a base pair with cytosine (C) in DNA) (see, e.g., Wahl, G.M. and S.L.Berger (1987) Methods enzymol.152: 399; Kimmel, A.R. (1987) Methods enzymol.152: 507).

"inhibitor" refers to an agent that reduces the expression or activity of a target gene or protein, respectively. An "antagonist" may be an inhibitor, but more specifically refers to an agent that binds to a receptor and thereby reduces or eliminates binding to other molecules.

As used herein, an "inhibitory nucleic acid" is a double-stranded RNA, RNA interference, miRNA, siRNA, shRNA or antisense RNA, or a portion thereof or mimetic thereof, which when administered to a mammalian cell results in decreased expression of a target gene. Typically, the nucleic acid inhibitor comprises at least a portion of the target nucleic acid molecule or ortholog thereof, or comprises at least a portion of the complementary strand of the target nucleic acid molecule. Typically, expression of the target gene may be reduced by 10%, 25%, 50%, 75% or even 90% to 100%.

"in vitro Lgr5 activity" refers to the level of expression or activity of Lgr5 in a population of in vitro cells. It can be measured in cells derived, for example, from Lgr5-GFP expressing mice (e.g., B6.129P2-Lgr5tm1(cre/ERT2) Cle/J mice (also known as Lgr5-EGFP-IRES-creERT2 or Lgr5-GFP mice, Jackson Lab cat # 008875)) by: cells were isolated as single cells, stained with Propidium Iodide (PI), and analyzed for Lgr5-GFP expression by flow cytometry. Inner ear epithelial cells from wild-type (non-Lgr 5-GFP) mice subjected to the same culture and analysis procedures were used as negative controls. Typically, two cell populations are shown in a bivariate plot (with GFP/FITC as one variable), which includes GFP positive and GFP negative populations. Lgr5 positive cells were identified by gating the GFP positive cell population. The percentage of Lgr5 positive cells was measured by gating the GFP positive cell population relative to the GFP negative population and the negative control. The number of Lgr5 positive cells was calculated by multiplying the total number of cells by the percentage of Lgr5 positive cells. For cells derived from non-Lgr 5-GFP mice, Lgr5 activity can be measured using anti-Lgr 5 antibodies or quantitative PCR on the Lgr5 gene.

As used herein, "in vivo Lgr5 activity" refers to the level of expression or activity of Lgr5 in a subject. It can be measured, for example, by removing the inner ear of the animal and measuring Lgr5 protein or Lgr5 mRNA. The anti-Lgr 5 antibody may be used to measure the fluorescence intensity determined by imaging the cochlear sample, thereby measuring the yield of Lgr5 protein, using fluorescence intensity as a measure of the presence of Lgr 5. Western blot may be used with anti-Lgr 5 antibodies, where cells may be harvested from treated organs to determine the increase in Lgr5 protein. Quantitative PCR or RNA in situ hybridization can be used to measure the relative change in Lgr5mRNA production, where cells can be harvested from the inner ear to determine changes in Lgr5 mRNA. Alternatively, the expression of Lgr5 may be measured using a GFP reporter transgene system driven by the Lgr5 promoter, where the presence or intensity of GFP fluorescence may be detected directly using flow cytometry, imaging, or indirectly using anti-GFP antibodies.

"increase" also refers to an increase of at least 1 fold, e.g., 1,2,3,4, 5,6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 fold or more, e.g., as compared to the level of a reference standard.

By "increase" is meant an increase of at least 5%, e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, e.g., as compared to a reference level.

By "intraaural administration" is meant administration of the composition to the middle or inner ear of a subject by direct injection of the composition.

"intracochlear" administration refers to the injection of the composition directly into the cochlea through the tympanic membrane and the round window membrane.

By "intra-vestibular" administration is meant injection of the composition directly into the vestibular apparatus through the tympanic membrane and round window membrane.

"isolated" refers to a substance that is separated to a different degree than the components normally associated with it that are found in its original state. "isolated" refers to the degree of separation from the original source or environment.

"Lgr 5" is a shorthand for the leucine rich repeat-containing G protein-coupled receptor 5, also known as G protein-coupled receptor 49(GPR49) or G protein-coupled receptor 67(GPR 67). It is a protein encoded by the Lgr5 gene in humans.

"Lgr 5 activity" is defined as the level of activity of Lgr5 in a population of cells. In the in vitro population, Lgr5 activity may be measured in an in vitro Lgr5 activity assay. In the in vivo population, Lgr5 activity may be measured in an in vivo Lgr5 activity assay.

"Lgr 5" as used herein⁺A cell "or" Lgr5 positive cell "is a cell that expresses Lgr 5. "Lgr 5" as used herein^－Cell "refers to non-Lgr 5⁺The cell of (1).

As used herein, "lineage tracing" refers to the use of mouse germline that is capable of fate tracing any cells that express a target gene upon reporter induction. This may include hair cells or supporting cell genes (Sox2, Lgr5, actin VIIa, Pou4f3, etc.). For example, lineage tracing can use Lgr5-EGFP-IRES-creERT2 mice hybridized to reporter mice that, when induced, allow the fate of cells expressing Lgr5 when induced to be followed. As another example, Lgr5 cells can be isolated as single cells and cultured in a stem cell proliferation assay to generate colonies, which are then differentiated in a differentiation assay and analyzed for cell fate by staining hair cells and/or supporting cell proteins and co-localization of reporter factors with hair cell or supporting cell staining to determine the fate of Lgr5 cells. In addition, lineage tracing can be performed in cochlear explants to follow the fate of the supporting cells or hair cells within the intact organ after treatment. For example, the fate of Lgr5 cells may be determined by isolating the cochlea from Lgr5-EGFP-IRES-creERT2 mice that are hybridized to reporter mice and inducing reporter factors in Lgr5 cells prior to or during treatment. Then, the cell fate of the organ can be analyzed by the following methods: hair cells and/or support cell proteins are stained and the co-localization of reporter factors to hair cell or support cell staining is determined to determine the fate of Lgr5 cells. In addition, lineage tracing can be performed in vivo to follow the fate of support cells or hair cells within the intact organ after treatment. For example, the fate of Lgr5 cells can be determined by inducing a reporter factor in Lgr5-EGFP-IRES-creERT2 mice that are crossed with reporter mice, treating the animals, and subsequently isolating the cochlea. Then, the cell fate of the organ can be analyzed by the following methods: hair cells and/or support cell proteins are stained and the co-localization of reporter factors to hair cell or support cell staining is determined to determine the fate of Lgr5 cells. Lineage tracing can be performed using alternative target reporter factors as standard in the art.

"mammal" means any mammal, including but not limited to humans, mice, rats, sheep, monkeys, goats, rabbits, hamsters, horses, cows, or pigs.

As used herein, the "mean release time" is the time in the release assay for half of the agent to be released from the carrier into phosphate buffered saline.

As used herein, "native morphology" means that the tissue architecture substantially reflects that in healthy tissue. For example, the "natural morphology" of the cochlea means: the hair cells are surrounded by supporting cells in a rosette-like pattern formed by lateral inhibition of Notch, the hair cells do not touch each other, 2 to 3 cell layers form the Corti organ epithelium, and the cells do not break through the otter board (i.e. do not break through the boundary between the endolymph and the perilymph).

As used herein, "non-human mammal" refers to any mammal that is not a human.

As used herein, "Notch activity assay" refers to an assay that determines Notch activity in a population using standard techniques, e.g., using, e.g., qPCR to determine expression of Hes5/Hes 1.

As used herein in the context, the term "number" of cells may be 0,1 or more cells.

As used herein, "Corti organ" refers to the sensory cells (inner and outer hair cells) of the hearing organ located in the cochlea.

"organoid" or "epithelial organoid" refers to a cluster or aggregate of cells that is similar to an organ or portion of an organ and has a cell type associated with that particular organ.

A "population" of cells refers to any number of cells greater than 1, but preferably at least 1 × 10³Individual cell, at least 1 × 10⁴Individual cell, at least 1 × 10⁵Individual cell, at least 1 × 10⁶Individual cell, at least 1 × 10⁷Individual cell, at least 1 × 10⁸Individual cell, at least 1 × 10⁹Individual cell or at least 1 × 10¹⁰And (4) cells.

As used herein, "progenitor cell" refers to a cell that, like a stem cell, has a tendency to differentiate into a particular type of cell, but has been more specialized than a stem cell and is driven to differentiate into its "target" cell.

As used herein, the "proliferation phase" is the period of the differentiation inhibitor having an effective sternness driver concentration and a differentiation inhibitory concentration.

"reference" refers to standard or control conditions (e.g., no treatment with a test agent or combination of test agents).

As used herein, a "release assay" refers to a test of the rate of release of an agent from a biocompatible matrix into a saline environment through a dialysis membrane. An exemplary release assay can be performed by: 30 microliters of the composition was placed in 1ml of phosphate buffered saline in a saline dialysis bag with an appropriate cut-off value, and the dialysis bag was placed in 10ml of phosphate buffered saline at 37 ℃. The dimensions of the dialysis membrane can be selected based on the size of the agent to allow the agent to be evaluated to exit the membrane. For the release of small molecules, a cut-off value of 3.5kDa to 5kDa can be used. The agent may be a sternness driver, differentiation inhibitor or other agent. The release rate of the composition may vary over time and may be measured in1 hour increments.

As used herein, a "representative microscopy sample" describes a sufficient number of fields of view in a cell culture system, a portion of extracted tissue, or an entire extracted organ, that the average feature size or number measured can reasonably be considered to represent the average feature size or number when all relevant fields of view are measured. For example, to assess hair cell counts over a range of frequencies on the Corti machine, ImageJ software (NIH) can be used to measure the total length of the cochlear whole sample and the length of the individual count segments. The total number of inner hair cells, outer hair cells and supporting cells can be counted in all or part of any one or several of the 1200 to 1400 μm four cochlear segments (apical, mid-basal and basal segments), at least 3 fields of 100 μm field size will be reasonably considered as representative microscopy samples. A representative microscopy sample may include measurements over a field of view, which may be measured as the number of cells per given distance. Representative microscopy samples can be used to assess morphology, such as cell-cell contacts, cochlear structures, and cellular components (e.g., bundles, synapses).

A "rosette-like pattern" is a characteristic arrangement of cells in the cochlea with < 5% of hair cells adjacent to other hair cells.

The term "sample" refers to a volume or mass obtained, provided and/or analyzed. In some embodiments, the sample is or comprises a tissue sample, a cell sample, a fluid sample, or the like. In some embodiments, the sample is taken from (or is) a subject (e.g., a human or animal subject). In some embodiments, the tissue sample is or comprises brain, hair (including roots), buccal swabs, blood, saliva, semen, muscle, or from any internal organ or cancer, pre-cancerous cells, or tumor cells associated with any of these. The fluid may be, but is not limited to, urine, blood, ascites, pleural fluid, spinal fluid, and the like. Body tissue may include, but is not limited to, brain, skin, muscle, endometrial, uterine and cervical tissue or cancers, precancerous or tumor cells associated with any of these. In one embodiment, the body tissue is brain tissue or a brain tumor or a brain cancer. One of ordinary skill in the art will appreciate that in some embodiments, a "sample" is a "primary sample" in that it is obtained from a source (e.g., a subject); in some embodiments, a "sample" is the result of processing a native sample, for example, to remove certain components that may be contaminating and/or to isolate or purify certain target components.

"self-renewal" refers to the process by which a stem cell divides to produce one (asymmetrically dividing) or two (symmetrically dividing) daughter cells that have a developmental potential that is indistinguishable from the parent cell. Self-renewal involves proliferation and maintenance of an undifferentiated state.

"siRNA" refers to double stranded RNA. Most preferably, the siRNA is 18,19,20,21, 22, 23 or 24 nucleotides in length and has a2 base extension overhang at its 3' end. These dsRNAs can be introduced into a single cell or culture system. These sirnas are used to down-regulate mRNA levels or promoter activity.

"Stem cells" refers to pluripotent cells that have the ability to self-renew and differentiate into multiple cell lineages.

As used herein, a "stem cell differentiation assay" is an assay that determines the differentiation capacity of stem cells. In an exemplary stem cell differentiation assay, the cell number of the initial cell population was obtained from 3 to 7 day old Atoh1-GFP mice by: the Corti sensory epithelium was isolated, the epithelium was dispersed into single cells, and the cells were passed through a 40 μm cell filter. Approximately 5000 cells were embedded in 40. mu.l of culture medium (e.g.matrigel (coming) and placed in the center of wells of a 24-well plate with 500. mu.l of the appropriate medium, Growth Factor and reagents tested. Suitable media and growth factors include Advanced DMEM/F12(1X N2, 1X B27, 2mM Glutamax, 10mM HEPES, 1mM N-acetylcysteine and 100U/ml penicillin/100. mu.g/ml streptomycin) with media supplements and growth factors (50ng/ml EGF, 50ng/ml bFGF and 50ng/ml IGF-1) and the reagents evaluated, which were added to each well. Cells were incubated at 37 ℃ and 5% CO₂The culture medium was changed every 2 days for 10 days in the standard cell culture chamber. These cells are then cultured by removing the stem cell proliferation assay reagents and replacing them with basal media and molecules that drive differentiation. Suitable basal media are Advanced DMEM/F12 supplemented with 1X N2, 1X B27, 2mM Glutamax, 10mM HEPES, 1mM N-acetylcysteine and 100U/ml penicillin/100. mu.g/ml streptomycin, and suitable molecules to drive differentiation are 3. mu.MCHIR 99021 and 5. mu.M DAPT for 10 days, with the media being changed every 2 days. Flow cytometry for GFP can be used to measure the number of hair cells in a population. Expression levels of hair cell markers (e.g., Myo7a) can be measured using qPCR to further assess hair cell differentiation levels, where the levels are normalized using a suitable and unregulated reference gene or housekeeping gene (e.g., Hprt). Hair cell differentiation levels can also be assessed by immunostaining for hair cell markers (e.g., myosin 7a, vgout 3, Espin, PMCA2, Ribeye, conjugated phalloidin, Atoh1, Pou4f3, etc.). The level of hair cell differentiationCan be assessed by western blotting against myosin 7a, vgout 3, Espin, PMCA, Prestin, Ribeye, Atoh1, Pou4f 3.

As used herein, a "stem cell assay" is an assay as follows: wherein a cell or population of cells is tested against a series of criteria to determine whether the cell or population of cells is a stem cell or enriched for a stem cell or stem cell marker. In stem cell assays, the cells/cell populations are tested for stem cell characteristics, such as expression of stem cell markers, and further optionally for stem cell function, including the ability to self-renew and differentiate.

As used herein, a "stem cell proliferator" is a composition that induces an increase in a cell population that has the ability to self-renew and differentiate.

As used herein, a "stem cell proliferation assay" is an assay to determine the ability of an agent to induce the production of stem cells from a starting cell population. In an exemplary stem cell proliferation assay, the number of cells of the initial cell population is obtained from 3 to 7 days old Lgr5-GFP mice (e.g., B6.129P2-Lgr5tm1(cre/ERT2) Cle/J mice, also known as Lgr5-EGFP-IRES-creERT2 or Lgr5-GFP mice, Jackson Lab cat # 008875) by: the Corti sensory epithelium was isolated and dispersed into single cells. Approximately 5000 cells were embedded in 40. mu.l of culture medium (e.g.matrigel (Corning, grown factor Reduced)) and placed in the center of wells of a 24-well plate with 500. mu.l of the appropriate medium, growth factors and reagents tested. Suitable media and growth factors include Advanced DMEM/F12(1X N2, 1X B27, 2mM Glutamax, 10mM HEPES, 1mM N-acetylcysteine and 100U/ml penicillin/100. mu.g/ml streptomycin) with media supplements and growth factors (50ng/ml EGF, 50ng/ml bFGF and 50ng/ml IGF-1) and the reagents evaluated, which were added to each well. Cells were incubated at 37 ℃ and 5% CO₂The culture medium was changed every 2 days for 10 days in the standard cell culture chamber. Lgr5⁺The number of cells was identified as Lgr5 in an in vitro Lgr5 activity assay⁺The number of cells (a) is counted to quantify the amount. Lgr5⁺The proportion of cells was identified by using the cell populationLgr5⁺Is quantified by dividing the number of cells in (a) by the total number of cells present in the cell population. Mean of clusters Lgr5⁺Activity is quantified by measuring the mean mRNA expression level of Lgr5 for this population, normalized using an appropriate and unregulated reference gene or housekeeping gene (e.g., Hprt). The number of hair cells in a population can be measured by: staining with hair cell markers (e.g., myosin VIIa), or using endogenous reporter factors for hair cell genes (e.g., Pou4f3-GFP, Atoh1-nGFP), and analysis using flow cytometry. The proportion of hair cells is quantified by dividing the number of cells in the cell population identified as hair cells by the total number of cells present in the cell population. Lgr5 activity can be measured by qPCR.

As used herein, a "stem cell marker" can be defined as a gene product (e.g., protein, RNA, etc.) that is specifically expressed in a stem cell. One type of stem cell marker is a gene product that directly and specifically supports the maintenance of stem cell characteristics. Examples include Lgr5 and Sox 2. Other stem cell markers can be identified using assays described in the literature. To determine whether a gene is necessary to maintain stem cell characteristics, gain-of-function and loss-of-function studies can be used. In gain-of-function studies, overexpression of specific gene products (stem cell markers) will help maintain stem cell characteristics. Whereas in loss of function studies, removal of stem cell markers will cause loss of stem cell characteristics or induce stem cell differentiation. Another type of stem cell marker is a gene that is expressed only in stem cells but does not necessarily have a specific function to maintain the characteristics of stem cells. Markers of this type can be identified by comparing gene expression-specific characteristics of sorted stem cells and non-stem cells using microarray and qPCR assays and the like. Stem Cell markers of this type can be found in the literature (e.g., Liu Q. et al, Int J Biochem Cell biol. 2015. 3; 60:99-111.http:// www.ncbi.nlm.nih.gov/pubmed/25582750). Potential stem cell markers include Ccdc121, Gdf10, Opcm1, Phex, and the like. Expression of stem cell markers (e.g., Lgr5 or Sox2) in a given cell or population of cells can be measured using assays such as qPCR, immunohistochemistry, western blotting, and RNA hybridization. Expression of stem cell markers can also be measured using transgenic cell expression reporter factors (e.g., Lgr5-GFP or Sox2-GFP) capable of indicating expression of a given stem cell marker. The activity of reporter factor expression can then be measured using flow cytometry analysis. Fluorescence microscopy can also be used to directly visualize the expression of the reporter factor. Expression of stem cell markers can also be determined using microarray analysis for whole gene expression profiling. The gene expression profile of a given cell population or purified cell population can be compared to the gene expression profile of stem cells to determine the similarity between the two cell populations. Stem cell function can be measured by colony formation assays or sphere formation assays, self-renewal assays, and differentiation assays. In a colony (or sphere) formation assay, stem cells should be capable of forming colonies on a cell culture surface (e.g., a cell culture dish) or embedded in a cell culture matrix (e.g., matrigel), or capable of forming spheres when cultured in suspension, when cultured in a suitable medium. In colony/sphere formation assays, individual stem cells are seeded in a suitable medium at low cell density and allowed to proliferate for a given period of time (7 to 10 days). The colonies formed were then counted and scored for expression of stem cell markers as an indicator of the sternness of the primary cells. Optionally, the formed colonies are then picked and passaged to test their self-renewal and differentiation potential. In a self-renewal assay, cells should maintain expression of a stem cell marker (e.g., Lgr5) during at least one (e.g., 1,2,3,4, 5, 10, 20, etc.) cell division when cultured in a suitable medium. In a stem cell differentiation assay, cells should be capable of producing hair cells when cultured in an appropriate differentiation medium, and hair cells can be identified by expression of hair cell markers as measured by qPCR, immunostaining, western blotting, RNA hybridization, or flow cytometry.

As used herein, a "dry driver" is an induced LGR5⁺Cell proliferation, upregulation of Lgr5 in cells, or maintenance of Lgr5 expression in cellsIn general, a sternness driver upregulates at least one biomarker for postnatal stem cells.

The "subject" includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, the subject is a mammal, particularly a primate, particularly a human. In some embodiments, the subject is a livestock animal, e.g., a cow, sheep, goat, cow, pig, and the like; poultry, such as chickens, ducks, geese, turkeys, and the like; and domestic animals, particularly pets, such as dogs and cats. In some embodiments (e.g., particularly in a research setting), the subject mammal will be, for example, a rodent (e.g., mouse, rat, hamster), rabbit, primate, or pig (e.g., an inbred pig), among others.

As used herein in connection with cochlear epithelium, "support cells" include epithelial cells within the organ of Corti that are not hair cells. This includes inner column cells, outer column cells, inner finger cells, Deiter cells, hansen cells, burtset cells, and/or claudi cells.

"synergistic" or "synergistic effect" refers to an effect that is greater than the sum of each effect taken alone, i.e., greater than the additive effect.

As used herein, a "Tgf β inhibitor" is a composition that reduces the activity of Tgf β.

A "tissue" is an aggregate of similar cells from the same source that collectively perform a particular function, including, for example, cochlear tissue, such as the organ of Corti.

By "trans-tympanic" administration is meant direct injection of the composition into the middle ear, across the tympanic membrane.

As used herein in connection with a cell population, a "treatment" is directed to the delivery of a substance to the population to produce a result. In the case of an in vitro population, the substance may be delivered directly (or even indirectly) to the population. In the case of an in vivo population, the substance may be delivered to the subject by administration.

"Valproic acid" (VPA) has the formula C₈H₁₆O₂Also named as 2-valproic acid. The chemical structure is as follows:

"Wnt activation" as used herein in association with a composition is activation of the Wnt signaling pathway.

Unless otherwise indicated, the use of "or" means "and/or. As used herein, the term "comprising" and variations thereof (e.g., "comprises" and "comprising") are not intended to exclude other additives, components, integers or steps. As used herein, the terms "about" and "approximately" are used equivalently. Any number used in this application, whether about/about, is intended to cover any normal fluctuations as understood by one of ordinary skill in the art. In certain embodiments, the term "about" or "approximately" refers to a range of values that, in either direction (greater or less) fall within the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lower of the referenced value, unless otherwise stated or clearly evident from the context (except where the number would exceed 100% of the possible values).

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, a "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration as being acceptable for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gum tragacanth; malt; gelatin; talc; cocoa butter, wax, animal and vegetable fats, paraffin, siloxane, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a phosphate buffer solution; and any other compatible materials used in pharmaceutical formulations.

"pharmaceutically acceptable salts" include acid addition salts and base addition salts.

"pharmaceutically acceptable acid addition salts" refers to those salts that retain the biological effectiveness and properties of the free base which are not biologically or otherwise undesirable, and are prepared with inorganic acids (such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like) as well as organic acids (such as, but not limited to, acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, heptonic acid, gluconic acid, glucuronic acid, glutamic acid, and the like), Glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like).

"pharmaceutically acceptable base addition salts" refers to those salts that retain the biological effectiveness and properties of the free acid which are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. For example, inorganic salts include, but are not limited to, ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to: salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, danitol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenybenzylamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Example organic bases used in certain embodiments include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, detackifying agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The compositions described herein may be formulated in any manner suitable for the desired delivery route, such as trans-tympanic injection, trans-tympanic stylet or catheter, and injectable depot. In general, the formulations comprise all physiologically acceptable compositions (including derivatives or prodrugs, solvates, stereoisomers, racemates or tautomers thereof) as well as any physiologically acceptable carriers, diluents and/or excipients.

Detailed Description

Example embodiments of the present invention will be described below.

The present disclosure relates to methods for preventing, reducing or treating the incidence and/or severity of inner ear disorders and hearing impairment involving inner ear tissue, particularly inner ear hair cells, progenitors thereof, and optionally vascular striations and associated auditory nerves. Of particular interest are conditions that result in permanent hearing loss and/or reduced hair cell function that may result from a reduction in hair cell numbers. Also of interest are conditions that arise as adverse side effects of ototoxic therapeutic agents (including cisplatin and its analogs, aminoglycoside antibiotics, salicylates and their analogs, or loop diuretics). In certain embodiments, the present disclosure relates to inducing, promoting or enhancing the growth, proliferation or regeneration of inner ear tissue, particularly inner ear support cells and hair cells.

In particular, the compounds provided herein can be used for the preparation of pharmaceutical preparations for the prevention and/or treatment of acute and chronic ear diseases and hearing loss, dizziness and balance problems, in particular for the prevention and/or treatment of sudden hearing loss, acoustic trauma, hearing loss due to prolonged noise exposure, presbycusis, trauma during implantation of an inner ear prosthesis (insertion trauma), dizziness due to diseases of the inner ear region, Meniere's disease-related dizziness and/or dizziness as a symptom of Meniere's disease, tinnitus and hearing loss due to antibiotics and cytostatics and other drugs.

Advantageously, the compositions disclosed herein are capable of activating pathways and mechanisms known to be involved in inducing stem cell properties, such as those used to generate "induced pluripotent stem cells" (e.g., in combination with Wnt stimulation, HDAC inhibition, TGF- β inhibition, RAR activation, and/or DKK1 inhibition). When a population of cochlear support cells is treated with such a composition, the treated support cells exhibit stem cell-like behavior, whether the population is in vivo or in vitro, because the treated support cells have the ability to proliferate and differentiate, more specifically, into cochlear hair cells. Preferably, the compositions induce and maintain the production of daughter stem cells by the support cells, which are capable of dividing through many generations and retain a high proportion of the capacity of the resulting cells to differentiate into hair cells. In certain embodiments, the stem cell marker expressed by a proliferating stem cell may comprise Lgr5, Sox2, operml, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3, Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt3l, Utf1, Tcl1, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3, D1, Smad2, Smad2/3, smasmasad 39 4, Smad5 and/or 7.

In some embodiments, the compositions of the present disclosure can be used to maintain or even transiently increase the sternness (i.e., self-renewal) of a pre-existing support cell population prior to significant hair cell formation. In some embodiments, the pre-existing support cell population comprises inner column cells, outer column cells, inner finger cells, Deiter cells, hansen cells, bertson cells, and/or claudi cells. Immunostaining on representative microscopy samples can be usedColor morphology analysis (including cell counts) and lineage tracing to confirm amplification of one or more of these cell types. In some embodiments, the pre-existing support cells comprise Lgr5⁺A cell. Immunostaining morphology analysis (including cell counts) and qPCR and RNA hybridization can be used to confirm Lgr5 upregulation in cell populations.

Advantageously, the methods and compositions of the present disclosure achieve these goals without the use of genetic manipulation. The germ line manipulations used in many academic studies are not ideal therapeutic methods for treating hearing loss. In general, the therapy preferably comprises administration of small molecules, peptides, antibodies or other non-nucleic acid molecules or nucleic acid delivery vehicles without concomitant gene therapy. In certain embodiments, the therapy comprises administration of a small organic molecule. Preferably, hearing protection or restoration is achieved by using a (non-genetic) therapeutic agent that is injected into the middle ear and diffuses into the cochlea.

The cochlea is very dependent on all the cell types present, and the architecture of these cells is important to its function. As supporting cells play an important role in neurotransmitter circulation and cochlear mechanics. Thus, maintaining a rosette-like pattern within the Corti device may be important to function. Cochlear mechanics of the basement membrane activates hair cell transduction. Ectopic hair cells have been generated by Atoh1 virus transduction, but are less likely to contribute to the auditory response due to sensory-related misalignments of these cells and mismatches with the apical membrane (tectomy membrane). In addition, these cells appear more similar to vestibular hair cells or non-mammalian hair cells. Thus, more signaling is necessary for cochlear hair cell development than with Atoh1 or Notch inhibition alone. Due to the high sensitivity of cochlear mechanics, it is also desirable to avoid cell clumping. In summary, maintaining the correct distribution and relationship of hair cells and supporting cells along the basement membrane (even after proliferation) may be a desirable feature for hearing because supporting cell function and correct mechanics are necessary for normal hearing.

In native cochlea, patterning of hair cells and supporting cells occurs through Notch lateral inhibition, where cells that become hair cells signal to nearby supporting cells to inhibit Atoh1 (a gene essential for hair cell fate), thereby producing a rosette-like epithelium. In one embodiment of the present disclosure, the cell density of hair cells in a cochlear cell population is increased in a manner that maintains even the characteristic rosette-like pattern of cochlear epithelium.

According to one aspect of the present disclosure, the cell density of hair cells can be increased in a cochlear cell population that includes hair cells and supporting cells. The cochlear cell population may be an in vivo population (i.e., consisting of the cochlear epithelium of the subject), or the cochlear cell population may be an in vitro (ex vivo) population. If the population is an in vitro population, the increase in cell density can be determined by reference to representative microscopic samples of the population taken before and after any treatment. If the population is an in vivo population, the increase in cell density can be determined indirectly by determining the effect of the increase in hair cell density associated with improved hearing on the hearing of the subject.

In one embodiment, a support cell placed in a stem cell proliferation assay forms a banded synapse in the absence of neuronal cells.

In the native cochlea, patterning of hair cells and supporting cells occurs in a parallel fashion to the basement membrane. In one embodiment of the disclosure, proliferation of the support cells in the cochlear cell population is amplified by way of the characteristic basement membrane of the cochlear epithelium.

In one such embodiment, when the composition is administered to cochlear tissue, the number of consecutive hair cells in the expanded cochlear cell population is less than 5% of the hair cells in the population. For another example, in one such embodiment, the number of consecutive hair cells in the expanded cochlear cell population is less than 4% of the hair cells in the population. For another example, in one such embodiment, the number of consecutive hair cells in the expanded cochlear cell population is less than 3% of the hair cells in the population. For another example, in one such embodiment, the number of consecutive hair cells in the expanded cochlear cell population is less than 2% of the hair cells in the population. For another example, in one such embodiment, the number of consecutive hair cells in the expanded cochlear cell population is less than 1% of the hair cells in the population. In some embodiments, the composition expands inner ear support cells to produce additional hair cells, wherein the epithelial tissue resembles a native morphology with no more than 5% contiguous hair cells when viewed under a microscope.

In some embodiments, the composition expands inner ear support cells in an animal over 2 weeks of age to produce additional hair cells, wherein the epithelial tissue resembles a native morphology with no more than 5% of adjacent hair cells in a representative microscopy sample.

In some embodiments, the composition results in > 5% of hair cells in a representative microscopy sample being in contact with both supporting cells and neurons.

In some embodiments, the composition results in less than 5% of hair cells that border other hair cells in a representative microscopy sample.

In one embodiment, the number of support cells in the initial cochlear cell population is selectively expanded by treating the initial cochlear cell population with a composition of the present disclosure (e.g., a composition containing an effective concentration of a sternness driver and an effective concentration of a differentiation inhibitor) to form an intermediate cochlear cell population, and wherein the ratio of support cells to hair cells in the intermediate cochlear cell population is greater than the ratio of support cells to hair cells in the initial cochlear cell population. The expanded cochlear cell population can be, for example, an in vivo population, an ex vivo population, or even an in vitro explant. In one such embodiment, the ratio of support cells to hair cells in the intermediate cochlear cell population is greater than the ratio of support cells to hair cells in the initial cochlear cell population. For example, in one such embodiment, the ratio of support cells to hair cells in the intermediate cochlear cell population is 1.1 times the ratio of support cells to hair cells in the initial cochlear cell population. For another example, in one such embodiment, the ratio of support cells to hair cells in the intermediate cochlear cell population is 1.5 times the ratio of support cells to hair cells in the initial cochlear cell population. For another example, in one such embodiment, the ratio of support cells to hair cells in the intermediate cochlear cell population is 2 times greater than the ratio of support cells to hair cells in the initial cochlear cell population. For another example, in one such embodiment, the ratio of support cells to hair cells in the intermediate cochlear cell population is 3 times greater than the ratio of support cells to hair cells in the initial cochlear cell population. In each of the foregoing embodiments, the ability of a composition of the present disclosure described in this paragraph to expand a cochlear cell population can be determined by a stem cell proliferation assay.

In one embodiment, the number of stem cells in a cochlear cell population is expanded by treating the cochlear cell population with a composition of the present disclosure (e.g., a composition containing an effective concentration of a sternness driver and an effective concentration of a differentiation inhibitor) to form an intermediate cochlear cell population, wherein the cell density of the stem cells in the intermediate cochlear cell population is greater than the cell density of the stem cells in the initial cochlear cell population. The treated cochlear cell population may be, for example, an in vivo population, an ex vivo population, or even an in vitro explant. In one such embodiment, the cell density of the stem cells in the treated cochlear cell population is at least 1.1 times greater than the cell density of the stem cells in the initial cochlear cell population. For example, in one such embodiment, the cell density of the stem cells in the treated cochlear cell population is at least 1.25 times the cell density of the stem cells in the initial cochlear cell population. For example, in one such embodiment, the cell density of the stem cells in the treated cochlear cell population is at least 1.5 times the cell density of the stem cells in the initial cochlear cell population. For another example, in one such embodiment, the cell density of the stem cells in the treated cochlear cell population is at least 2 times greater than the cell density of the stem cells in the initial cochlear cell population. For another example, in one such embodiment, the cell density of the stem cells in the treated cochlear cell population is at least 3 times greater than the cell density of the stem cells in the initial cochlear cell population. The cochlear cell population in vitro can expand significantly more than the in vivo population; for example, in certain embodiments, the cell density of the stem cells in the expanded in vitro stem cell population may be at least 4,5, 6,7, 8, 9, 10, 15, 20, 25, 35, 40, 45,50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or even 3000 times the cell density of the stem cells in the initial cochlear cell population. In each of the foregoing embodiments, the ability of the compositions of the present disclosure described in this paragraph to expand a cochlear cell population can be determined by a stem cell proliferation assay.

According to one aspect of the disclosure, treatment of a population of cochlear support cells with a composition of the disclosure (e.g., a composition containing an effective concentration of a sternness driver and an effective concentration of a differentiation inhibitor) increases the activity of Lgr5 of the population. For example, in one embodiment, the composition is capable of increasing and maintaining the activity of Lgr5 in a cochlear support cell population in vitro by at least a factor of 1.2. For another example, in one such embodiment, the composition is capable of increasing the activity of Lgr5 in a cochlear support cell population in vitro by at least a factor of 1.5. For another example, in one such embodiment, the composition is capable of increasing the activity of Lgr5 in a cochlear support cell population in vitro by a factor of 2,3, 5, 10, 100, 500, 1,000, 2,000, or even 3,000. An increase in Lgr5 activity may also be observed in the in vivo population, but the observed increase may be slightly more slight. For example, in one embodiment, the composition is capable of increasing activity of Lgr5 in a cochlear support cell population by at least 5% in vivo. For another example, in one such embodiment, the composition is capable of increasing the activity of Lgr5 in a cochlear support cell population by at least 10% in vivo. For another example, in one such embodiment, the composition is capable of increasing the activity of Lgr5 in a cochlear support cell population by at least 20% in vivo. For another example, in one such embodiment, the composition is capable of increasing the activity of Lgr5 in a cochlear support cell population by at least 30% in vivo. In each of the foregoing embodiments, this ability of the composition to increase Lgr5 activity may be, for example, in vitro Lgr5⁺Activity assays are demonstrated, but in vivo populations, e.g., in vivo Lgr5⁺Activity assayThis was confirmed by isolating organs and using immunostaining for morphological analysis, endogenous fluorescent protein expression of Lgr5 (e.g., Lgr5, Sox2), and qPCR for Lgr 5.

By treating Lgr 5-containing compositions (e.g., compositions containing an effective concentration of a sternness driver and an effective concentration of a differentiation inhibitor) with a composition of the present disclosure⁺The cell-supporting cochlear cell population (whether in vivo or in vitro), in addition to increasing the activity of Lgr5 in the population, can also increase Lgr5 in the cochlear cell population⁺Number of supporting cells. In general, the cell density of the stem/progenitor support cells can be expanded relative to the initial cell population by one or more of several mechanisms. For example, in one such embodiment, a newly generated Lgr5 with increased stem cell propensity (i.e., greater ability to differentiate into hair cells) may be generated⁺Supporting the cell. As another example, in one such embodiment, there are no child Lgr5⁺Cells are generated by cell division, but induce pre-existing Lgr5⁺Supporting differentiation of the cells into hair cells. As another example, in one such embodiment, there are no child Lgr5⁺Cells are generated by cell division, but instead Lgr5 is introduced^－The support cells are activated to a higher level of Lgr5 activity, and these activated support cells are then able to differentiate into hair cells. Regardless of the mechanism, in one embodiment, the compositions of the present disclosure are capable of modulating Lgr5 in a population of cochlear support cells isolated in vitro⁺The cell density of the supporting cells is increased by at least 5-fold. For another example, in one such embodiment, the composition is capable of modulating Lgr5 in an in vitro cochlear support cell population⁺The cell density of the supporting cells is increased by at least 10-fold. For another example, in one such embodiment, the composition is capable of modulating Lgr5 in an in vitro cochlear support cell population⁺The cell density of the supporting cells is increased at least 100-fold, at least 500-fold, at least 1000-fold, or even at least 2,000-fold. Lgr5⁺An increase in the cell density of the supporting cells may also be observed in the in vivo population, but the observed increase may be slightly more slight. For example, in one embodimentThe composition can promote growth of Lgr5 in cochlear support cell population in vivo⁺The cell density of the supporting cells is increased by at least 5%. For another example, in one such embodiment, the composition is capable of releasing Lgr5 from cochlear support cell populations in vivo⁺The cell density of the supporting cells is increased by at least 10%. For another example, in one such embodiment, the composition is capable of releasing Lgr5 from cochlear support cell populations in vivo⁺The cell density of the supporting cells is increased by at least 20%. For another example, in one such embodiment, the composition is capable of releasing Lgr5 from cochlear support cell populations in vivo⁺The cell density of the supporting cells is increased by at least 30%. This increase in the in vivo population of the composition is Lgr5⁺The ability to support cells can be demonstrated in, for example, a stem cell proliferation assay or an appropriate in vivo assay. In one embodiment, the compositions of the present disclosure are capable of increasing Lgr5 in the cochlea by inducing expression of Lgr5 in cells without detectable levels or with low detectable levels of Lgr5 protein⁺The number of cells while maintaining the native morphology. In one embodiment, the compositions of the present disclosure are capable of increasing Lgr5 in the cochlea by inducing expression of Lgr5 in cells without detectable levels or with low detectable levels of Lgr5 protein⁺Number of cells while maintaining native morphology and not producing cell aggregates.

Except for the addition of Lgr5⁺In addition to supporting the cell density of cells, in one embodiment, the compositions of the present disclosure are capable of increasing Lgr5 in cochlear cell populations⁺Cell to hair cell ratio. In one embodiment, Lgr5 in an initial cochlear cell population is selectively expanded by treating the initial cochlear cell population with a composition of the present disclosure (e.g., a composition containing an effective concentration of a sternness driver and an effective concentration of a differentiation inhibitor)⁺Supporting the number of cells to form an expanded cell population, and wherein Lgr5 is present in the expanded cochlear cell population⁺The number of support cells is at least equal to the number of hair cells. The expanded cochlear cell population can be, for example, an in vivo population, an ex vivo population, or even an in vitro explant. In one such embodiment, the expanded cochlear cell populationLgr5⁺The ratio of supporting cells to hair cells is at least 1: 1. For example, in one such embodiment, Lgr5 is present in an expanded cochlear cell population⁺The ratio of supporting cells to hair cells is at least 1.5: 1. For another example, in one such embodiment, Lgr5 is present in an expanded cochlear cell population⁺The ratio of supporting cells to hair cells is at least 2: 1. For another example, in one such embodiment, Lgr5 is present in an expanded cochlear cell population⁺The ratio of supporting cells to hair cells is at least 3: 1. For another example, in one such embodiment, Lgr5 is present in an expanded cochlear cell population⁺The ratio of supporting cells to hair cells is at least 4: 1. For another example, in one such embodiment, Lgr5 is present in an expanded cochlear cell population⁺The ratio of supporting cells to hair cells is at least 5: 1. In each of the foregoing embodiments, the ability of the compositions of the present disclosure described in this paragraph to expand a cochlear cell population can be determined by a stem cell proliferation assay.

In certain embodiments, the composition will sense Lgr5 on the epithelium⁺The proportion of cells in the total cells is increased by at least 10%, 20%, 50%, 100%, 250%, 500%, 1000% or 5000%.

In certain embodiments, the composition increases Lgr5⁺The cells are maintained until the cells reach at least 10%, 20%, 30%, 50%, 70% or 85% of the cells on the sensory epithelium (e.g., the organ of Corti).

In general, it is preferable to avoid excessive proliferation of the supporting cells in the cochlea. In one embodiment, the compositions of the present disclosure are capable of expanding a cochlear cell population without producing protrusions of new cells, such as cell aggregates, that exceed the natural surface of the cochlea. In some embodiments, the cochlear tissue has a native morphology 30 days after placement of the composition on the round window membrane. In some embodiments, 30 days after placing the composition on the round window membrane, the cochlear tissue has a native morphology and is free of cell aggregates. In some embodiments, 30 days after placing the composition on the round window membrane, the cochlear tissue has a native morphology with at least 10%, 20% of the Corti apparatus30%, 50%, 75%, 90%, 95%, 98% or even at least 99% of Lgr5⁺None of the cells are part of a cell aggregate.

In addition to the expansion of the bulk support cell population and specific Lgr5 described above⁺In support of extracellular, compositions of the present disclosure (e.g., compositions containing an effective concentration of a sternness driver and an effective concentration of a differentiation inhibitor) are capable of maintaining the ability to differentiate into hair cells in progeny cells. In the in vivo population, this maintenance of capacity can be indirectly observed by an improvement in the hearing of the subject. In the in vitro population, this maintenance of capacity can be observed directly by an increase in the number of hair cells relative to the starting population, or indirectly by measuring LGR5 activity, SOX2 activity, or one or more other stem cell markers identified elsewhere herein.

In one embodiment, the composition increases the overall cochlear support cell population and specific Lgr5⁺The ability to support the sternness of a cell population may be compared to isolated Lgr5⁺An increase in Lgr5 activity (determined using an Lgr5 activity assay) in an in vitro population of cells is correlated. As previously described, in one such embodiment, the composition is capable of increasing the activity of Lgr5 of stem cells in the intermediate cell population by an average of 5-fold relative to the activity of Lgr5 of cells in the initial cell population. For another example, in one such embodiment, the composition is capable of increasing the activity of Lgr5 of a stem cell gene in the intermediate cell population by a factor of 10 relative to the activity of Lgr5 of cells in the initial cell population. For another example, in one such embodiment, the composition is capable of increasing the activity of Lgr5 of stem cells in the intermediate cell population by a factor of 100 relative to the activity of Lgr5 of cells in the initial cell population. For another example, in one such embodiment, the composition is capable of increasing the activity of Lgr5 of stem cells in the intermediate cell population by a factor of 1000 relative to the activity of Lgr5 of cells in the initial cell population. In each of the above embodiments, the increase in stem cell activity in the cell population can be determined by immunostaining of the target gene or in vitro measurement of endogenous fluorescent protein expression and analyzing its relative intensity by imaging analysis or flow cytometry,Or in vitro using qPCR for the target stem cell gene. The identity of the resulting stem cell population may optionally be further determined by stem cell assays including stem cell marker expression assays, colony formation assays, self-renewal assays and differentiation assays defined in stem cell assays.

In some embodiments, the method, when applied to an adult mammal, produces adult mammal Lgr5 in S phase⁺A population of cells.

In one embodiment, the in vivo Lgr5 of the cell population in the organ of Corti, relative to the baseline value of the population not exposed to the composition, following application of the composition to the round window of a mouse⁺The activity increased 1.3-fold, 1.5-fold, up to 20-fold. In some embodiments, the average in vivo Lgr5 of cells in Corti organ relative to baseline values for a population not exposed to the composition after application of the composition to the round window of a mouse⁺The activity increased 1.3-fold, 1.5-fold, up to 20-fold.

In certain embodiments, the composition increases Lgr5⁺The cells are maintained until the cells reach at least 10%, 7.5%, 10%, up to 100% of the support cell population in number.

In some cases, a sternness driver can also induce differentiation of the supporting cells into hair cells if an effective differentiation-inhibiting concentration of a differentiation-inhibiting agent is not present. Examples of sternness drivers that can drive proliferation and differentiation include GSK3 β inhibitors and Wnt agonists. In certain embodiments, stem cell proliferation may be enhanced by the addition of a modulator of a pathway that regulates cell cycle or plasticity (e.g., p27) or a modulator of the Tgf β pathway.

In some embodiments, a dry driver may be used to drive Lgr5⁺Proliferation of stem cells. In some cases, a sternness driver may also induce Lgr5 if an effective differentiation-inhibiting concentration of a differentiation-inhibiting agent is not present⁺Examples of desiccating drivers that can drive proliferation and differentiation include GSK3 β inhibitorsAnd Wnt agonists. In some embodiments, the differentiation inhibitor is also a sternness driver. In some embodiments, the differentiation inhibitor is a Notch agonist and is also a sternness driver. In some embodiments, the differentiation inhibitor is valproic acid, which may be a sternness driver. If the differentiation inhibitor is also a sternness driver, the concentration of the differentiation inhibitor during the differentiation phase should be lower than the effective differentiation inhibiting concentration.

In certain embodiments, a combination of (i) a GSK3 β inhibitor and/or a Wnt agonist and (ii) a notch agonist and/or an HDAC inhibitor is used, applied to Lgr5 obtained from the inner ear of a mouse⁺When cells are present, the population of stem cells can be increased at least 3-fold over the population of stem cells prior to the administering step.

In certain embodiments, the composition is capable of sequestering Lgr5 in the cochlea⁺The percentage of cells is increased by 5%, 10%, 25%, 50% or 80%. in certain embodiments, a combination of (i) a GSK3 β inhibitor and/or a Wnt agonist and (ii) a notch agonist and/or a HDAC inhibitor is used, which is capable of targeting Lgr5 in the cochlea⁺The percentage of cells is increased by 5%, 10%, 25%, 50% or 80%.

Dry driving agent

Exemplary GSK3 β inhibitors of the present disclosure are listed in table 1.

TABLE 1 GSK3beta inhibitors

Classes of GSK3 β inhibitors for use in the various embodiments of the compositions and methods disclosed herein include, but are not limited to, those listed in column a of table 1. Specific GSK3 β inhibitors for use in the various embodiments of the compositions and methods disclosed herein include, but are not limited to, those listed in column B of table 1. Classes of Wnt agonists useful in the various embodiments of the compositions and methods disclosed herein include, but are not limited to, those listed in column a of table 2. Specific Wnt agonists for use in the various embodiments of the compositions and methods disclosed herein include, but are not limited to, those listed in column B of table 2. Classes of notch agonists useful in various embodiments of the compositions and methods disclosed herein include, but are not limited to, those listed in column a of table 3. Specific notch agonists for use in the various embodiments of the compositions and methods disclosed herein include, but are not limited to, those listed in column B of table 3. Classes of HDAC inhibitors useful in the various embodiments of the compositions and methods disclosed herein include, but are not limited to, those listed in column a of table 4. Specific HDAC inhibitors useful in the various embodiments of the compositions and methods disclosed herein include, but are not limited to, those listed in column B of table 4. All agents listed in table 1B, table 2B, table 3B, table 4B are understood to include derivatives or pharmaceutically acceptable salts thereof. All categories listed in table 1A, table 2A, table 3A, table 4A are understood to include the agents that constitute the category as well as derivatives or pharmaceutically acceptable salts thereof. Members of these categories also include, but are not limited to, those described in appendix A, pages 51-55, and appendix A, pages 90-102.

GSK3 β inhibitors also include, but are not limited to, the following agents: the agent reduces the activity of GSK3 β by more than 5%, 10%, 20%, 30% or 50% when an ear cell line or primary cells obtained from ear tissue is exposed to a pharmaceutically acceptable concentration of an inhibitor and the activity is assessed by western blotting or other standard methods in the literature. As used herein, a pharmaceutically acceptable concentration is that concentration of the active agent in the formulation that is non-toxic and can be delivered to the target tissue. In certain embodiments, the compositions comprise a GSK3 β inhibitor in combination with a notch agonist and/or an HDAC inhibitor, which GSK3 β inhibitor reduces GSK3 β activity by more than 5%, 10%, 20%, 30% or 50% when the conditions described in this paragraph are used. The "high potency GSK3 β inhibitor" is the following agent: the agent reduces the activity of GSK3 β by more than 50% when an ear cell line or primary cells obtained from ear tissue is exposed to a pharmaceutically acceptable concentration of an inhibitor and the activity is assessed by western blotting or other standard methods in the literature.

Wnt agonists

Exemplary Wnt agonists in the present disclosure are listed in table 2.

TABLE 2 Wnt agonists

Wnt agonists also include, but are not limited to, the following agents: when an ear cell line or primary cells obtained from ear tissue is exposed to a pharmaceutically acceptable concentration of agonist and activity is assessed by western blotting or other standard methods in the literature, the agent increases Wnt activity by more than 5%, 10%, 20%, 30% or 50%. In certain embodiments, the composition comprises a Wnt agonist in combination with a notch agonist and/or a HDAC inhibitor, which increases Wnt activity by more than 5%, 10%, 20%, 30% or 50% when conditions described in this paragraph are used. A "high potency Wnt agonist" is the following agent: when an ear cell line or primary cells obtained from ear tissue is exposed to a pharmaceutically acceptable concentration of agonist and activity is assessed by western blotting or other standard methods in the literature, the agent increases Wnt activity by more than 50%.

Differentiation inhibitors

Notch agonists

Exemplary Notch agonists in the present disclosure are listed in table 3.

TABLE 3 Notch agonists

Column A	B column
		Natural receptor ligands	Jagged 1
	Jagged 2
			Delta-like 1
	Delta-like 2
			Delta-like 3
	Delta-like 4
			DSL peptides
	Delta 1
			Delta D
Receptor antibodies	Notch 1 antibodies
		HDAC inhibitors	VPA
	TSA
			Tubastatin A
	Compound 7

Notch agonists also include, but are not limited to, the following: the agent increases Notch activity by more than 5%, 10%, 20%, 30% or 50% when ear cell lines or primary cells obtained from ear tissue are exposed to a pharmaceutically acceptable concentration of agonist and activity is assessed by western blotting or other standard methods in the literature. In certain embodiments, the compositions comprise a Notch agonist in combination with a GSK3 β inhibitor or a Wnt agonist, which increases Notch activity by more than 5%, 10%, 20%, 30%, or 50% when conditions described in this paragraph are used. A "high potency Notch agonist" is the following agent: the agent increases Notch activity by more than 50% when an ear cell line or primary cells obtained from ear tissue are exposed to a pharmaceutically acceptable concentration of agonist and activity is assessed by western blotting or other standard methods in the literature.

HDAC inhibitors

Exemplary HDAC inhibitors of the present disclosure are listed in table 4.

TABLE 4 HDAC inhibitors

HDAC inhibitors also include, but are not limited to, the following agents: when an ear cell line or primary cells obtained from ear tissue are exposed to an inhibitor and activity is assessed by western blot or other standard methods in the literature, the agent reduces the activity of HDAC by more than 5%, 10%, 20%, 30% or 50%. In certain embodiments, the composition comprises an HDAC inhibitor that reduces HDAC activity by more than 5%, 10%, 20%, 30%, or 50% when conditions described in this paragraph are used in combination with a GSK3 β inhibitor or a Wnt agonist. "high potency HDAC inhibitors" are the following agents: when an ear cell line or primary cells obtained from ear tissue is exposed to a pharmaceutically acceptable concentration of an inhibitor and activity is assessed by western blot or other standard methods in the literature, the agent reduces HDAC activity by more than 50%.

Representative methods for assessing HDAC inhibitors can be found in the following documents:

Mak BC,Takemaru K,Kenerson HL,Moon RT,Yeung RS(2003)."The tuberin-hamartin complex negatively regulates beta-catenin signaling activity".J.Biol.Chem.278(8):5947–51.doi:10.1074/jbc.C200473200.PMID 12511557.

Nakamura T,Hamada F,Ishidate T,Anai K,Kawahara K,Toyoshima K,AkiyamaT(1998)."Axin,an inhibitor of the Wnt signalling pathway,interacts with beta-catenin,GSK-3beta and APC and reduces the beta-catenin level".Genes Cells 3(6):395–403.doi:10.1046/j.1365-2443.1998.00198.x.PMID 9734785.

von Kries JP,Winbeck G,Asbrand C,Schwarz-Romond T,Sochnikova N,Dell'Oro A,Behrens J,Birchmeier W(2000)."Hot spots in beta-catenin forinteractions with LEF-1,conductin and APC".Nat.Struct.Biol.7(9):800–7.doi:10.1038/79039.PMID 10966653.

Schwarz-Romond T,Asbrand C,Bakkers J,Kühl M,Schaeffer HJ,Huelsken J,Behrens J,Hammerschmidt M,Birchmeier W(2002)."The ankyrin repeat proteinDiversinrecruits Casein kinase Iepsilon to the beta-catenin degradationcomplex and acts in bothcanonical Wnt and Wnt/JNK signaling".Genes Dev.16(16):2073–84.doi:10.1101/gad.230402.PMC 186448.PMID 12183362.

Wang L,Lin HK,Hu YC,Xie S,Yang L,Chang C(2004)."Suppression ofandrogen receptor-mediated transactivation and cell growth by the glycogensynthase kinase 3 beta in prostate cells".J.Biol.Chem.279(31):32444–52.doi:10.1074/jbc.M313963200.PMID 15178691.

Davies G,Jiang WG,Mason MD(2001)."The interaction between beta-catenin,GSK3beta and APC after motogen induced cell-cell dissociation,andtheir involvement in signal transduction pathways in prostate cancer".Int.J.Oncol.18(4):843–7.doi:10.3892/ijo.18.4.843.PMID 11251183.

Kishida S,Yamamoto H,Hino S,Ikeda S,Kishida M,Kikuchi A(1999)."DIXdomains of Dvl and axin are necessary for protein interactions and theirability to regulate beta-catenin stability".Mol.Cell.Biol.19(6):4414–22.PMC104400.PMID 10330181.

Hong YR,Chen CH,Cheng DS,Howng SL,Chow CC(1998)."Human dynamin-likeprotein interacts with the glycogen synthase kinase 3beta".Biochem.Biophys.Res.Commun.249(3):697–703.doi:10.1006/bbrc.1998.9253.PMID9731200.

Wu,Xiaoyang；Shen QT；Oristian DS；Lu CP；Zheng Q；Wang HW；Fuchs E(2011)."Skin Stem Cells Orchestrate Directional Migration by Regulating Microtubule-ACF7 Connections through GSK3b".Cell 144.doi:10.1016/j.cell.2010.12.033.

Li Y,Bharti A,Chen D,Gong J,Kufe D(1998)."Interaction of glycogensynthase kinase 3beta with the DF3/MUC1 carcinoma-associated antigen andbeta-catenin".Mol.Cell.Biol.18(12):7216–24.PMC 109303.PMID 9819408.

Li Y,Kuwahara H,Ren J,Wen G,Kufe D(2001)."The c-Src tyrosine kinaseregulates signaling of the human DF3/MUC1 carcinoma-associated antigen withGSK3 beta and beta-catenin".J.Biol.Chem.276(9):6061–4.doi:10.1074/jbc.C000754200.PMID 11152665.

Guo X,Ramirez A,Waddell DS,Li Z,Liu X,Wang XF(2008)."Axin and GSK3-control Smad3 protein stability and modulate TGF-signaling".Genes Dev.22(1):106–20.doi:10.1101/gad.1590908.PMC 2151009.PMID 18172167.

Foltz DR,Santiago MC,Berechid BE,Nye JS(2002)."Glycogen synthasekinase-3beta modulates notch signaling and stability".Curr.Biol.12(12):1006–11.doi:10.1016/S0960-9822(02)00888-6.PMID 12123574.

Espinosa L,Inglés-Esteve J,Aguilera C,Bigas A(2003)."Phosphorylationby glycogen synthase kinase-3 beta down-regulates Notch activity,a link forNotch and Wnt pathways".J.Biol.Chem.278(34):32227–35.doi:10.1074/jbc.M304001200.PMID 12794074.

Watcharasit P,Bijur GN,Zmijewski JW,Song L,Zmijewska A,Chen X,JohnsonGV,Jope RS(2002)."Direct,activating interaction between glycogen synthasekinase-3beta and p53 after DNA damage".Proc.Natl.Acad.Sci.U.S.A.99(12):7951–5.doi:10.1073/pnas.122062299.PMC 123001.PMID 12048243.

Dai F,Yu L,He H,Chen Y,Yu J,Yang Y,Xu Y,Ling W,Zhao S(2002)."Humanserum and glucocorticoid-inducible kinase-like kinase(SGKL)phosphorylatesglycogen syntheses kinase 3beta(GSK-3beta)at serine-9through directinteraction".Biochem.Biophys.Res.Commun.293(4):1191–6.doi:10.1016/S0006-291X(02)00349-2.PMID 12054501.

in some embodiments, the differentiation inhibitor is selected to have a solubility relative to the drykinesis that facilitates release of the differentiation inhibitor in an aqueous environment faster than the drykinesis. In some embodiments, the differentiation inhibitor has a solubility in phosphate buffered saline that is 5, 10, 50, 100, 500, 1000, or 5000 times greater than the solubility of the dryness driver.

TGF-beta inhibitors

In certain embodiments, the one or more additional agents comprise a TGF β type I receptor inhibitor. Exemplary TGF- β inhibitors are shown in table 5.TGF β type I receptor inhibitors include, but are not limited to, 2- (3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) -1, 5-naphthyridine, [3- (pyridin-2-yl) -4- (4-quinolinyl) ] -1H-pyrazole, and 3- (6-methylpyridin-2-yl) -4- (4-quinolinyl) -1-phenylthiocarbamoyl-1H-pyrazole, which is commercially available from Calbiochem (San Diego, ca). Other small molecule inhibitors include, but are not limited to, SB-431542 (see, e.g., Halder et al, 2005; Neolasia 7(5): 509-.

TABLE 5 TGF-. beta.inhibitors

Additional therapeutic agents

In certain embodiments, the administering step comprises administering or causing to be administered one or more additional agents (e.g., agents other than (i) and (ii)) to the population of stem cells. In certain embodiments, the one or more additional agents comprise a ROS inhibitor or scavenger. ROS scavengers include, but are not limited to, enzymes such as catalase, glutathione peroxidase, and ascorbate peroxidase. In addition, vitamins A, E and C are known to have scavenger activity. Minerals such as selenium and manganese may also be effective compounds for scavenging ROS.

ROS inhibitors include, but are not limited to, alpha-lipoic acid, superoxide dismutase mimics, or catalase mimics. The superoxide dismutase mimetic or catalase mimetic may be MnTBAP (Mn (III) tetrakis (4-benzoic acid) porphyrin chloride) (produced by Calbiochem), ZnTBAP (Zn (III) tetrakis (4-benzoic acid) porphyrin chloride), SC-55858 (dichloro (2R,3R,8R, 9R-dicyclohexyl-1, 4,7,10, 13-pentaazacyclopentadecane) manganese (II) ], Euk-1343,3' -methoxysarin Mn (III)) (produced by Eukarion), M40403 (dichloro [ (4aR,13aR,17aR,21aR) -1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20, 21-eicosahydro-7, 11-nitrilo-7H-dibenzo [1,4,7,10] tetraazacyclo-heptadecane-Kacarbene, kappaN13, kappaN18, kappaN21, kappaN22] manganese (produced by Metaphore), AEOL 10112, AEOL 10113, and AEOL 10150 (manganese (III) meta-tetrakis (di-N-ethylimidazole) porphyrin) (all AEOL compounds are produced by Incara Pharmaceuticals). Alternatively, the ROS inhibitor may be an iron chelator. Among the iron chelators, desferrioxamine or DFO may be of prime importance as it is FDA approved for the treatment of iron excess in thalassemia. Furthermore, the ROS inhibitor may be a composition consisting of a mixture of iron chelators.

ROS inhibitors may be radioprotective agents and include compounds such as uric acid, thionine sulfate, diethyl maleate, vitamin E, vitamin C, cysteine (e.g., N-acetylcysteine) or glutathione, metronidazole, and retinoids (e.g., vitamin A). Other ROS scavengers can be found.

In certain embodiments, the one or more additional agents comprise vitamin C or a derivative thereof. In certain embodiments, the one or more additional agents comprise a TGF β type I receptor inhibitor. TGF β type I receptor inhibitors include, but are not limited to, 2- (3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) -1, 5-naphthyridine, [3- (pyridin-2-yl) -4- (4-quinolinyl) ] -1H-pyrazole, and 3- (6-methylpyridin-2-yl) -4- (4-quinolinyl) -1-phenylthiocarbamoyl-1H-pyrazole, which is commercially available from Calbiochem (San Diego, ca). Other small molecule inhibitors include, but are not limited to, SB-431542 (see, e.g., Halder et al, 2005; Neolasia 7(5): 509-.

In one embodiment, the ALK5 inhibitor, 2- (3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) 1, 5-naphthyridine, is used with the methods described herein. This inhibitor, also referred to herein as ALK5 inhibitor II, is commercially available from Calbiochem (catalog No. 616452, San Diego, ca). In one embodiment, the inhibitor is SB431542, which is an ALK-4, -5, -7 inhibitor, commercially available from Sigma (product number 54317, Saint Louis, Mo.). SB431542 is also expressed by the following chemical name: 4- [4- (1, 3-benzodioxol-5-yl) -5- (2-pyridyl) -1H-imidazol-2-yl ] -benzamide, 4- [4- (3, 4-methylenedioxyphenyl) -5- (2-pyridyl) -1H-imidazol-2-yl ] -benzamide, or 4- (5-benzo [1,3] dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl) -benzamide hydrate.

Small molecule inhibitors of TGF- β signaling can be classified based on the basic backbone of the molecule. For example, TGF- β signaling inhibitors may be based on dihydropyrrolopyrazole-based frameworks, imidazole-based frameworks, pyrazolopyridine-based frameworks, pyrazole-based frameworks, imidazopyridine-based frameworks, triazole-based frameworks, pyridopyrimidine-based frameworks, pyrrolopyrazole-based frameworks, isothiazole-based frameworks, and oxazole-based frameworks.

TGF- β signalling inhibitors are described, for example, in the following documents: callahan, J.F., et al, J.Med.chem.45,999-1001 (2002); sawyer, J.S. et al, J.Med.chem.46,3953-3956 (20031; Gellibert, F. et al, J.Med.chem.47,4494-4506 (2004); Tojo, M. et al, Cancer Sci.96:791- (ii) a U.S. patent application publication 2004/0147574; U.S. patent application publication 2007/0066632; U.S. patent application publication 2003/0028905; U.S. patent application publication 2005/0032835; U.S. patent application publication 2008/0108656; U.S. patent application publication 2004/015781; U.S. patent application publication 2004/0204431; U.S. patent application publication 2006/0003929; U.S. patent application publication 2007/0155722; U.S. patent application publication 2004/0138188 and U.S. patent application publication 2009/0036382; the contents of each of which are incorporated herein by reference in their entirety.

Oligonucleotide TGF- β signalling modulators, such as siRNA and antisense oligonucleotides, are described in: us patent 5,731,424; us patent 6,124,449; U.S. patent application publications 2008/0015161, 2006/0229266, 2004/0006030, 2005/0227936, and 2005/0287128, the contents of each of which are incorporated by reference herein in their entirety. Other antisense nucleic acids and sirnas can be obtained by methods known to those of ordinary skill in the art.

Exemplary TGF- β signaling inhibitors include, but are not limited to: AP-12009 (TGF-beta receptor type II antisense oligonucleotides), Lerdelimumab (CAT 152, anti-TGF-beta receptor type II antibody), GC-1008 (antibodies against all isoforms of human TGF-beta), ID11 (antibodies against all isoforms of murine TGF-beta), soluble TGF-beta receptor type II, dihydropyrroloimidazole analogs (e.g., SKF-104365), triarylimidazole analogs (e.g., SB-202620(4- (4- (4-fluorophenyl) -5- (pyridin-4-yl) -1H-imidazol-2-yl) benzoic acid) and SB-203580(4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- (4-pyridyl) -1H-imidazole)), RL-0061425, 1, 5-naphthyridinaminothiazole and pyrazole derivatives (e.g. 4- (6-methyl-pyridin-2-yl) -5- (1, 5-naphthyridin-2-yl) -1, 3-thiazol-2-amine and 2- [3- (6-methyl-pyridin-2-yl) -1H-pyrazol-4-yl ] -1, 5-naphthyridine), SB-431542(4- (5-benzo [1,3] dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl) -benzamide), GW788388(4- (4- (3- (pyridin-2-yl)) -1H-pyrazol-4-yl) Pyridin-2-yl) -N- (tetrahydro-2H-pyran-4-yl) benzamide), a-83-01(3- (6-methyl-2-pyridyl) -N-phenyl-4- (4-quinolyl)) -1H-pyrazole-1-thioamide), Decorin, Lefty 1, Lefty 2, follistatin, Noggin, Chordin, Cerberus, Gremlin, Inhibin (Inhibin), BIO (6-bromo-indirubin-3' -oxime), Smad proteins (e.g., Smad6, Smad7), and cystatin C.

TGF- β signaling inhibitors also include molecules that inhibit TGF- β receptor type I. Inhibitors of the TGF-. beta.receptor type I are described in the following references: byfield, S.D. and Roberts, A.B., Trends Cell biol.14,107-111 (2004); sawyer j.s. et al, bioorg.med.chem.lett.14,3581-3584 (2004); sawyer, J.S. et al, J.Med.chem.46,3953-3956 (2003); byfield, s.d., et al, mol. pharmacol.65,744-752 (2004); gellibert, F. et al, J.Med.chem.47,4494-4506 (2004); yingling, j.m., et al, Nature rev.drug disc.3,1011-1022 (2004); dumont, N, et al, Cancer Cell 3,531-536 (2003); tojo, M, et al, Cancer Sci.96:791-800 (2005); WO 2004/026871; WO 2004/021989; WO 2004/026307; WO 2000/012497; us patent 5,731,424; us patent 5,731,144; us patent 7,151,169; US 2004/00038856 and US 2005/0245508; the contents of each of which are incorporated herein by reference in their entirety.

Combination of reagents

In certain embodiments, the composition comprises an agent (or derivative or pharmaceutically acceptable salt thereof) in the categories of column a of table 1 and an agent (or derivative or pharmaceutically acceptable salt thereof) in the categories of column a of table 3. In certain embodiments, the composition comprises an agent (or derivative or pharmaceutically acceptable salt thereof) in the categories of column a of table 1 and an agent (or derivative or pharmaceutically acceptable salt thereof) in the categories of column a of table 4. In certain embodiments, the composition comprises an agent (or derivative or pharmaceutically acceptable salt thereof) in the categories of column a of table 2 and an agent (or derivative or pharmaceutically acceptable salt thereof) in the categories of column a of table 3. In certain embodiments, the composition comprises an agent (or derivative or pharmaceutically acceptable salt thereof) in the categories of column a of table 2 and an agent (or derivative or pharmaceutically acceptable salt thereof) in the categories of column a of table 4.

In certain embodiments, the composition comprises an agent of column B of table 1 (or a derivative or pharmaceutically acceptable salt thereof) and an agent of column B of table 3 (or a derivative or pharmaceutically acceptable salt thereof). In certain embodiments, the composition comprises an agent of column B of table 1 (or a derivative or pharmaceutically acceptable salt thereof) and an agent of column B of table 4 (or a derivative or pharmaceutically acceptable salt thereof). In certain embodiments, the composition comprises an agent of column B of table 2 (or a derivative or pharmaceutically acceptable salt thereof) and an agent of column B of table 3 (or a derivative or pharmaceutically acceptable salt thereof). In certain embodiments, the composition comprises an agent of column B of table 2 (or a derivative or pharmaceutically acceptable salt thereof) and an agent of column B of table 4 (or a derivative or pharmaceutically acceptable salt thereof).

In certain embodiments, the compositions comprise a combination of agents comprising (i) a GSK3- β inhibitor and/or a Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof) and (ii) a notch agonist and/or a HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof), wherein the agents are from tables 1-4, respectively.

In certain embodiments, the compositions comprise (i) a GSK3- β inhibitor (or derivative or pharmaceutically acceptable salt thereof) selected from an aminopyrimidine, an inorganic atom, or a thiadiazolidinedione and (ii) a notch agonist and/or an HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof) from tables 3-4. In certain embodiments, the compositions comprise (i) a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) selected from a GSK3- β inhibitor, a Wnt ligand or a Wnt-related protein and (ii) a notch agonist and/or an HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof) from tables 3-4. In certain embodiments, the compositions comprise (i) a Notch agonist (or derivative or pharmaceutically acceptable salt thereof) selected from an HDAC inhibitor or a natural receptor ligand and (ii) a GSK3- β inhibitor and/or a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) from tables 1-2. In certain embodiments, the compositions comprise (i) an HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof) selected from a hydroxamate, fatty acid or benzamide and (ii) a GSK3- β inhibitor and/or a Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof) from tables 1-2. In certain embodiments, the compositions comprise a combination of agents comprising (i) a GSK3- β inhibitor and/or a Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof) and (ii) a notch agonist and/or a HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof), wherein the agents are from tables 1-4, respectively.

In certain embodiments, the composition comprises (i) a GSK3- β inhibitor (or derivative or pharmaceutically acceptable salt thereof) selected from CHIR99021, lithium or NP031112(Tideglusib) and (ii) a notch agonist and/or HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof) from tables 3-4. In certain embodiments, the composition comprises (i) a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) selected from CHIR99021, Wnt3a or R-spondin1 and (ii) a notch agonist and/or an HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof) from tables 3-4. In certain embodiments, the compositions comprise (i) a Notch agonist (or derivative or pharmaceutically acceptable salt thereof) selected from valproic acid, SAHA, Jagged 1, Delta-like 1, or Delta-like 4 and (ii) a GSK3- β inhibitor and/or a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) from tables 1-2. In certain embodiments, the composition comprises (i) an HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof) selected from valproic acid, SAHA (vorinostat) or Tubastatin a and (ii) a GSK3- β inhibitor and/or a Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof) from tables 1-2. In certain embodiments, the compositions comprise a combination of agents comprising (i) a GSK3- β inhibitor and/or a Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof) and (ii) a notch agonist and/or a HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof), wherein the agents are from tables 1-4, respectively.

In certain embodiments, the compositions comprise (i) a GSK3- β inhibitor (or derivative or pharmaceutically acceptable salt thereof) selected from an aminopyrimidine, an inorganic atom, or a thiadiazolidinedione and (ii) a high potency notch agonist and/or a high potency HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof) from tables 3-4. In certain embodiments, the compositions comprise (i) a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) selected from a GSK3- β inhibitor, a Wnt ligand or a Wnt-related protein and (ii) a high potency notch agonist and/or a high potency HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof) from tables 3-4. In certain embodiments, the compositions comprise (i) a Notch agonist (or a derivative or pharmaceutically acceptable salt thereof) selected from an HDAC inhibitor or a natural receptor ligand and (ii) a high potency GSK3- β inhibitor and/or a high potency Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof) from tables 1-2. In certain embodiments, the composition comprises an HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof) selected from a hydroxamate, a fatty acid, or a benzamide and (ii) a high potency GSK3- β inhibitor and/or a high potency Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof) from tables 1-2.

In certain embodiments, the compositions comprise (i) a GSK3- β inhibitor (or derivative or pharmaceutically acceptable salt thereof) selected from CHIR99021, lithium or NP031112(Tideglusib) and (ii) a high potency notch agonist and/or a high potency HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof) from tables 3-4. In certain embodiments, the composition comprises (i) a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) selected from CHIR99021, Wnt3a or R-spondin1 and (ii) a high potency notch agonist and/or a high potency HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof) from tables 3-4. In certain embodiments, the compositions comprise (i) a Notch agonist (or derivative or pharmaceutically acceptable salt thereof) selected from valproic acid, SAHA, Jagged 1, Delta-like 1, or Delta-like 4 and (ii) a high potency GSK3- β inhibitor and/or a high potency Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) from tables 1-2. In certain embodiments, the composition comprises an HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof) selected from valproic acid, SAHA (vorinostat) or Tubastatin a and (ii) a high potency GSK3- β inhibitor and/or a high potency Wnt agonist (or a derivative or pharmaceutically acceptable salt thereof) from tables 1-2.

In certain embodiments, the compositions comprise a combination of agents comprising (i) a GSK3- β inhibitor and/or a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) and a second agent different from the first agent, which is (ii) a notch agonist and/or an HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof).

In certain embodiments, the compositions comprise a combination of agents comprising (i) a high potency GSK3- β inhibitor and/or a high potency Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) and (ii) a high potency notch agonist and/or a high potency HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof). In certain embodiments, the compositions comprise a combination of agents comprising (i) a high potency GSK3- β inhibitor and/or a high potency Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) and (ii) a notch agonist and/or an HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof). In certain embodiments, the compositions comprise a combination of agents comprising (i) a GSK3- β inhibitor and/or a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) and (ii) a high potency notch agonist and/or a high potency HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof).

In certain embodiments, the composition comprises a dry propellant at a concentration of 5, 10, 20, 50, 100, 200, 500, 1000, or 5000 times the effective dry propellant. In certain embodiments, the composition further comprises a differentiation inhibitor at a5, 10, 20, 50, 100, 200, 500, 1000, or 5000-fold effective differentiation inhibitory concentration. Optionally, any of the above compositions may comprise one or more agents that target ROS or Tgf β. Optionally, any of the above compositions may comprise one or more neurotrophins.

Delivery characteristics of combined agents

In some embodiments, a dry driver may be used to drive Lgr5⁺Proliferation of stem cells. In some cases, if there is no valid score presentDifferentiation inhibitor with chemoinhibitory concentration, and Lgr5 induced by xerosis driver⁺In some embodiments, a prior proliferation phase of a Wnt agonist or a GSK3 β inhibitor at an effective sternness driver concentration and a Notch agonist or an HDAC inhibitor at an effective differentiation inhibitory concentration may be present, followed by a differentiation phase of a Wnt agonist or a GSK3 β inhibitor at an effective sternness driver concentration and a differentiation inhibitor at an effective differentiation inhibitory concentration, followed by a differentiation phase of a Wnt agonist or a GSK3 β inhibitor at an effective sternness driver concentration but no effective differentiation inhibitory concentration.

In some embodiments, the desired proliferation period is 1,2, 4, 8, 16, 24, 48, 72, 96, or 192 hours. In some embodiments, the composition maintains an effective release rate of the dry propellant throughout the desired proliferation period. In some embodiments, the composition maintains an effective release rate of the dry propellant for at least 1 hour. In some embodiments, the desired dry propellant release rate is 10, 20, 50, 100, 500, or 1000 times the effective release rate of the dry propellant during the desired proliferative period.

In some embodiments, the desired proliferation period is 1,2, 4, 8, 16, 24, 48, 72, 96, or 192 hours. In some embodiments, the composition placed on the round window membrane of the mouse maintains an effective dry driver concentration throughout the cochlea during the desired proliferative phase. In some embodiments, the composition placed on the round window membrane of a mouse maintains an effective dry driver concentration in the cochlea for at least 1 hour. In some embodiments, the composition placed on the round window membrane of a mouse maintains an effective dry driver concentration in the cochlea for at least 2,4, 8, 16, 24, 48, 72, 96, or 192 hours.

In some embodiments, the desired proliferation period is 1,2, 4, 8, 16, 24, 48, 72, 96, or 192 hours. In some embodiments, the composition maintains an effective release rate of the differentiation inhibitor throughout the desired proliferative phase. In some embodiments, the composition maintains an effective release rate of the differentiation inhibitor for at least 1 hour. In some embodiments, the composition placed on the round window membrane of a mouse maintains an effective release rate of the differentiation inhibitor for at least 2,4, 8, 16, 24, 48, 72, 96, or 192 hours.

In some embodiments, the desired proliferation period is 1,2, 4, 8, 16, 24, 48, 72, 96, or 192 hours. In some embodiments, the composition placed on the round window membrane of the mouse maintains an effective differentiation-inhibiting concentration of the differentiation-inhibiting agent throughout the cochlea during the desired proliferative phase. In some embodiments, the composition placed on the round window membrane of a mouse maintains an effective differentiation inhibitory concentration in the cochlea for at least 1 hour. In some embodiments, the composition placed on the round window membrane of a mouse maintains an effective differentiation inhibitory concentration in the cochlea for at least 2,4, 8, 16, 24, 48, 72, 96, or 192 hours.

In some embodiments, the desired proliferation period is 1,2, 4, 8, 16, 24, 48, 72, 96, or 192 hours. In some embodiments, the composition may release both the sternness driver and the differentiation inhibitor. It may be advantageous to have a differentiation inhibitor to reduce Lgr5 that the sicca driver would like to proliferate the therapy⁺The extent to which Notch activity in target cells is reduced. In some embodiments, the composition has a release rate of the sternness drivers and differentiation inhibitors throughout the proliferation period such that: if the mass of agent released within 1 hour is placed in 30. mu.l and added to a Notch activity assay in cell culture, its Notch activity will be greater than20, 30, 40, 50, 60, 70, 80 or 90 of Notch activity in the native state when the agent is not applied.

In some embodiments, the desired proliferation period is 1,2, 4, 8, 16, 24, 48, 72, 96, or 192 hours. In some embodiments, the composition may release both the sternness driver and the differentiation inhibitor. In some embodiments, a desired composition placed on a mouse round window membrane simultaneously releases an amount of a dry driver and a differentiation inhibitor to maintain a Notch activity of 20, 30, 40, 50, 60, 70, 80, or 90 greater than the Notch activity in the native state without application of the agent.

In some embodiments, the desired proliferation period is 1,2, 4, 8, 16, 24, 48, 72, 96, or 192 hours. In some embodiments, the composition may release both the sternness driver and the differentiation inhibitor. It may be advantageous to have the differentiation inhibitor reduce Lgr5 that the sicca driver would like to proliferate the therapy⁺The extent to which Notch activity in target cells is reduced. In some embodiments, the composition has a release rate of the sternness drivers and differentiation inhibitors throughout the proliferation period such that: if the mass of agent released within 1 hour is placed in 30 μ l and added to a Notch activity assay in cell culture, its Notch activity will be 2,3,4,5, 10, 20, 50, 100, 500, 100 times greater than the Notch activity obtained when the composition contains an equivalent amount of dry drivers but does not contain any differentiation inhibitors.

In some embodiments, the desired proliferation period is 1,2, 4, 8, 16, 24, 48, 72, 96, or 192 hours. In some embodiments, the composition may release both the sternness driver and the differentiation inhibitor. It may be advantageous to have the differentiation inhibitor reduce Lgr5 that the sicca driver would like to proliferate the therapy⁺The extent to which Notch activity in target cells is reduced. In some embodiments, Lgr5 in the cochlea when a composition comprising a sternness driver and a differentiation inhibitor is placed on the round window membrane of a mouse⁺The Notch activity of the cells is greater than when the composition contains an equivalent amount of the sternness driver but does not contain any differentiation inhibitor2,3,4,5, 10, 20, 50, 100, 500, 100 times the Notch activity obtained.

In some embodiments, the desired differentiation period is 1,2, 4, 8, 16, 24, 48, 72, 96 days. In some embodiments, the composition does not achieve an effective release rate of the differentiation inhibitor at any time during the desired differentiation phase. In some embodiments, the composition does not achieve an effective release rate of the differentiation inhibitor for more than 1 day. In some embodiments, the composition does not achieve an effective release rate of the differentiation inhibitor for at least 2,4, 8, 16, 24, 48, 72, or 96 days.

In some embodiments, the desired differentiation period is 1,2, 4, 8, 16, 24, 48, 72, 96 days. In some embodiments, the composition placed on the round window membrane of a mouse does not maintain an effective differentiation-inhibiting concentration of a differentiation-inhibiting agent at any time during the desired differentiation phase. In some embodiments, the composition placed on the mouse round window membrane does not maintain an effective differentiation inhibitory concentration for more than 1 day. In some embodiments, the composition does not maintain an effective differentiation inhibitory concentration for at least 2,4, 8, 16, 24, 48, 72, or 96 days.

In some embodiments, the dry drivers need to be released for a longer period of time than the differentiation inhibitors. In some embodiments, the average release time of the dry driver is 2,4, 8, 16, or 32 times the average release time of the differentiation inhibitor.

In certain embodiments, the population of stem cells comprises support cells. In certain embodiments, the supporting cell is Lgr5⁺A cell. In certain embodiments, the population of stem cells comprises postnatal cells. In certain embodiments, the hair cells are inner ear hair cells. In some embodiments, the hair cells are external ear hair cells.

In certain embodiments, the stem cells comprise progenitor cells.

In certain embodiments, the administering step comprises administering or causing to be administered to the population of stem cells a notch agonist that is also an HDAC inhibitor. In certain embodiments, the administering step comprises administering or causing to be administered a notch agonist comprising a synthetic molecule to the stem cell population. In certain embodiments, the administering step comprises administering or causing valproic acid (VPA) (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to be administered to the population of stem cells (e.g., wherein VPA is a notch agonist and is also an HDAC inhibitor).

In certain embodiments, the administering step comprises administering or causing to be administered a Wnt agonist that is also a GSK3- β inhibitor to a population of stem cells. In certain embodiments, the administering step comprises administering or causing to be administered a Wnt agonist comprising a synthetic molecule to the population of stem cells. In certain embodiments, the administering step comprises administering or causing to be administered CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the stem cell population (e.g., wherein CHIR99021 is a GSK 3-beta inhibitor).

In certain embodiments, the administering step comprises administering or causing to be administered a notch inhibitor to the stem cell population. In certain embodiments, the notch inhibitor comprises: DAPT, LY411575, MDL-28170, Compound E, RO 4929097; DAPT (N- [ (3, 5-difluorophenyl) acetyl group]-L-alanyl-2-phenyl]Glycine-1, 1-dimethylethyl ester); l-685458((5S) - (tert-butoxycarbonylamino) -6-phenyl- (4R) hydroxy- (2R) benzylhexanoyl) -L-lysyl-L-phenylalaninamide); BMS-708163 (Avagacestat); BMS-299897(2- [ (1R) -1- [ [ (4-chlorophenyl) sulfonyl group](2, 5-difluorophenyl) amino]Ethyl-5-fluorobenzenebutyric acid); m-0752; YO-01027; MDL28170 (Sigma); LY411575(N-2((2S) -2- (3, 5-difluorophenyl) -2-hydroxyacetyl) -N1- ((7S) -5-methyl-6-oxo-6, 7-dihydro-5H-dibenzo [ b, d)]Aza derivatives-7-yl) -1-alaninamide); ELN-46719 (2-hydroxy-pentanoic acid amide analog of LY 411575); PF-03084014((S) -2- ((S) -5, 7-difluoro-1, 2,3, 4-tetrahydronaphthalen-3-ylamino) -N- (1- (2-methyl-1- (neopentylamino) propan-2-yl) -1H-imidazol-4-yl) pentanamide); compound E ((2S) -2- { [ (3, 5-dichlorophenyl) acetyl]Amino } -N- [ (3S) -1-methyl esterRadical-2-oxo-5-phenyl-2, 3-dihydro-1H-1, 4-benzodiazepine-3-yl]Propionamide; and Semagacestat (LY 450139; (2S) -2-hydroxy-3-methyl-N- ((1S) -1-methyl-2- { [ (1S) -3-methyl-2-oxo-2, 3,4, 5-tetrahydro-1H-3-benzazepine-1-yl]Amino } -2-oxoethyl) butanamide), or a pharmaceutically acceptable salt thereof (e.g., in a pharmaceutically acceptable form (e.g., a salt)).

In certain embodiments, the administering step comprises administering or causing to be administered (i) CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) and (ii) VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the population of stem cells (e.g., wherein (i) comprises CHIR99021 and (ii) comprises VPA). In certain embodiments, the administering step further comprises administering or causing to be administered DAPT (e.g., wherein DAPT is a notch inhibitor) to the population of stem cells.

In certain embodiments, the administering step is performed by performing one or more injections into the ear (e.g., trans-tympanic injection). In certain embodiments, the one or more injections are injections into the middle ear. In certain embodiments, the one or more injections are injections into the inner ear. In certain embodiments, performing one or more injections comprises: anesthetizing the tympanic membrane and/or surrounding tissue, placing a needle through the tympanic membrane into the middle ear, and injecting (i) and/or (ii).

In certain embodiments, the administering step comprises administering the notch agonist and/or the HDAC inhibitor in a pulsatile manner and administering the GSK3- β inhibitor and/or the Wnt agonist in a sustained manner. In certain embodiments, the administered formulation is sterile. In certain embodiments, the administered formulation is pyrogen-free. In certain embodiments, the pharmaceutically acceptable formulation is administered as described in the appendix, pages 21-36. In certain embodiments, the formulation is a combination of (i) a GSK3 β inhibitor and/or a Wnt agonist and (ii) a notch agonist and/or an HDAC inhibitor, administered as described in the pharmaceutically acceptable formulations, pages 21-36 of the appendix.

In certain embodiments, the stem cell population is of a subject in vivo, and the method is treatment of hearing loss and/or vestibular dysfunction (e.g., wherein production of inner ear hair cells from the expanded stem cell population results in partial or complete restoration of hearing loss and/or improved vestibular function). In certain embodiments, the stem cell population is of a subject in vivo, and the method further comprises delivering the drug to the subject (e.g., for treating a disease and/or disorder not associated with hearing loss and/or vestibular dysfunction) at a concentration higher than a maximum dose known to be safe for the subject (e.g., a maximum dose known to be safe when delivered without the method to produce inner ear hair cells) (e.g., due to a reduction or elimination of dose limiting ototoxicity of the drug).

In certain embodiments, the methods further comprise high-throughput screening using the produced inner ear hair cells. In certain embodiments, the methods comprise using the inner ear hair cells produced to screen the molecules for toxicity to the inner ear hair cells. In certain embodiments, the methods comprise using the produced inner ear hair cells to screen for the ability of a molecule to improve survival of inner ear hair cells (e.g., inner ear hair cells exposed to the molecule).

In another aspect, the present disclosure relates to a method of producing an expanded stem cell population, the method comprising administering or causing to be administered both (i) a GSK3- β inhibitor and/or a Wnt agonist, and (ii) a notch agonist and/or a HDAC inhibitor, to a stem cell population (e.g., of an in vitro, ex vivo or in vivo sample/subject) thereby expanding stem cells in the stem cell population and producing an expanded stem cell population⁺A cell. In certain embodiments, the population of stem cells comprises postnatal stem cells. In certain embodiments, the stem cell population comprisesContains epithelial stem cells. In certain embodiments, the stem cells comprise progenitor cells.

In certain embodiments, the administering step comprises administering or causing to be administered to the population of stem cells a notch agonist that is also an HDAC inhibitor. In certain embodiments, the administering step comprises administering or causing to be administered a notch agonist comprising a synthetic molecule to the stem cell population. In certain embodiments, the administering step comprises administering or causing to be administered VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the population of stem cells (e.g., wherein VPA is a notch agonist, also an HDAC inhibitor).

In certain embodiments, the administering step comprises administering or causing to be administered a Wnt agonist that is also a GSK3- β inhibitor to a population of stem cells. In certain embodiments, the administering step comprises administering or causing to be administered a Wnt agonist comprising a synthetic molecule to the population of stem cells. In certain embodiments, the administering step comprises administering or causing to be administered CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the stem cell population (e.g., wherein CHIR99021 is a GSK 3-beta inhibitor).

In certain embodiments, the administering step is performed by performing one or more injections into the ear (e.g., trans-tympanic injection into the middle and/or inner ear).

In certain embodiments, the administering step comprises administering the notch agonist and/or the HDAC inhibitor in a pulsatile manner and administering the GSK3- β inhibitor and/or the Wnt agonist in a sustained manner.

In certain embodiments, the hair cells are inner ear stem cells and/or support cells.

In certain embodiments, the method further comprises high throughput screening using the generated expanded stem cell population. In certain embodiments, the method further comprises using the generated stem cells to screen the stem cells and/or progeny thereof for toxicity of the molecule. In certain embodiments, the methods further comprise using the generated stem cells to screen for the ability of the molecule to improve survival of the stem cells and/or progeny thereof.

In another aspect, the present disclosure relates to a method of treating a subject suffering from or at risk of developing hearing loss and/or vestibular dysfunction, the method comprising: identifying a subject who has experienced or is at risk of developing hearing loss and/or vestibular dysfunction, administering or causing to be administered to said subject (i) and (ii): (i) a GSK3- β inhibitor and/or a Wnt agonist, and (ii) a notch agonist and/or a HDAC inhibitor, thereby treating or preventing hearing loss and/or vestibular dysfunction in a subject.

In certain embodiments, the stem cell population comprises Lgr5⁺A cell. In certain embodiments, the population of stem cells comprises postnatal cells. In certain embodiments, the population of stem cells comprises epithelial stem cells. In certain embodiments, the stem cells comprise progenitor cells.

In certain embodiments, the administering step comprises administering or causing to be administered to the subject a notch agonist that is also an HDAC inhibitor. In certain embodiments, the administering step comprises administering or causing to be administered to the subject a notch agonist comprising a synthetic molecule. In certain embodiments, the administering step comprises administering or causing to be administered VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the subject (e.g., wherein VPA is a notch agonist, also an HDAC inhibitor).

In certain embodiments, the step of administering comprises administering or causing to be administered to the subject a Wnt agonist that is also a GSK3- β inhibitor. In certain embodiments, the administering step comprises administering or causing to be administered to the subject a Wnt agonist comprising a synthetic molecule. In certain embodiments, the administering step comprises administering or causing to be administered CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the subject (e.g., wherein CHIR99021 is a GSK 3-beta inhibitor).

In certain embodiments, the administering step is performed by performing one or more injections into the ear (e.g., trans-tympanic injection into the middle and/or inner ear).

In certain embodiments, the methods comprise administering a notch agonist and/or an HDAC inhibitor in a pulsatile manner and administering a GSK3- β inhibitor and/or a Wnt agonist in a sustained manner.

In another aspect, the present disclosure relates to a kit comprising: (a) a set of one or more compositions, the set comprising (i) and (ii): (i) a GSK3- β inhibitor and/or a Wnt agonist, and (ii) a notch agonist and/or a HDAC inhibitor, each of the one or more compositions provided in a pharmaceutically acceptable carrier; and (b) instructions for using the set of one or more compositions to treat an inner ear disorder.

In certain embodiments, the set of one or more compositions further comprises a TGF inhibitor. In certain embodiments, the set of one or more compositions further comprises a ROS scavenger. In certain embodiments, the ROS scavenger is vitamin C or a derivative thereof. In certain embodiments, the set of one or more compositions is in an injectable form (e.g., by injection via a syringe). In certain embodiments, the group of one or more compositions is in a form that can be injected into the middle ear.

In another aspect, the disclosure relates to a pharmaceutical composition comprising a GSK3- β inhibitor and a notch agonist in lyophilized form.

In another aspect, the disclosure relates to a pharmaceutical composition comprising a hydrated form of a GSK3- β inhibitor and a notch agonist.

In certain embodiments, the GSK3- β inhibitor is CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)). In certain embodiments, the notch agonist is VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)).

In another aspect, the present disclosure relates to a method of producing inner ear hair cells, the method comprising: expanding stem cells in a naive stem cell population (e.g., a stem cell population of a sample/subject in vitro, ex vivo, or in vivo) to produce an expanded stem cell population (e.g., such that the expanded population is at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold greater than the naive stem cell population); and exposing the expanded stem cell population to a GSK 3-beta inhibitor and/or a Wnt agonist, and optionally to a notch inhibitor, thereby promoting production of inner ear hair cells from the expanded stem cell population.

In another aspect, the present disclosure relates to a method of producing inner ear hair cells, the method comprising: administering CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to a cell population in the inner ear of the subject, thereby promoting production of inner ear hair cells.

In another aspect, the present disclosure relates to a method of producing inner ear hair cells, the method comprising: expanding postnatal LGR5 in a naive stem cell population (e.g., a population of stem cells of a sample/subject in vitro, ex vivo, or in vivo)⁺Cells producing expanded LGR5⁺A population of cells (e.g., such that the expanded population is at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold greater than the initial population of stem cells); the amplified LGR5⁺The cell population results in the production of inner ear hair cells. In certain embodiments, the stem cells comprise progenitor cells.

In another aspect, the present disclosure relates to a method of treating a disease or disorder, the method comprising: amplification of postnatal Lgr5 in an initial population of (in vivo) subjects⁺Epithelial cells, producing expanded Lgr5⁺Epithelial cell populations (e.g., such that the expanded population is the initial postnatal Lgr5⁺At least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold of the epithelial cell population).

In certain embodiments, Lgr5⁺The cells differentiate into hair cells. In certain embodiments, a notch inhibitor is used to induce differentiation. Notch pathway modulators include, but are not limited to, those listed in reference 69 or disclosed in U.S. patent 8,377,886, which is incorporated herein by reference in its entirety.

RO 4929097; DAPT (N- [ (3, 5-difluorophenyl) acetyl group]-L-alanyl-2-phenyl]Glycine-1, 1-dimethylethyl ester); l-685458((5S) - (tert-butoxycarbonylamino) -6-phenyl- (4R) hydroxy- (2R) benzylhexanoyl) -L-lysyl-L-phenylalaninamide); BMS-708163 (Avagacestat); BMS-299897(2- [ (1R) -1- [ [ (4-chlorophenyl) sulfonyl group](2, 5-difluorophenyl) amino]Ethyl-5-fluorobenzenebutyric acid); m-0752; YO-01027; MDL28170 (Sigma); LY411575(N-2((2S) -2- (3, 5-difluorophenyl) -2-hydroxyacetyl) -N1- ((7S) -5-methyl-6-oxo-6, 7-dihydro-5H-dibenzo [ b, d)]Aza derivatives-7-yl) -1-alaninamide); ELN-46719 (2-hydroxy-pentanoic acid amide analog of LY 411575); PF-03084014((S) -2- ((S) -5, 7-difluoro-1, 2,3, 4-tetrahydronaphthalen-3-ylamino) -N- (1- (2-methyl-1- (neopentylamino) propan-2-yl) -1H-imidazol-4-yl) pentanamide); compound E ((2S) -2- { [ (3, 5-dichlorophenyl) acetyl]Amino } -N- [ (3S) -1-methyl-2-oxo-5-phenyl-2, 3-dihydro-1H-1, 4-benzodiazepine-3-yl]Propionamide; and Semagacestat (LY 450139; (2S) -2-hydroxy-3-methyl-N- ((1S) -1-methyl-2- { [ (1S) -3-methyl-2-oxo-2, 3,4, 5-tetrahydro-1H-3-benzazepine-1-yl]Amino } -2-oxoethyl) butanamide), or a pharmaceutically acceptable salt thereof.

Administration of

The round window membrane is the biological barrier in the inner ear space and is the major obstacle for the topical treatment of hearing impairment. The administered drug must overcome the membrane to reach the inner ear space. The drug can be placed locally to the round window membrane in an operable manner (e.g., injected through the tympanic membrane) and then can permeate through the round window membrane. The material that permeates through the round window is usually distributed in the perilymph, thereby reaching the hair cells and the supporting cells.

In certain embodiments, the pharmaceutical formulation is adapted for topical application of the drug to the round window membrane. The pharmaceutical formulation may also include a membrane permeation enhancer that supports the agent described herein across the round window membrane. Thus, liquid, gel or foam formulations may be used. The active ingredients may also be administered orally or using a combination of delivery routes.

The delivery of drugs to the ear tympanic cavity (IT) is increasingly used for clinical and research purposes. Some groups have used microcatheters or microcatheter needles to administer drugs in a sustained manner, but most have administered them as single or repeated IT injections (up to 8 injections over a period of up to two weeks) 8.

Intratympanic administered drugs are believed to enter the inner ear fluid primarily by crossing the Round Window (RW) membrane. Calculations show that the primary factor controlling the amount of drug entering the ear and the distribution of the drug along the length of the ear is the time the drug remains in the middle ear space. A single "single dose" (one-shot) "administration or administration of an aqueous solution for several hours results in a sharply varying drug gradient of the administered substance along the length of the cochlea and, as the drug is subsequently distributed throughout the ear, a rapid drop in concentration in the basal ganglia of the cochlea.

Other injection methods include: osmotic pumps, or in combination with implanted biomaterials, more preferably injection or infusion. Biomaterials that can help control the release kinetics and distribution of drugs include hydrogel materials, degradable materials. One class of materials most preferably used includes in situ gelling materials. All potential materials and methods mentioned in these references are incorporated herein by reference^11,13-58. Other materials include collagen or other natural materials, including fibrin, gelatin, and decellularized tissue. Gelatin sponge (gelfoam) may also be suitable.

Delivery may also be enhanced by alternative means, including but not limited to agents added to the delivered composition, such as penetration enhancers, or delivery may be by ultrasound, electroporation, or high-speed jet devices.

The methods described herein can also be used with inner ear cell types that can be generated using a variety of methods known to those skilled in the art, including those described in PCT application WO2012103012a 1.

With respect to human and veterinary therapy, the amount of a particular agent administered may depend on a variety of factors, including: the condition being treated and the severity of the condition; the activity of the particular agent used; the age, weight, general health, sex, and diet of the patient; time of administration, route of administration, and rate of excretion of the particular agent used; the duration of treatment; drugs combined or used in combination with the particular agent used; the judgment of the prescribing physician or veterinarian; and similar factors known in the medical and veterinary arts.

The agents described herein can be administered in a therapeutically effective amount to a subject in need of treatment. Administration of the compositions described herein may be by any suitable route of administration, particularly by intratympanic administration. Other approaches include: ingestion, or parenteral, e.g., intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intranasal, subcutaneous, sublingual, transdermal, or inhalation or insufflation, or topical administration by intraaural instillation for absorption through the membranes of the ear canal skin and tympanic membrane. Such administration may be in single or multiple oral doses, a defined number of ear drops, or as a bolus, multiple injection, or as a short or long term infusion. Implantable devices (e.g., implantable infusion pumps) can also be used to periodically parenterally deliver equivalent or different doses of a particular formulation over a period of time. For such parenteral administration, the compounds are preferably formulated as sterile solutions in water or other suitable solvent or solvent mixture. The solution may contain other substances, such as salts, sugars (in particular glucose or mannitol) to make the solution isotonic with blood; buffers such as acetic acid, citric acid and/or phosphoric acid and their sodium salts; and a preservative. The preparation of suitable (preferably sterile) parenteral formulations is described in detail in the "compositions" section above.

The compositions described herein can be administered in a variety of ways sufficient to deliver the composition to the inner ear. Delivering the composition to the inner ear comprises: the composition is applied to the middle ear such that the composition can spread across the round window to the inner ear, and is applied to the inner ear by direct injection through the round window membrane. These methods include, but are not limited to: otic administration is carried out with a trans-tympanic catheter or a stylet, or parenteral administration is carried out by, for example, intra-aural, trans-tympanic or intra-cochlear injection.

In particular embodiments, the compositions and formulations of the present disclosure are administered topically, meaning that they are not administered systemically.

In one embodiment, the composition is administered to a subject by otic administration using a syringe and needle device. A needle of appropriate size is used to pierce the tympanic membrane, and a stylet or catheter containing the composition is inserted through the pierced tympanic membrane into the middle ear of the subject. The device may be inserted such that it is in contact with or in close proximity to the round window. Exemplary devices for otic administration include, but are not limited to: trans-tympanic stylet, trans-tympanic catheter, round window microcatheter (small catheter for drug delivery to round window), and Silverstein Microwicks^TM(a small tube with a "core" passing through the tube to a round window so that it can be manipulated by the subject or medical professional).

In another embodiment, the composition is administered to a subject using a syringe and needle device using trans-tympanic injection, behind the tympanic membrane injection into the middle and/or inner ear. The formulation may be administered directly to the round window membrane by trans-tympanic injection, or may be administered directly to the cochlea by intracochlear injection or to the vestibular organ by intracochlear injection.

In some embodiments, the delivery device is a device designed to apply the composition to the middle and/or inner ear. By way of example only: GYRUS Medical Gmbh provides a miniature otoscope for visualization of the round window niche and drug delivery; arenberg has described in U.S. patents 5,421,818, 5,474,529, and 5,476,446 a medical device for delivering fluid to the inner ear structure, the disclosure of each of which is incorporated herein by reference. U.S. patent application 08/874,208, the disclosure of which is incorporated herein by reference, describes a surgical method of implanting a fluid delivery conduit to deliver a composition to the inner ear. U.S. patent application publication 2007/0167918, the disclosure of which is incorporated herein by reference, further describes a combined ear aspirator and drug dispenser for transtympanic fluid sampling and drug application.

In some embodiments, the compositions disclosed herein are administered once to a subject in need thereof. In some embodiments, a composition disclosed herein is administered more than once to a subject in need thereof. In some embodiments, a second, three, four, or five administrations of a composition disclosed herein are performed after a first administration of a composition disclosed herein.

The number of times the composition is administered to a subject in need thereof will depend upon the judgment of the medical professional, the condition, the severity of the condition, and the subject's response to the agent. In some embodiments, the compositions disclosed herein are administered once to a subject in need thereof with a mild acute condition. In some embodiments, a composition disclosed herein is administered more than once to a subject in need thereof with a moderate or severe acute condition. In the case where the condition of the subject is not improved, the composition may be administered for a prolonged period, i.e., for a prolonged period, including throughout the life of the subject, to ameliorate or control or limit the symptoms of the disease or condition in the subject, at the discretion of the physician.

Where the subject's condition does improve, the composition may continue to be administered, at the discretion of the physician; alternatively, the dose of drug administered may be temporarily reduced or temporarily stopped for a period of time (i.e., a "drug holiday"). The length of the drug holiday varies between 2 days and 1 year, which includes, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dose reduction during the drug holiday can be 10% to 100%, including by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once the hearing and/or balance of the subject is improved, a maintenance dose can be administered, if desired. Subsequently, the dose and/or frequency of administration is optionally reduced to a level that allows the improved disease, disorder or condition to be retained, depending on the symptoms. In certain embodiments, the subject is in need of chronic intermittent treatment at the time of any recurrence of symptoms.

Preparation

The bioactive compositions described herein (sometimes referred to herein as "bioactive agents" or more simply as "agents") can be formulated in any manner suitable for the desired delivery route to a population of cells in vitro or in vivo, such as trans-tympanic injection, trans-tympanic stylet and catheter, and injectable depot. Typically, such formulations include the biologically active composition in a physiologically acceptable form, including free acid forms, free base forms, acid addition salts, base addition salts, other derivatives thereof (e.g., prodrugs or solvates thereof), racemates, optically active materials or tautomers thereof, and any physiologically acceptable carriers, diluents and/or excipients.

Solid formulations of the compositions described herein, such as dragees, capsules, pills, and granules, can optionally be scored or prepared with coatings and shells (e.g., enteric coatings and other coatings). Solid dosage forms may also be formulated to provide slow or controlled release of the ion channel modulating compound. Thus, the solid formulation may include any material that can provide the desired release profile of the ion channel modulating compound, including but not limited to hydroxypropylmethylcellulose in various ratios, or other polymer matrices, liposomes, and/or microspheres.

Pharmaceutically acceptable carriers may include, but are not limited to: water; ethanol; oils, including those derived from petroleum, animal, vegetable, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like; excipients, such as methylcellulose, carrageenan, and the like.

Liquid dosage forms may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition, the liquid dosage forms may contain inert diluents commonly used in the art, including but not limited to: water or other solvents; solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol; oils (in particular cottonseed, groundnut, corn, germ, olive, castor and sesame oils); glycerol; tetrahydrofuryl alcohol; polyethylene glycol; fatty acid esters of sorbitan; and mixtures thereof.

Suspension formulations include, but are not limited to: ethoxylated isostearyl alcohols; polyoxyethylene sorbitol and sorbitan esters; microcrystalline cellulose; aluminum metahydroxide; bentonite; agar; gum tragacanth; and mixtures thereof.

Proper fluidity of liquids, suspensions and other formulations of ion channel modulating compounds can be maintained by: use of coating materials (e.g., lecithin); for the dispersion, the desired particle size is maintained; or use of a surfactant.

The formulation may also include an anti-fouling agent for preventing microbial contamination. The anti-fouling agents may include, but are not limited to, antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, antibiotics, and the like.

The formulation may also be sterilized, for example, by: immediately prior to use of the formulation, filtration is through a bacterial-retaining filter, or a sterilizing agent in the form of a sterile solid formulation capable of being dissolved in sterile water or some other sterile medium is added.

The preparation may also be endotoxin free. As used herein, the term "endotoxin-free" refers to a composition or formulation containing up to a trace amount (i.e., an amount that has no adverse physiological effect on a subject) of endotoxin, preferably an undetectable amount of endotoxin. By "substantially endotoxin free" is meant less endotoxin per cell dose than FDA allowed for biologicals, i.e. 5EU/kg body weight total endotoxin per day for an average of 70kg of human subjects at a total cell dose of 350 EU. In one embodiment, the term "endotoxin-free" means at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% endotoxin-free. Endotoxins are toxins associated with certain bacteria, usually gram-negative bacteria, but endotoxins may also be present in gram-positive bacteria, such as listeria monocytogenes. The most common endotoxins are Lipopolysaccharides (LPS) or Lipooligosaccharides (LOS) present in the outer membrane of various gram-negative bacteria, which are central pathogenic features of the ability of these bacteria to cause disease. Small amounts of endotoxin in the human body can produce adverse physiological effects such as fever, reduction in blood pressure, inflammation and activation of blood coagulation. Therefore, it is often desirable to remove most or all traces of endotoxin from pharmaceutical product containers, as even small amounts may have an adverse effect on humans.

The pharmaceutical compositions described herein are formulated such that the active ingredient contained therein is bioavailable when the composition is administered to a subject. For example, the pharmaceutical compositions described herein may be prepared by combining the Notch activator and/or HDAC inhibitor and GSK3b inhibitor and/or WNT activator and/or small molecule with a suitable pharmaceutically acceptable carrier, diluent or excipient and may be formulated in solid, semi-solid, liquid gel and microsphere forms, including those suitable for otic administration by trans-tympanic stylet or catheter or for parenteral administration (e.g., by intra-aural, trans-tympanic or intra-cochlear injection). However, in certain embodiments, the subject compounds may simply be dissolved or suspended in sterile water.

Coated, gel or encapsulated formulations of Notch activators and/or HDAC inhibitors and GSK3b inhibitors and/or WNT activators or derivatives and/or small molecules may also be formulated to deliver pulsatile, sustained or extended release. For example, one method of pulsatile release may be achieved by layering multiple coatings of the agent or derivative and/or small molecule, or by adding the agent, derivative and/or small molecule to different regions of the formulation with different release times.

Injectable depot formulations can be prepared by forming a microencapsulation matrix of the composition in a biodegradable polymer. Examples of biodegradable polymers include, but are not limited to, polylactide-polyglycolide, poly (orthoester), and poly (anhydride). The ratio of composition to polymer and the nature of the particular polymer used may affect the rate of release of the Notch activator and/or HDAC inhibitor and GSK3b inhibitor and/or WNT activator or derivative and/or small molecule from the composition. Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions.

The pharmaceutical composition may further comprise one or more components that enhance the bioavailability of the active ingredients of the composition (e.g., penetration enhancers), stabilizers, and one or more components that provide slow or controlled release of the Notch activator and/or HDAC inhibitor and GSK3b inhibitor and/or WNT activator derivative and/or small molecule in the composition (e.g., biocompatible polymers and/or gels).

In particular embodiments, a composition comprising a penetration enhancer will facilitate delivery of the composition across a biological barrier (e.g., round window) separating the middle and inner ear, thereby efficiently delivering a therapeutically effective amount of the composition to the inner ear. Effective delivery to the cochlea, Corti organ, and/or vestibular organ is desirable because these tissues, when treated or contacted by the compositions described herein, carry supporting cells that promote the regeneration of sensory hair cells.

"penetration enhancer" or "permeability enhancer" includes: polyols such as polyethylene glycol (PEG), glycerol (glycerin), maltitol, sorbitol, and the like; diethylene glycol monoethyl etherAzone, benzalkonium chloride (ADBAC), cetylpyridinium chloride, cetylmethyl ammonium bromide, dextran sulfate, lauric acid, menthol, methoxysalicylate, oleic acid, phosphatidylcholine, polyoxyethylene, polysorbate 80, sodium glycocholate, sodium lauryl sulfate, sodium salicylate, sodium taurocholate, sodium taurodeoxycholate, sulfoxide, sodium deoxycholate, sodium glucose deoxycholate, sodium taurocholate; and surfactants such as sodium lauryl sulfate, laureth-9, cetylpyridinium chloride and polyoxyethylene monoalkyl ether; benzoic acids such as sodium salicylate and methoxysalicylate; fatty acids such as lauric acid, oleic acid, undecanoic acid, and methyl oleate; (ii) a Fatty alcohols, e.g. octanol and nonanol, lauryl nitrogenKetones, cyclodextrins, thymol, limonene, urea, chitosan and other natural and synthetic polymers.

Other penetration enhancers include, but are not limited to, those described in U.S. patent application publication US 20110166060.

Suitable polyols for inclusion in the solutions described herein include glycerol and sugar alcohols (e.g., sorbitol, mannitol or xylitol), polyethylene glycol and derivatives thereof. In some embodiments, the composition further comprises a preservative. Acceptable preservatives, such as benzalkonium chloride and Edetate Disodium (EDTA), are included in the compositions described herein at concentrations sufficient to produce an effective antimicrobial effect in the range of about 0.0001% to 0.1% by weight of the composition.

In certain embodiments, the compositions of the present disclosure further comprise a stabilizer to increase the therapeutic useful life of the composition in vivo. Exemplary stabilizers include fatty acids, fatty alcohols, long chain fatty acid esters, long chain ethers, hydrophilic derivatives of fatty acids, polyvinylpyrrolidone, polyvinyl ethers, polyvinyl alcohols, hydrocarbons, hydrophobic polymers, hygroscopic polymers, and combinations thereof. In other embodiments, the stabilizing agent is selected to alter the hydrophobicity of the formulation (e.g., oleic acid, wax) or improve the mixing of various components in the formulation (e.g., ethanol), affect the moisture content of the formulation (e.g., PVP or polyvinylpyrrolidone), affect the mobility of the phases (substances with melting points above room temperature, such as long chain fatty acids, alcohols, esters, ethers, amides, etc., or mixtures thereof; waxes), and/or improve the compatibility of the formulation with the encapsulating material (e.g., oleic acid or waxes). In other embodiments, the stabilizing agent is present in an amount sufficient to inhibit degradation of the Notch activator and/or HDAC inhibitor and GSK3b inhibitor and/or WNT activator derivatives and small molecules in the composition. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1mM to about 10mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

In certain embodiments, the compositions of the present disclosure are formulated as controlled release formulations. Generally, controlled release drug formulations achieve control of drug release with respect to the site and time of release in vivo. Controlled release includes immediate release, delayed release, sustained release, extended release, variable release, pulsatile release and bimodal release.

Advantages of controlled release include: less frequent dosing; more efficient drug utilization; locally delivering the drug by placing a delivery device or formulation at the treatment site in the body; and the opportunity to administer and release two or more different drugs, each with a unique release profile, or the opportunity to release the same drug at different rates or for different durations through a single dose unit.

Controlled release formulations can be prepared by formulating the compositions with biocompatible polymers, viscosity agents, gels, coatings, foams, xerogels, microparticles, hydrogels, nanocapsules and thermoreversible gels or combinations thereof. In certain embodiments, the release properties of the polymer or gel that is biodegradable are often controlled by the particular combination of polymers or gels used to formulate the composition. These methods are well known in the art.

Exemplary polymers suitable for use in formulating the bioactive compositions of the present disclosure include, but are not limited to: polyamides, polycarbonates, polyalkylene (polyethylene glycol (PEG)), polymers of acrylates and methacrylates, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly (ortho) esters, poly (butyric acid), poly (valeric acid), copoly (lactide-caprolactone), polysaccharides, proteins, poly (hyaluronic acid), polycyanoacrylates, and blends, mixtures or copolymers thereof.

In one embodiment, the bioactive composition of the present disclosure is formulated in an ABA-type or BAB-type triblock copolymer or mixture thereof, wherein the a blocks are relatively hydrophobic and comprise a biodegradable polyester or poly (orthoester) and the B blocks are relatively hydrophilic and comprise polyethylene glycol (PEG). The biodegradable hydrophobic a polymer block comprises a polyester or poly (orthoester), wherein the polyester is synthesized from monomers selected from the group consisting of D, L-lactide, D-lactide, L-lactide, D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, -caprolactone, -hydroxycaproic acid, γ -butyrolactone, γ -hydroxybutyric acid, -valerolactone, -hydroxyvaleric acid, hydroxybutyric acid, malic acid, and copolymers thereof.

Exemplary viscosity agents suitable for use in formulating the compositions described herein include, but are not limited to: hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate, gum arabic (acacia), agar, agarose, magnesium aluminum silicate, sodium alginate, sodium stearate, fucans, bentonite, carbomer, carrageenan, carbopol, xanthan gum, cellulose, microcrystalline cellulose (MCC), carob, chitin, carboxymethylated chitosan, cartilage, dextrose, furcellaran, gelatin, ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, karaya gum, xanthan gum, tragacanth, ethyl cellulose, ethyl hydroxyethyl cellulose, ethyl methyl cellulose, hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl cellulose, poly (hydroxyethyl methacrylate), oxidized polygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly (methoxyethyl methacrylate), poly (methoxyethoxyethyl) ester, hydroxypropyl cellulose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, or polyvinylpyrrolidone (PVP: povidone).

Suitable gelling agents for preparing the gel formulation include, but are not limited to: cellulose, cellulose derivatives, cellulose ethers (e.g., carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose), guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid), silicates, starch, tragacanth, carboxyvinyl polymers, carrageenan, paraffin, petrolatum, and any combination or mixture thereof.

A variety of suitable biocompatible polymers can be used to deliver the compounds to the ear. Preferably, the polymer may form a gel. Examples of suitable gel-forming biocompatible polymers include hyaluronic acid, hyaluronate, lecithin gel, (poly) alanine derivatives, pluronic, poly (ethylene glycol), poloxamers, chitosan, xyloglucan, collagen, fibrin, polyesters, poly (lactide), poly (glycolide) or copolymers thereof PLGA, sucrose acetate isobutyrate, and glycerol monooleate.

Hyaluronic acid is a naturally occurring biocompatible polysaccharide that binds water and forms a degradable gel with high viscosity. Polyethylene glycol (PEG) is a biocompatible, hydrophilic polymer.

Thermoset polymers are also suitable, which are fluid at low temperatures, but more viscous at higher temperatures. A common reversible thermoset system is poloxamer. When dissolved, the solution can remain liquid at low temperatures, but when the temperature is increased, a more viscous solid-like implant can be formed.

Chitosan is biocompatible and has antibacterial properties, and chitosan-glycerophosphate solutions are capable of forming reversible thermoset gels.

The gel may also be formed from an enzymatically degradable polypeptide polymer. The polypeptide bonds in the polypeptide polymer are more hydrolytically stable than the ester bonds in the PEG/PLGA polymer system, and the polypeptide may also include a biodegradable polymer having a biodegradable polypeptide block attached to a second polymer block to form a graft or linear polymer. Examples of polypeptide polymers are poly (alanine) and derivatives thereof. The polypeptide carrier can also be a protein matrix called fibrin. Fibrinogen is a naturally occurring protein that, when bound to thrombin (another naturally occurring protein), forms a biological matrix called fibrin.

Other biocompatible polymers include starch, cellulose, gelatin-pluronic, Tetronic, the latter two being poly (ethylene oxide)/poly (propylene oxide) materials. Other materials that may be used include chondroitin sulfate and the general class of mucopolysaccharides (e.g., glycosaminoglycans) and other biocompatible polymers having properties similar to hyaluronic acid.

In some cases, the biocompatible polymer may be crosslinked. Various crosslinking agents for biodegradable materials are known in the art. Preferably, the crosslinking is accomplished such that the final crosslinked material of the delivery unit is substantially non-toxic (e.g., by using thermal crosslinking, gamma irradiation, ultraviolet irradiation, chemical crosslinking, etc.). Generally, the degree of crosslinking is inversely related to the degree of swelling or water absorption of the formed polymer structure. The degree of swelling or water absorption regulates the drug delivery rate of the polymer structure.

In some embodiments, the drug is administered across the tympanic membrane, e.g., by puncturing, injection, or the like. In some embodiments, the drug is administered across the eardrum without piercing it, e.g., absorbed across the eardrum.

In some embodiments, it is desirable that the formulation is sufficiently viscous to remain in the application area for an appropriate period of time, e.g., 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 5 hours, 12 hours, 1 day, 2 days, 7 days. The desired area may be outside of, but in contact with, the eardrum, e.g., when the subject is upright, it may be desirable for the solution layer to remain in contact with the eardrum (e.g., not flow out of the ear); even if some of the adhesive formulation leaves the ear canal, in some embodiments, the adhesive film layer may be brought into contact with the eardrum. Similarly, in embodiments, administration is across the tympanic membrane.

It will be appreciated that in some embodiments, the liquid-gel transition may be adjusted to become rapid and reproducible when loaded with a drug, thereby altering the formulation.

For a carrier poloxamer 407, gelation can decrease with increasing concentration of poloxamer 407, at least in the absence of drug. The addition of some drugs, such as hydrophilic drugs, including VPA and pVc at concentrations greater than 88mg/ml and 14mg/ml, respectively, inhibited gelation of the poloxamer 407 solution. Thus, gels were prepared using 18% (w/w) poloxamer solutions with VPA and pVc concentrations equal to or less than 88mg/ml and 14mg/ml, respectively. An appropriate volume of hydrophobic drug (including CHIR, Repsox, and TTNPB) from its DMSO stock solution was added to the poloxamer 407 solution containing the hydrophilic drug and mixed with suction at 4 ℃. The concentrations of drugs CHIR, Repsox and TTNPB in the stock solutions were maintained at 55.6mg/ml to 69.5mg/ml, 23mg/ml to 28.75mg/ml and 35mg/ml, respectively, to ensure that the total DMSO concentration in the final formulation was less than 5% to 6%. Higher concentrations of DMSO significantly reduced the gelation temperature of the formulation. The final formulation was a viscous liquid at storage temperature (4 ℃) and formed a semi-solid gel above its liquid-gel transition temperature (37 ℃).

In some cases, the composition has a viscosity of less than 100,000 centipoise (cps) at 25 ℃. The composition also has a minimum yield stress sufficient to retain the formulation on the tympanic membrane. Yield stress is the amount of force that causes a solid material to behave like a liquid, which will continue to deform without further increase in stress. The minimum yield stress depends on the thickness of the applied gel, but is independent of the geometry of the gel and the ambient temperature. The minimum yield stress of the composition means that the applied gel has a thickness of 4mm and a density of 1 g/L. The yield stress (σ 0) is σ 0 ═ ρ gh, where ρ is the density, g is the acceleration of gravity, and h is the layer thickness. Typically, the minimum yield stress is about 39 pascals (Pa).

The viscosity generator is typically a polymer or other chemical moiety that increases the viscosity of the fluid. When included in the composition, suitable viscosity-generating agents allow the composition to transition from a liquid-like state (e.g., flowable) at 25 ℃ to a solid-like state (e.g., a gel) after contact with the tympanic membrane, and can be non-biodegradable (e.g., not broken down by chemicals or enzymes naturally present in the mammal, or biodegradable). The composition includes an amount of a viscosity generator effective to produce a viscosity of the composition of less than 100,000cps (e.g., less than 90,000cps, 60,000cps, 30,000cps, 20,000cps, or 10,000cps) at 25 ℃, and generally effective to produce a minimum yield stress of 39Pa upon application to the tympanic membrane. Typically, the composition comprises from 0.05% to 50% of the viscosity generating agent (e.g., from 0.15% to 25%, from 5% to 45%, from 10% to 40%, from 12% to 37%, from 15% to 35%, from 17% to 33%, or from 20% to 30% of the viscosity generating agent).

Exemplary viscosity generators include gellan (GELRITE or kelgel), carbopol 940 with hydroxypropyl methylcellulose (HPMC), N-isopropylacrylamide (NiPAAm) and N-alkylacrylamide with sodium acrylate, polyacrylic acid with polyethylene glycol (PEG) or polymethacrylic acid with PEG, cellulose acetate hydrogen phthalate latex (CAP), sodium alginate, and nonionic surfactants such as Poloxamers (PLURIONICs) and polyoxyamines (TETRONIC) reversible temperature-dependent gelling systems. Gellan is a natural polymer secreted by Pseudomonas elodea (Pseudomonas elodea) and is an anionic deacetylated extracellular polysaccharide. The tetrasaccharide repeat unit consists of one alpha-L-rhamnose, one beta-D-glucuronic acid and two beta-D-glucose moieties. The in situ gelation mechanism of gellan is cation-induced (e.g., the presence of calcium ions) and temperature-dependent (e.g., physiological temperature). Gelation is thermally reversible. Carbopol 940 with HPMC gelled in situ in a pH dependent manner. CARBOPOL (CARBOPOL) is the gelling agent, while HPMC is used to enhance the viscosity of the gel. NiPAAm with sodium acrylate and N-alkylacrylamide is a terpolymer hydrogel capable of undergoing reversible sol-gel conversion based on temperature. Sodium acrylate and N-alkylacrylamides are used to modify the properties of the hydrogel, in particular the transition temperature.

It is believed that polyacrylic acid with PEG or polymethacrylic acid with PEG gels based on the formation of hydrogen bonds. Polyacrylic acid can be dissolved in hydroalcoholic solutions, and after injection, the alcohol can diffuse out, resulting in polymer precipitation and gelation of the solution. CAP is a nanoparticle system that gels in a pH-dependent manner. The active compound (pharmacological agent) is partially adsorbed on the surface of the polymer particles. Sodium alginate gels in the presence of calcium or other multivalent ions.

Poloxamers are triblock copolymers formed from triblock copolymers of poly (oxyethylene) -poly (oxypropylene) -poly (oxyethylene), i.e., hydrophilic poly (oxyethylene) blocks and hydrophobic poly (oxypropylene) blocks. Poloxamers are a class of block copolymer surfactants having a propylene oxide block hydrophobic portion and an ethylene oxide hydrophilic portion. Poloxamers are commercially available (e.g.,polyols available from BASF Corporation). Alternatively, poloxamers can be synthesized using known techniques.

The formulations described herein are suitable for inhalation or intravenous, intraperitoneal, intramuscular, subcutaneous, mucocutaneous, oral, rectal, transdermal, topical, buccal, intradermal, intragastric, intradermal, intranasal, intraoral, transdermal or sublingual administration. In certain embodiments, administration is by injection into the middle ear and topical application to the eardrum. The formulation may also be administered directly to the inner ear. The formulation may be administered by a variety of carriers including biomaterials, solutions, devices (including but not limited to hearing aids, cochlear implants, headsets and earplugs).

The pharmaceutical compositions may be prepared and administered in the form of transdermal delivery systems (patch films), drops, pills, tablets, film-coated tablets, multi-layered tablets, gels, ointments, syrups, granules, suppositories, emulsions, dispersions, microcapsules, nanoparticles, microparticles, capsules, powders or injectable solutions. The pharmaceutical preparations are preferably in the form of liposomes, emulsions and gels, and combinations thereof.

Embodiments described herein also include pharmaceutical compositions prepared using at least one compound described herein or a salt thereof.

The pharmaceutical composition may also contain pharmacologically acceptable carriers, excipients and/or solvents.

These formulations are suitable for inhalation or intravenous, intraperitoneal, intramuscular, subcutaneous, mucocutaneous, oral, rectal, transdermal, topical, buccal, intradermal, intragastric, intradermal, intranasal, intraoral, transdermal or sublingual administration. In certain embodiments, administration is by injection into the middle ear and topical application to the eardrum.

The pharmaceutical compositions may be prepared and administered in the form of transdermal delivery systems (patch films), drops, pills, tablets, film-coated tablets, multi-layered tablets, gels, ointments, syrups, granules, suppositories, emulsions, dispersions, microcapsules, capsules, powders or injectable solutions. The pharmaceutical preparations may be in the form of liposomes, emulsions and gels.

For the preparation of suppositories, low melting waxes, fatty acid esters and glycerides may be used. Pharmaceutical compositions for any route of administration of the beta-carbolines comprise a sufficient therapeutically effective amount of the beta-carboline, and if necessary an inorganic or organic solid or liquid pharmaceutically acceptable carrier. Pharmaceutical compositions suitable for enteral or parenteral administration include tablets or gelatin capsules or aqueous solutions or suspensions as described above.

The pharmaceutical compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting and/or emulsifying agents, salts for regulating the osmotic pressure and/or buffers. The pharmaceutical composition of the present invention may contain other active ingredients, if necessary. These pharmaceutical compositions may be prepared by any method known in the art, for example by conventional methods such as mixing, granulating, packaging and lyophilizing solutions, and may be about 0.01% to 100%, preferably 0.1% to 50% of the lyophilizate, containing up to 100% of the beta-carboline.

Certain compositions of the present invention for topical administration may be other pharmaceutically acceptable substances and/or substances. In certain embodiments of the invention, such a topical excipient is selected: if it is disintegrating (verbrerich) at, in or in the ear canal, it does not increase the delivery of beta-carboline and optionally other active ingredients into the blood circulation system or central nervous system. For example, it may be desirable for the topical excipient not to have significant repellency to enhance transdermal transport through the mucosa into the systemic circulatory system. Such carriers include hydro carboxylic acids such as anhydrous absorbent hydrophilic petrolatum (petrolatum) and anhydrous lanolin (e.g. Aquaphor), as well as means based on water-oil emulsions such as lanolin and cold creams. Certain embodiments include substantially non-exclusive carriers and generally include water-soluble support materials, as well as materials based on oil-in-water emulsions (creams or hydrophilic ointments) and water-soluble base materials such as polyethylene glycol-based excipients and aqueous solutions gelled with various agents, such as methylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose.

All delivery methods and materials mentioned in U.S. Pat. Nos. 5,837,681 and 6,593,290 are incorporated herein by reference.

If desired, the compositions to be administered may also contain minor amounts of non-toxic auxiliary carrier or excipient materials such as wetting agents, emulsifying or solubilizing agents, antioxidants, antimicrobials, pH buffering agents and the like, for example, sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate and the like. The actual methods of preparing these dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington, the science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 20 th edition, 2000. In any event, the composition or formulation to be administered contains an amount of the active compound effective to reduce the symptoms of the subject being treated. One skilled in the art will appreciate that the agents described herein may also be configured for targeted delivery to a subset of the tissues or cells of a subject. Typically, targeted delivery is achieved by constructing the compound of the agent with targeting moieties. These moieties include lipids, liposomes, and molecular ligands for binding other molecules in vivo.

Any derivative form of the agent (e.g., synthetic or natural compounds) or conjugates thereof can be prepared as acid or base salts, as well as free acid or free base forms. These compositions are encompassed in certain embodiments described herein if used to prevent or treat auditory dysfunction.

In some embodiments, the compositions herein also target support cells that do not express Lgr 5. In certain embodiments, in addition to promoting proliferation of the support cells, the agents described herein affect differentiation of the support cells into cell types that can help enhance hearing. In some embodiments, the agents described herein also affect the survival of the supporting cells.

The amount of agent required for therapeutic use may vary not only with the particular agent and salt selected, but also with factors such as the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or clinician. The required dose may conveniently be presented in a single dose, or in divided doses administered at appropriate intervals, for example two, three, four or more sub-doses per day. The sub-dose itself may be further divided, for example, into a plurality of discrete, loosely spaced administrations, such as multiple intakes of a bolus dose or a liquid dose.

Furthermore, the agents and their corresponding acid or base salts may be formulated as liquid (preferably aqueous) formulations for storage and administration, as well as dry formulations which may be used, for example, as powders for administration or reconstituted into liquid form immediately prior to administration to a subject. The pharmaceutically administrable liquid composition can be prepared, for example, by the following method: dissolving, dispersing, etc., the particular agent and optional pharmaceutical adjuvants in an aqueous carrier. Aqueous carriers include water (particularly for injection into humans), alcohol/water solutions, and emulsions and suspensions. Pharmaceutically acceptable aqueous carriers include sterile, buffered, isotonic saline solutions. The carrier may comprise sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or a non-volatile oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Non-aqueous solvents may also be used, including propylene glycol, ethanol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). The nutritional, pharmaceutical and veterinary compositions of the agents, whether dry or liquid, may also preferably be formulated for oral administration.

As used herein, a coating (also referred to as a film-forming agent) is a solution consisting of a solvent, a monomer or polymer, an active agent, and optionally one or more pharmaceutically acceptable excipients. After application to tissue, the solvent evaporates, leaving a thin coating consisting of the monomer or polymer and the active agent. By way of non-limiting example, the coating includes collodion (e.g., flexible collodion, USP) and a solution comprising the saccharide siloxane copolymer and a crosslinker. The coatings contemplated for use herein are flexible so that they do not interfere with the propagation of pressure waves through the ear. In addition, the coating may be applied as a liquid (i.e., a solution, suspension, or emulsion), semi-solid (i.e., a gel, foam, paste, or jelly), or aerosol.

Examples of suitable foamable carriers for use in the compositions disclosed herein include, but are not limited to: alginates and derivatives thereof, carboxymethylcellulose and derivatives thereof, collagen, polysaccharides including, for example, dextran derivatives, pectin, starch, modified starches (e.g., starches having additional carboxyl and/or carboxamide groups and/or having hydrophilic side chains), cellulose and derivatives thereof, agar and derivatives thereof (e.g., agar stabilized with polyacrylamide), polyoxyethylene, ethylene glycol methacrylate, gelatin, gums (e.g., xanthan gum, guar gum, karaya gum, gellan gum, gum arabic, tragacanth gum, and locust bean gum), or combinations thereof. The formulation may also optionally contain a foaming agent to promote foam formation, including surfactants or external propellants. Examples of suitable foaming agents include cetyltrimethylammonium bromide, lecithin, soaps, silicones, and the like. Commercially available surfactants, e.g. Tween^TMAnd are also suitable.

In particular embodiments, gel formulations useful for practicing the methods described herein include, but are not limited to: glycerolic gels, glycerol-derived compounds, conjugated or crosslinked gels, matrices, hydrogels, and polymers, as well as gelatin and its derivatives, alginates, and alginate-based gels, as well as various natural and synthetic hydrogels and hydrogel-derived compounds.

In some embodiments, the concentration of each pharmaceutically active ingredient (i.e., the Notch activator and/or HDAC inhibitor and the GSK3b inhibitor and/or WNT activator or derivatives, small molecules, pharmaceutically acceptable salts, prodrugs, solvates, stereoisomers, racemates or tautomers) of the compositions described herein is about 0.01% to about 90%, about 0.01% to about 50%, about 0.1% to about 70%, about 0.1% to about 50%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10% or about 0.1% to about 5% by weight of the composition.

In some embodiments, each pharmaceutically active agent of the compositions described herein as an active ingredient, or a pharmaceutically acceptable salt, prodrug, solvate, stereoisomer, racemate or tautomer thereof, is present at a concentration of from about 1% to about 50%, from about 5% to about 50%, from about 10% to about 40%, or from about 10% to about 30% by weight of the composition.

In some embodiments, the pharmaceutically active ingredient of the formulations described herein, as an active agent or a pharmaceutically acceptable salt, prodrug, solvate, stereoisomer, racemate or tautomer thereof, is at a concentration of from about 0.1mg/ml to about 70mg/ml, from about 0.5mg/ml to about 50mg/ml, from about 0.5mg/ml to about 20mg/ml, from about 1mg/ml to about 70mg/ml, from about 1mg/ml to about 50mg/ml, from about 1mg/ml to about 20mg/ml, from about 1mg/ml to about 10mg/ml, or from about 1mg/ml to about 5mg/ml of the volume of the formulation.

In one embodiment, the formulations disclosed herein additionally provide for the immediate release, or release within 1 minute, or within 5 minutes, or within 10 minutes, or within 15 minutes, or within 30 minutes, or within 60 minutes, or within 90 minutes, of one or more pharmaceutically active ingredients (i.e., the Notch activator and/or HDAC inhibitor and GSK3b inhibitor and/or WNT activator, or other small molecules, pharmaceutically acceptable salts, prodrugs, solvates, stereoisomers, racemates or tautomers thereof) from the composition. In other embodiments, a therapeutically effective amount of at least one pharmaceutically active ingredient (i.e., a Notch activator and/or HDAC inhibitor and a GSK3b inhibitor and/or WNT activator or derivatives, small molecules, pharmaceutically acceptable salts, prodrugs, solvates, stereoisomers, racemates or tautomers thereof) is released from the composition immediately or within 1 minute, or within 5 minutes, or within 10 minutes, or within 15 minutes, or within 30 minutes, or within 60 minutes or within 90 minutes.

In other embodiments, the composition is configured as an extended release formulation. In certain embodiments, diffusion of at least one pharmaceutically active ingredient (including a Notch activator and/or HDAC inhibitor and a GSK3b inhibitor and/or WNT activator), small molecule, pharmaceutically acceptable salt, prodrug, solvate, stereoisomer, racemate or tautomer thereof, from the formulation occurs over a period of 5 minutes, or 15 minutes, 30 minutes, or 1 hour, or 4 hours, or 6 hours, or 12 hours, or 18 hours, or 1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 10 days, or 12 days, or 14 days, or 18 days, or 21 days, or 25 days, or 30 days, or 45 days, or 2 months, or 3 months, or 4 months, or 5 months, or 6 months, or 9 months, or 1 year. In other embodiments, the therapeutically effective amount of at least one pharmaceutically active ingredient (including the Notch activator and/or HDAC inhibitor and GSK3b inhibitor and/or WNT activator) is released from the formulation over a period of 5 minutes, or 15 minutes, 30 minutes, or 1 hour, or 4 hours, or 6 hours, or 12 hours, or 18 hours, or 1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 10 days, or 12 days, or 14 days, or 18 days, or 21 days, or 25 days, or 30 days, or 45 days, or 2 months, or 3 months, or 4 months, or 5 months, or 6 months, or 9 months, or 1 year.

In other embodiments, the formulation provides an immediate release formulation and an extended release agent. In particular embodiments, the formulation comprises an immediate release formulation and an extended release formulation in a ratio of 0.25:1, or a ratio of 0.5:1, or a ratio of 1:2, or a ratio of 1:3 or a ratio of 1:4, or a ratio of 1:5, or a ratio of 1:7, or a ratio of 1:10, or a ratio of 1:15, or a ratio of 1: 20. In another embodiment, the formulation provides immediate release of the first pharmaceutically active ingredient (i.e., the small molecule, pharmaceutically acceptable salt, prodrug, solvate, stereoisomer, racemate or tautomer thereof) and extended release of the second pharmaceutically active ingredient or other therapeutic agent. In some embodiments, the formulation provides an immediate release formulation and an extended release formulation of the first pharmaceutically active ingredient and the second pharmaceutically active ingredient in a 0.25:1 ratio, or a 0.5:1 ratio, or a 1:2 ratio, or a 1:3 or a 1:4 ratio, or a 1:5 ratio, or a 1:7 ratio, or a 1:10 ratio, or a 1:15 ratio, or a 1:20 ratio.

Combinations of immediate release, delayed release, and/or extended release compositions or formulations can be combined with other agents disclosed herein as well as excipients, diluents, stabilizers, carriers, and other components. Thus, depending on the components of the composition, the desired thickness or viscosity, or the selected mode of delivery, alternative aspects of the embodiments disclosed herein are correspondingly combined with immediate release, delayed release, and/or extended release embodiments.

F. Administration of

In certain embodiments, the pharmaceutical formulation is adapted for topical application of the drug to the round window membrane. The pharmaceutical formulation may also include a membrane permeation enhancer that supports the agent described herein across the round window membrane. Thus, liquid, gel, or foam formulations may be used in these embodiments. The active ingredients may also be administered orally or using a combination of delivery routes.

The long acting formulation with extended drug release and/or improved stability may be in the form of: the agents described herein are complexed or covalently conjugated (through reversible or irreversible bonding) to macromolecules such as water-soluble polymers selected from PEG and polypropylene glycol homopolymers and polyoxyethylene polyols, i.e. those that are soluble in water at room temperature. Alternatively, the agents mentioned herein may be complexed or conjugated to polymers to prolong their drug release and/or half-life. Examples of polyethylene polyols and polyoxyethylene polyols that may be used for this purpose include polyoxyethylene glycerol, polyethylene glycol, polyoxyethylene sorbitol, polyoxyethylene glucose, and the like. The glycerol backbone of polyoxyethylene glycerol is the same backbone that occurs in, for example, monoglycerides, diglycerides and triglycerides in animals and humans. The agents mentioned herein may be encapsulated in and/or conjugated to a material. To deliver more than one agent, one or more delivery vehicles may be used. The delivery kinetics of the multiple agents may be the same or different. For example, in one case, it may be beneficial to include a short burst of drug and have the other drug released for a longer period of time.

Liquid formulations include solutions, suspensions, emulsions and sprays. For example, injection solutions for parenteral injection, water or water-propylene glycol.

Pharmaceutical compositions suitable for topical administration in the middle ear include aqueous solutions or suspensions that can be prepared separately or together with a carrier prior to administration in the middle ear, for example in the case of lyophilized formulations containing the compositions of the present disclosure. The pharmaceutical compositions also include gels, which are biodegradable or non-biodegradable, aqueous or non-aqueous, or based on microspheres. Examples of such gels and other suitable materials include poloxamers, hyaluronate, xyloglucan, chitosan, polyesters, polylactide, polyglycolide and their copolymers PLGA polymers, polyanhydrides, polycaprolactone sucrose, and glycerol monooleate.

The pharmaceutical compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting and/or emulsifying agents, salts for regulating the osmotic pressure and/or buffers. If desired, the pharmaceutical compositions may contain other active ingredients in addition to those previously described herein.

These pharmaceutical compositions may be prepared by any method known in the art, for example by conventional methods such as mixing, granulating, packaging and lyophilizing the solution.

The pharmaceutical composition may also comprise agents and/or excipients that facilitate controlled delivery as described in US patent application US 20110166060.

In certain embodiments, the pharmaceutical compositions of the present invention are formulated for topical administration. Suitable carriers for otogenic administration are organic or inorganic substances which are pharmaceutically acceptable and do not react with the agents, and/or other active compounds, for example saline, alcohols, vegetable oils, benzyl alcohols, alkyl glycols, polyethylene glycols, triacetin, gelatin, carbohydrates (e.g. lactose or starch), magnesium oxide (magnesite, chalk), stearates (wax), talc and petrolatum (vaseline). The above-mentioned compositions may be sterilized and/or contain adjuvants, such as lubricants, preservatives (e.g. thimerosal, e.g. 50% by weight), stabilizers and/or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, colorants and/or flavorings. These compositions may contain one or more other active ingredients, if desired. The otogenic compositions of the present invention may comprise different materials and/or substances, including other biologically active substances, such as antibiotics, anti-inflammatory agents, such as steroids, cortisone, analgesics, antipyrine, benzocaine, procaine, and the like.

The delivery of drugs to the ear tympanic cavity (IT) is increasingly used for clinical and research purposes. Some groups have used microcatheters or microcatheter needles to administer drugs in a sustained manner, but most have administered them as single or repeated IT injections (up to 8 injections over a period of up to two weeks).

Intratympanic administered drugs are believed to enter the inner ear fluid primarily by crossing the Round Window (RW) membrane. Calculations show that the primary factor controlling the amount of drug entering the ear and the distribution of the drug along the length of the ear is the time the drug remains in the middle ear space. A single "single dose" administration or administration of an aqueous solution for several hours results in a steep drug gradient of the administered substance along the length of the cochlea and, as the drug is subsequently distributed throughout the ear, a rapid drop in concentration in the basal turn of the cochlea.

Methods for delivery (including related materials and variations thereof) and methods of studying the distribution and kinetics of drugs delivered to the inner ear are known in the art. For example, in one embodiment, a 20% (w/w) stock solution of poloxamer 407 gel (spectra chemical mfg. corp., Gardena, Calif, USA) is prepared by slowly adding it to cold 10mM phosphate buffered saline at pH 7.4. Other poloxamers or other materials may also be used. Additional buffer solution was added to obtain poloxamer 407 gel at a concentration of 17% w/w. The solution is liquid at refrigeration or at room temperature, but solidifies at body temperature. The gel may be colored with a dye such as evans blue dye (50ppm) and sterilized by filtration. Using aseptic technique, sterilized micronized dex (pure dex in crystalline powder form; Pfizer Inc., Kalamazoo, Mich., USA) was suspended with an appropriate amount of sterile poloxamer 407 solution to give a 4.5% solution. Formulation samples were stored under refrigerated conditions and resuspended immediately prior to administration.

Poloxamer 407 hydrogel was prepared using the "cold method". Briefly, a weighed amount of poloxamer 407 was added to 40ml of cold ultrapure water or cold PBS (pH 7.4) and stirred overnight at 4 ℃ on a magnetic stir plate to achieve complete dissolution. Solutions of poloxamer 407 were prepared at various concentrations ranging from 18% (w/w) to 25% (w/w). Hydrophilic drugs, including valproic acid (VPA) and phosphorylated ascorbic acid (PAC), were added to 5ml of poloxamer 407 solution and dissolved on a magnetic stir plate at 4 ℃. The weight ratio of poloxamer 407 to drug was varied to understand the effect of the drug on the gelling properties of poloxamer 407 and to determine the optimal formulation to gel at 37 ℃ with the maximum possible loading of hydrophilic drug. The gelation temperature of the formulation was determined by "visual tube inversion method". Briefly, a glass vial containing a solution of poloxamer 407 with or without a hydrophilic drug was placed in a water bath. The temperature was slowly increased and the temperature at which the flow of the solution stopped when the glass vial was tilted was recorded as the gelation temperature.

To encapsulate hydrophobic drugs, including CHIR99021 (CHIR), Repsox and TTNPB, an appropriate volume from its DMSO stock solution was added to the hydrophilic drug-containing poloxamer 407 solution and mixed with suction at 4 ℃. The maximum DMSO concentration to which the hydrophobic drug is added is limited to 5% to 6% (v/v) of the total gel volume. Higher concentrations of DMSO lower the gelation temperature of the gel. The gelation temperature of the formulation was determined by "visual tube inversion" method as described previously.

The bioactive agents of the present disclosure can be prepared as acid or base salts, as well as in the free acid or free base form. The amount of agent required for therapeutic use may vary not only with the particular agent and salt selected, but also with factors such as the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or clinician. The required dose may conveniently be presented in a single dose, or in divided doses administered as appropriate intervals.

Furthermore, the active agents and their corresponding acid or base salts may be formulated as liquid (preferably aqueous) preparations for storage and administration, as well as dry preparations which may be used, for example, as powders for administration or reconstituted into liquid form immediately prior to administration to a subject. The pharmaceutically administrable liquid composition can be prepared, for example, by the following method: dissolving, dispersing, etc., the particular agent and optional pharmaceutical adjuvants in an aqueous carrier. Aqueous carriers include water (particularly for injection into humans), alcohol/water solutions, and emulsions and suspensions. Pharmaceutically acceptable aqueous carriers include sterile, buffered, isotonic saline solutions. The carrier may comprise sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or a non-volatile oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Non-aqueous solvents may also be used, including propylene glycol, ethanol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate).

Measuring hearing ability

Hearing can be measured using behavioral audiometry and/or electronic audiometry.

Tests to diagnose hearing loss in humans may include, but are not limited to:

general screening tests. The doctor may require covering one ear at a time to see how well the individual hears speech at various volumes, and how they respond to other sounds.

And (5) testing a tuning fork. A tuning fork is a two-pronged metal device that produces sound when struck. A simple test using a tuning fork can help a physician to detect hearing loss. Tuning fork assessment may also reveal whether the hearing loss is due to damage to the vibrating portion of the middle ear (including the eardrum), to the sensor or nerves of the inner ear, or to both.

And (5) testing by using an audiometer. In these more thorough tests conducted by an audiologist, an individual wears headphones and hears sound to one ear at a time. Audiologists provide sounds of various tones and require an individual to indicate each time they hear a sound. Each tone is repeated at a faint level to find out when the individual is barely audible. Audiologists also issue various words to determine the hearing ability of an individual.

Other tests may include:

an Auditory Brainstem Response (ABR) test or a Brainstem Auditory Evoked Response (BAER) test, which examines the brain's response to sound. Since the test is independent of the person's reaction behavior, the person under test may fall asleep completely during the test.

Otoacoustic emission (OAE) is a test that examines the response of the inner ear to sound. Since the test is independent of the person's reaction behavior, the person under test may fall asleep completely during the test.

Behavioral audiometry evaluates how a test person responds to sound as a whole. Behavioral audiometry assessments tested the function of all parts of the ear. The person to be tested must be awake and actively respond to the sounds heard during the test.

For a child, the audiologist will, with parental approval, share results with the child's primary care physician and other experts, such as:

ear, nose and throat doctors (also known as otorhinolaryngologists), oculists (also known as ophthalmologists), genetically trained professionals (also known as clinical geneticists or genetic counselors).

Exemplary embodiments

The present disclosure also includes the following exemplary embodiments.

In some embodiments, a "stem cell" comprises a progenitor cell.

In some embodiments, an FGF1 agonist is used. In some embodiments, it may be substituted or added to the Notch activator and/or HDAC inhibitor. In some embodiments, a molecule that activates the promoter of the FGF1 gene (e.g., FGF-1B) is selected.

In certain embodiments, the cell populations described herein can be delivered to the inner ear to occupy the inner ear and/or enhance hearing. It has previously been shown that cell populations survive transplantation into the inner ear as described in patent CN103361300A and the paper.

In addition, human amniotic epithelial cells have been shown to survive for up to three weeks after transplantation into the inner ear of guinea pigs, and expression of important proteins remains homeostatic (Yuge, 1, et al, (2004): 77 (9)) 1452-1471 ".

In certain embodiments, Lgr5 from the inner ear is administered using a combination of at least one WNT activator and at least one NOTCH inhibitor⁺And (4) cell differentiation.

In certain embodiments, the polyoxyethylene-polyoxypropylene triblock copolymer or derivative thereof may be used to deliver the molecules or factors described herein to the middle ear and/or for controlled release.

In certain embodiments, the method comprises determining a baseline level of hearing at one or more frequencies prior to administration of the composition, and determining a subsequent hearing level at the same one or more frequencies after administration of the composition, and administering one or more additional doses of the composition until the desired hearing level at the one or more frequencies is restored.

In certain embodiments, subsequent hearing levels are determined one week, two weeks, three weeks, one month, two months, three months, four months, six months, and/or twelve months after administration of the composition.

The following methods are presented herein: wherein the mammal is a child, adolescent or adult, for example over the age of 1,2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 or 13 years.

The following methods are presented herein: wherein the mammal is an adult human of at least 40 years of age, e.g., at least 45, 50, 55, 60, 65, 70 years of age.

Methods of delivering a composition to a mammal are given herein.

Methods are presented in which the mammal is a human.

Described herein is the use of cell populations for ototoxicity assays, e.g., using Lgr5 in 96 or 384 well plates (or other plate formats)⁺Screening cells for agents that enhance production of support cells or proteins expressed by support cells (or screening for drugs that inhibit protein expression) and/or differentiation thereof, or identifying agents that produce ototoxic hair cells or enhance hair cell survival, which can help maintain hearing after hair cell damage or hair cells after regeneration, or screening for killing of specific cell populations if necessary (Lgr 5)⁺Cells or progeny thereof, including hair cells). Lgr5 produced herein may also be used⁺The cells serve as a population of cells for reprogramming or programming protocols.

In certain embodiments, γ -secretase inhibitors are used in addition to GSK3b inhibitors to promote differentiation of the supporting cells into hair cells.

The following methods are presented herein: amplification of Lgr5 with at least one wnt activator and at least one notch activator (and/or HDAC inhibitor)⁺The cells are supported and then differentiated into hair cells using at least one WNT activator (CHIR is used in the preferred embodiment) and one notch inhibitor.

A method of treating a subject suffering from or at risk of developing hearing loss or vestibular dysfunction, the method comprising: identifying a subject who has experienced or is at risk of developing hearing loss or vestibular dysfunction, administering to the ear of the subject a composition comprising one or more compounds that increase wnt expression or activity and inhibit HDAC (and/or activate NOTCH) in cells in the ear of the subject, thereby treating hearing loss or vestibular dysfunction in the subject.

A method of treating a subject suffering from or at risk of developing hearing loss or vestibular dysfunction, the method comprising: identifying a subject who has experienced or is at risk of developing hearing loss or vestibular dysfunction, administering a composition comprising CHIR99021 to the ear of the subject.

A method of treating a subject suffering from or at risk of developing hearing loss or vestibular dysfunction, the method comprising: selecting a subject in need of treatment, obtaining a population of cells capable of differentiating into hair cells, contacting the population of cells in vitro with an effective amount of a composition comprising one or more compounds that increase WNT expression or activity and inhibit HDAC (and/or activate NOTCH) for a time sufficient to induce at least some of the cells to express one or more WNT, or to reduce or limit expression of HDAC, and/or to express NOTCH or a homolog thereof, and administering the population of cells or a subset thereof to the ear of the subject.

In one embodiment, the cell populations described herein can be used to screen for gamma-secretase inhibitors that induce differentiation of inner ear stem cells into hair cells.

In one embodiment, the tube is placed in the tympanic membrane for continuous and/or repeated administration into the middle ear.

In particular embodiments, the composition comprises a biodegradable polymer.

In another embodiment, the composition comprises one or more small molecules that decrease the expression of Atoh1 gene.

In one embodiment, the composition comprises one or more small molecules that reduce the expression of Atoh1 protein.

In another embodiment, the composition comprises one or more small molecules that increase the expression of the Atoh1 gene.

In one embodiment, the composition comprises one or more small molecules that increase the expression of Atoh1 protein.

In a certain embodiment, the composition comprises one or more small molecules that increase the activity of the Atoh1 protein.

In other embodiments, the one or more small molecules that increase Atoh1 gene expression, increase Atoh1 protein expression, or increase Atoh1 protein activity are selected from the group consisting of CHIR99021, 1-azakinoparone, and (2' Z,3' E) -6-bromoindirubin-3 ' -oxime (BIO).

In particular embodiments, the composition comprises a biodegradable polymer.

In another embodiment, the composition is administered to the middle ear of the subject.

In another embodiment, the composition is applied on or near a round window film.

In one embodiment, the composition is administered to the inner ear of a subject.

In a particular embodiment, the composition is administered to the cochlea of a subject. In one embodiment, the composition is administered to a Corti organ of the subject.

In yet another embodiment, the composition is administered by trans-tympanic administration.

In another embodiment, the composition is administered with a transtympanic stylet.

In yet another embodiment, the composition is administered with a trans-tympanic catheter.

In yet another embodiment, the composition is administered by intracochlear injection.

In various embodiments, a portion of the present disclosure encompasses a method of promoting cochlear hair cell regeneration, comprising administering to the middle or inner ear of a subject an effective amount of a composition comprising a notch activator (and/or HDAC inhibitor) and a wnt activator for a time sufficient to promote cochlear hair cell proliferation, thereby promoting cochlear hair cell regeneration.

In one embodiment, the subject has partial or complete hearing loss or balance loss.

In particular embodiments, the subject has sensorineural hearing loss due to acute or chronic exposure to an ototoxic compound, acute or chronic exposure to noise, age-related hearing loss, genetically related hearing loss, or has auditory neuropathy.

In certain embodiments, the subject is at risk for developing sensorineural hearing loss or auditory neuropathy.

The methods and reagents described in the following documents are included herein in their entirety.

Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells.White PM,Doetzlhofer A,Lee YS,Groves AK,SegilN.Nature.2006 Jun 22；441(7096):984-7。

Lgr5-positive supporting cells generate new hair cells in thepostnatal cochlea.Bramhall NF,Shi F,Arnold K,Hochedlinger K,Edge AS.Stem CellReports.2014 Feb20；2(3):311-22.doi:10.1016/j.stemcr.2014.01.008.eCollection2014Mar 11。

Notch inhibition induces cochlear hair cell regeneration and recoveryof hearing after acoustic trauma.Mizutari K,Fujioka M,Hosoya M,Bramhall N,Okano HJ,Okano H,Edge AS.Neuron.2013Jan 9；77(1):58-69.doi:10.1016/j.neuron.2012.10.032.Erratum in:Neuron.2013Apr 24；78(2):403。

Generation of hair cells in neonatal mice byβ-catenin overexpressionin Lgr5-positive cochlear progenitors.Shi F,Hu L,Edge AS.Proc Natl Acad Sci US A.2013 Aug 20；110(34):13851-6.doi:10.1073/pnas.1219952110.Epub 2013Aug 5。

Notch signaling alters sensory or neuronal cell fate specification ofinner earstem cells.Jeon SJ,Fujioka M,Kim SC,Edge AS.J Neurosci.2011Jun 8；31(23):8351-8.doi:10.1523/JNEUROSCI.6366-10.2011。

Wnt-responsive Lgr5-expressing stem cells are hair cell progenitorsin the cochlea.Shi F,Kempfle JS,Edge AS.J Neurosci.2012Jul 11；32(28):9639-48.doi:10.1523/JNEUROSCI.1064-12.2012.PMID:

Spontaneous hair cell regeneration in the neonatal mouse cochlea invivo.Cox BC,Chai R,Lenoir A,Liu Z,Zhang L,Nguyen DH,Chalasani K,SteigelmanKA,Fang J,Rubel EW,Cheng AG,Zuo J.Development.2014Feb；141(4):816-29.doi:10.1242/dev.103036。

Those skilled in the art will recognize many variations on how to use and deliver the agents described herein or derivatives thereof. Combinations with other agents may also be used. Relevant patent documents describing other agents that may be used in combination with the agents described herein (and describing methods that may be used) include, but are not limited to: US 20130324542 a1, WO2011079841 a1, US 7498031B 2, US 6177434B 1, CA 2268331C, WO2008010852 a2, US 7387614B 2, WO1996040094 a1, WO2008076556 a 2.

Additional background information

Auditory hair cells of birds

Currently, hearing loss in mammals is permanent. Although hearing-impaired frogs, fish and birds will naturally recover their hearing, mammals lose this ability as early as 3 hundred million years, and scientists have not been successful in solving this problem to date. The ultimate goal of hair cell regeneration is to restore functional hearing. Because birds begin to perceive and produce chirps early in life, they provide an advantageous mode of study to investigate not only whether regeneration of lost hair cells reproduces auditory sensitivity, but also whether the periphery of such regeneration restores complex auditory perception and auditory production. They are rare instances where hair cell regeneration occurs naturally after hair cell loss, and the ability to correctly sense and produce complex acoustic signals is critical for reproduction and survival. Thus, there are biology that regenerates inner ear cells to enhance or restore hearing.

Importantly, acoustic overstimulation can lead to loss of sensory cells (hair cells) in the auditory epithelium. Damaged hair cells in the organ of Corti (mammalian auditory end organs) degenerate and are replaced by non-sensory cells (supporting cells), which constitute an irreversible scar. However, in birds, damaged auditory hair cells from acoustic trauma or ototoxic drugs can be replaced by new hair cells. As early as one day after noise exposure, the supporting cells in the damaged area of avian auditory epithelium incorporate the DNA-specific marker bromodeoxyuridine 7. Following acoustic challenge to the inner ear of birds, supporting cells located within the sensory epithelium divide near the luminal surface and refill the epithelium. These results indicate that the supporting cells are involved in scarring during hair cell degeneration and produce regenerating new cells.

Hearing loss and destruction of hair cells

The inner ear capillaries are critical for hearing and vestibular function. In mammals, the loss of sensory hair cells is permanent because there is no significant ability to regenerate these cells. Drugs such as aminoglycoside antibiotics and many antineoplastic drugs are often used, despite undesirable side effects. One such side effect is hearing loss due to death of sensory hair cells of the inner ear. Aminoglycosides are clinically used drugs that cause dose dependent sensorineural hearing loss (Smith et al, New Engl J Med. (1977)296:349-53) and are known to kill hair cells in the inner ear of mammals (Theopold, Acta Otolaryngol (1977)84: 57-64). In the united states, over 200 million people receive aminoglycoside therapy each year. The clinical efficacy of these drugs in the treatment of drug-resistant bacterial infections and their low cost generally explain their continued use and need. Cisplatin is a chemotherapeutic agent that, despite its toxic effects on inner ear hair cells, is still used because it is beneficial for life. High frequency hearing loss (>8kHZ) has been reported to be as high as 90% in children undergoing cisplatin therapy (Allen et al, Otolarynggol Head neural Surg (1998)118: 584-588). Relatively few studies have been made on the incidence of vestibular toxic effects of these drugs on patient populations. It is continuously reported in the literature that patients with aminoglycoside-induced vestibulotoxicity are estimated to be 3% to 6% (Dhandirddy et al, Arch Otolantrol Head NeckSurg (2005)131: 46-48). Other clinically important and commonly used drugs also have documented ototoxic effects, including loop diuretics (Greenberg, Am J Med Sci. (2000)319:10-24) and antimalarial quinine (Claessen et al, TropMed Int Health, (1998)3:482-9), salicylates (Matz, an Otol Rhinol Larynggol Suppl (1990)148: 39-41).

Studies over the past decades have discovered a number of key intracellular events that can lead to hair cell death. Several candidate protective agents have been evaluated, such as antioxidants, caspase inhibitors and jun kinase inhibitors (Kopke RD et al, Am JOtol 1997,18: 559-571; Liu W et al, Neuroreport 1998,9: 2609-2614; Yamasoba T. et al, BrainRes 1999,815:317 JI 325; Matsui et al, J Neurosci 2002,22: 1218-1227; Sugahara K et al, Hear Res 2006,221: 128-135). Some of these candidate otoprotectants have progressed to the human trial stage (Sha SH et al, N Engl J Med 2006,354: 1856-. Furthermore, in response to different forms of injury, different cell death pathways can be triggered, and many protective molecules provide incomplete hair cell protection, suggesting that multiple drug treatment approaches may provide the greatest benefit. Several examples of agents that have been explored to protect hair cells are included in U.S. patent application publication No. US20110135756a 1.

Hearing loss or impairment is a common occurrence in mammals, and impairment anywhere along the auditory pathway from the external auditory meatus to the central nervous system can result in hearing loss. Auditory devices can be divided into the outer middle ear, the inner ear, and the auditory nerve, as well as the central auditory pathway. Although there are some differences between species, the general characteristics of all mammals are common. Auditory stimuli are mechanically transmitted to the inner ear through the external auditory canal, tympanic membrane and ossicular chain. The middle ear and mastoid process are usually filled with air. Disorders of the external and middle ear usually produce conductive hearing loss by interfering with this mechanical transmission. Common causes of conductive hearing loss include obstruction of the external ear canal, which can be caused by occlusion or cerumen in the ear canal, thickening or perforation of the tympanic membrane, which can be caused by trauma or infection, fixation or resorption of components of the ossicular chain, and obstruction of the eustachian tube, resulting in fluid engorgement of the middle ear space. Auditory information is transformed from mechanical signals to neuro-conductive electrical impulses through the action of neuroepithelial cells (hair cells) and inner ear SGN. All central fibers of SGN form synapses in the cochlear nucleus of the pons. Auditory projections from the cochlear nucleus are bilateral, with the main nucleus located in the hypothalamus, medial geniculate and temporal lobe of the thalamus. The number of neurons involved in hearing increases dramatically from the cochlea to the auditory brainstem and auditory cortex. All auditory information is transformed by a limited number of hair cells, which are sensory receptors of the inner ear, where a relatively small number of so-called inner hair cells are of crucial importance, since they form synapses with about 90% of primary auditory neurons. In contrast, at the level of the cochlear nucleus, the number of involved neurons is in the hundreds of thousands. Thus, damage to relatively few cells in the auditory periphery can result in greater hearing loss. Thus, many causes of sensorineural loss can be attributed to injury in the inner ear. Such hearing loss may be progressive. Furthermore, hearing becomes significantly less acute as the anatomy of the ear changes with age of the animal.

During embryogenesis, the vestibular ganglia, spiral ganglia and auditory capsule originate from the same neurogenic ectoderm, the auditory basal plate. Thus, the vestibular and auditory systems share many features, including peripheral neuronal innervation of hair cells and central projection of the brainstem nuclei. Both systems are sensitive to ototoxins, including therapeutic drugs, antineoplastic agents, contaminants in food or drugs, and environmental and industrial contaminants. Ototoxic drugs include the widely used chemotherapeutic agent cisplatin and its analogs (Fleischman et al, 1975; Stadnicki et al, 1975; Nakai et al, 1982; Bertgren et al, 1990; Dublin, 1976; Hood and Berlin, 1986), the commonly used aminoglycoside antibiotics (e.g., gentamicin, for the treatment of infections caused by gram-negative bacteria) (Sera et al, 1987; Hinojosa and Lerner, 1987; Bareggi et al, 1990), quinine and its analogs, salicylates and its analogs, and loop diuretics.

The toxic effects of these drugs on auditory cells and spiral ganglion neurons are often the limiting factor in their therapeutic availability. For example, antibacterial aminoglycosides such as gentamicin, streptomycin, kanamycin, tobramycin, and The like, are known to have severe toxicity, particularly ototoxicity and nephrotoxicity, which reduces The availability of these antimicrobials (see The Pharmacological Basis of Therapeutics, 6 th edition, a. Goodman Gilman et al, eds.; Macmillan Publishing co., inc., New York, page 1169-. Aminoglycoside antibiotics are commonly used as broad-spectrum antimicrobial agents that are effective against, for example, gram-positive bacteria, gram-negative bacteria, and acid-resistant bacteria. Susceptible microorganisms include the genera Escherichia, Haemophilus, Listeria, Pseudomonas, Nocardia (Nocardia spp), Yersinia spp, Klebsiella spp, Enterobacter, Salmonella, Staphylococcus, Streptococcus, Mycobacterium, Shigella, and Serratia (Serratiaspp). Nevertheless, aminoglycosides are mainly used for the treatment of infections caused by gram-negative bacteria and are combined, for example, with penicillin to obtain a synergistic effect. All aminoglycoside antibiotics contain an amino sugar glycosidically linked as revealed by the common name of the family. Otitis media is a term used to describe middle ear infections, which are very common, especially in children. Typically, antibiotics are administered systemically to combat middle ear infections, e.g., in a responsive or prophylactic manner. Systemic administration of antibiotics to combat middle ear infections often results in longer delay times to achieve therapeutic levels in the middle ear, and high initial doses are required to achieve such levels. These disadvantages complicate the ability to achieve therapeutic levels and may preclude the use of some antibiotics together. Systemic administration is often most effective when the infection reaches a late stage, but may already cause permanent damage to the middle and inner ear structures. Ototoxicity is clearly a dose limiting side effect of antibiotic administration. For example, approximately 75% of patients receiving 2 grams of streptomycin per day for 60 to 120 days showed some vestibular impairment, while the incidence at 1 gram per day dropped to 25% (U.S. patent 5,059,591). Hearing impairment is observed in 4% to 15% of patients receiving 1 gram daily for more than one week, developing measurable hearing loss that slowly gets worse if treatment is continued and can lead to complete permanent deafness. Ototoxicity is also a serious dose-limiting side effect of cisplatin, a platinum coordination complex, which has proven effective in a variety of human cancers, including testicular, ovarian, bladder, and head and neck cancers. Cisplatin impairs the auditory and vestibular systems (Fleischman et al, 1975, Stadnicki et al, 1975, Nakai et al, 1982, Carenza et al, 1986, Sera et al, 1987, Bareggi et al, 1990); salicylic acids, such as aspirin, are the most commonly used therapeutic agents due to their anti-inflammatory, analgesic, anti-pyretic and anti-thrombotic effects. Unfortunately, they have ototoxic side effects, often leading to tinnitus ("ringing in the ear") and temporary hearing loss (Myers and Bernstein, 1965). However, if these drugs are used at high doses for a long time, the hearing impairment may persist and be irreversible as shown in clinical reports (Jarvis, 1966).

Related biology

Fish and birds produce inner ear hair cells, with the supporting cells in the cochlea acting as precursor cells. It is believed that hair cell regeneration is achieved by two methods: direct differentiation transformation, in which the supporting cells directly become hair cells; and mitotic regeneration, in which hair cell division is supported and one or both of the resulting cells develop into hair cells.

It has been determined that hair cell regeneration in the auditory and vestibular systems does occur in chickens and other non-mammals, but not in humans. This spontaneous regeneration results in the restoration of hearing and balance.

Other embodiments and related aspects

1. A method of promoting production of inner ear hair cells, the method comprising:

administering or causing to be administered to a population of stem cells a first composition comprising (i) and (ii):

(i) a GSK3 beta inhibitor or a derivative or a pharmaceutically acceptable salt thereof and/or a Wnt agonist or a derivative or a pharmaceutically acceptable salt thereof, and

(ii) (ii) a Notch agonist or derivative or pharmaceutically acceptable salt thereof and/or an HDAC inhibitor or derivative or pharmaceutically acceptable salt thereof, thereby expanding the stem cell population; and

exposing the expanded stem cell population to a second composition comprising a GSK3- β inhibitor and/or a Wnt agonist and optionally a notch inhibitor, thereby promoting production of inner ear hair cells from the expanded stem cell population.

The method of embodiment 1, wherein the stem cell population is an in vitro stem cell population.

The method of embodiment 1, wherein the stem cell population is an ex vivo stem cell population.

The method of embodiment 1, wherein the stem cell population is an in vivo stem cell population.

The method of embodiment 1, wherein the stem cell population is an in vivo stem cell population comprised by a subject, and the first composition is administered to the stem cell population by administering the first composition to the subject.

The method of any one of the preceding embodiments, wherein the first composition comprises a Wnt agonist.

The method of any of the preceding embodiments, wherein the first composition comprises a GSK3- β inhibitor.

The method of any one of the preceding embodiments, wherein the first composition comprises a notch agonist.

The method of any one of the preceding embodiments, wherein the first composition comprises an HDAC inhibitor.

The method of any one of the preceding embodiments, wherein the second composition comprises a Wnt agonist.

The method of any of the preceding embodiments, wherein the second composition comprises a GSK3- β inhibitor.

The method of any of the preceding embodiments, wherein the second composition comprises a notch agonist.

The method of any one of the preceding embodiments, wherein the second composition comprises an HDAC inhibitor.

The method of any one of the preceding embodiments, wherein the first and second compositions are the same composition.

The method of embodiment 1 or any one of embodiments 1A to 1L, wherein the first and second compositions are different compositions.

2. The method of any one of the preceding embodiments, wherein the population of stem cells comprises supporting cells.

3. The method of embodiment 2, wherein the support cell is Lgr5⁺A cell.

4. The method of any one of the preceding embodiments, wherein the stem cell population comprises postnatal cells.

5. The method of any one of the preceding embodiments, wherein the hair cells are inner ear hair cells.

6. The method of any one of the preceding embodiments, wherein the hair cells are external ear hair cells.

7. The method of any one of the preceding embodiments, wherein the administering step comprises administering or causing to be administered to a population of stem cells a notch agonist, which is also an HDAC inhibitor.

8. The method of any one of the preceding embodiments, wherein the administering step comprises administering or causing to be administered a notch agonist comprising a synthetic molecule to the stem cell population.

9. The method of any one of the preceding embodiments, wherein the administering step comprises administering or causing to be administered VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the population of stem cells (e.g., wherein VPA is a notch agonist and is also an HDAC inhibitor).

10. The method of any one of the preceding embodiments, wherein the step of administering comprises administering or causing to be administered to a population of stem cells a Wnt agonist that is also a GSK3- β inhibitor.

11. The method of any one of the preceding embodiments, wherein the step of administering comprises administering or causing to be administered a Wnt agonist comprising a synthetic molecule to a population of stem cells.

12. The method of any one of the preceding embodiments, wherein the administering step comprises administering or causing to be administered CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the stem cell population (e.g., wherein CHIR99021 is a GSK 3-beta inhibitor).

13. The method of any one of the preceding embodiments, wherein the administering step comprises administering or causing to be administered a notch inhibitor to the stem cell population.

14. The method of embodiment 13, wherein the notch inhibitor comprises DAPT (e.g., in a pharmaceutically acceptable form (e.g., a salt)).

15. The method of any one of the preceding embodiments, wherein the administering step comprises administering or causing to be administered (i) CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) and (ii) VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the population of stem cells (e.g., wherein (i) comprises CHIR99021 and (ii) comprises VPA).

16. The method of embodiment 15, wherein the step of administering further comprises administering or causing to be administered DAPT (e.g., wherein DAPT is a notch inhibitor) to the population of stem cells.

17. The method of any one of the preceding embodiments, wherein the administering step is performed by performing one or more injections into the ear (e.g., trans-tympanic injection).

18. The method of embodiment 17, wherein the one or more injections are into the middle ear.

19. The method of embodiment 17, wherein the one or more injections are injections into the inner ear.

20. The method of embodiment 17, wherein performing one or more injections comprises: anesthetizing the tympanic membrane and/or surrounding tissue, placing a needle through the tympanic membrane into the middle ear, and injecting (i) and/or (ii).

21. The method of any one of the preceding embodiments, wherein the administering step comprises administering or causing to be administered one or more additional agents (e.g., agents other than (i) and (ii)) to the population of stem cells.

22. The method of embodiment 21, wherein the one or more additional agents comprises a ROS inhibitor.

23. The method of embodiment 21 or 22, wherein the one or more additional agents comprise vitamin C or a derivative thereof.

24. The method of any one of embodiments 21-23, wherein the one or more additional agents comprises a TGF β type I receptor inhibitor.

25. The method of any one of the preceding embodiments, wherein the expanded stem cell population is at least three times larger than the stem cell population prior to the administering step.

26. The method of any one of the preceding embodiments, wherein the administering step comprises administering a notch agonist and/or an HDAC inhibitor in a pulsatile manner and a GSK3- β inhibitor and/or a Wnt agonist in a sustained manner.

27. The method of any one of the preceding embodiments, wherein the stem cell population is of an in vivo subject, and the method is a treatment of hearing loss and/or vestibular dysfunction (e.g., wherein production of inner ear hair cells from the expanded stem cell population results in partial or complete restoration of hearing loss and/or improved vestibular function).

28. The method of any one of the preceding embodiments, wherein the stem cell population is of a subject in vivo, and wherein the method further comprises delivering the drug to the subject (e.g., for treating a disease and/or disorder not associated with hearing loss and/or vestibular dysfunction) at a concentration higher than the known maximum safe dose for the subject (e.g., the known maximum safe dose when delivered without the method to produce inner ear hair cells) (e.g., due to a reduction or elimination of dose limiting ototoxicity of the drug).

29. The method of any one of the preceding embodiments, further comprising performing high-throughput screening using the produced inner ear hair cells.

30. The method of embodiment 29, comprising using the inner ear hair cells produced to screen molecules for toxicity to inner ear hair cells.

31. The method of embodiment 29, comprising using the produced inner ear hair cells to screen for the ability of a molecule to improve survival of inner ear hair cells (e.g., inner ear hair cells exposed to the molecule).

32. A method of producing an expanded stem cell population, the method comprising:

(iii) administering or causing to be administered both (i) and (ii) to a population of stem cells (e.g., a population of stem cells of an in vitro, ex vivo or in vivo sample/subject):

(i) a GSK 3-beta inhibitor and/or a Wnt agonist, and

(ii) a notch agonist and/or an HDAC inhibitor,

thereby expanding the stem cells in the stem cell population and producing an expanded stem cell population.

33. The method of embodiment 32, wherein the stem cell population comprises Lgr5⁺A cell.

34. The method of embodiment 33, wherein the stem cell population comprises postnatal stem cells.

35. The method of any one of embodiments 32 to 34, wherein the stem cell population comprises epithelial stem cells.

36. The method of any one of embodiments 32 to 35 wherein the step of administering comprises administering or causing to be administered to a population of stem cells a notch agonist, which notch agonist is also an HDAC inhibitor.

37. The method of any one of embodiments 32 to 36 wherein the step of administering comprises administering or causing to be administered a notch agonist comprising a synthetic molecule to the stem cell population.

38. The method of any one of embodiments 32 to 37, wherein the step of administering comprises administering or causing to be administered VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the population of stem cells (e.g., wherein VPA is a notch agonist and is also an HDAC inhibitor).

39. The method of any one of embodiments 32 to 38, wherein the step of administering comprises administering or causing to be administered to a population of stem cells a Wnt agonist that is also a GSK3- β inhibitor.

40. The method of any one of embodiments 32 to 39, wherein the step of administering comprises administering or causing to be administered a Wnt agonist comprising a synthetic molecule to a population of stem cells.

41. The method of any one of embodiments 32 to 40, wherein the administering step comprises administering or causing to be administered CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the population of stem cells (e.g., wherein CHIR99021 is a GSK 3-beta inhibitor).

42. The method of any one of embodiments 32-41, wherein the administering step is performed by performing one or more injections into the ear (e.g., trans-tympanic injection into the middle and/or inner ear).

43. The method of any one of embodiments 32 to 42, wherein the step of administering comprises administering a notch agonist and/or an HDAC inhibitor in a pulsatile manner and administering a GSK3- β inhibitor and/or a Wnt agonist in a sustained manner.

44. The method of any one of embodiments 32 to 43, wherein the stem cells are inner ear stem cells.

45. The method of any one of embodiments 32 to 44, further comprising high throughput screening using the generated expanded stem cell population.

46. The method of embodiment 45, comprising using the stem cells produced to screen the stem cells and/or progeny thereof for toxicity of the molecule.

47. The method of embodiment 45, comprising using the produced stem cells to screen for the ability of the molecule to improve survival of the stem cells and/or progeny thereof.

48. A method of treating a subject having or at risk of developing a hearing loss and/or vestibular dysfunction, the method comprising:

identifying a subject who has experienced or is at risk of developing a hearing loss and/or vestibular dysfunction,

(iii) administering or causing to be administered to the subject both (i) and (ii):

(i) a GSK 3-beta inhibitor and/or a Wnt agonist, and

(ii) a notch agonist and/or an HDAC inhibitor,

thereby treating or preventing hearing loss and/or vestibular dysfunction in said subject.

49. The method of embodiment 48, wherein said stem cell population comprises Lgr5⁺A cell.

50. The method of embodiment 48 or 49, wherein said stem cell population comprises postnatal cells.

51. The method of any one of embodiments 48 to 50, wherein said stem cell population comprises epithelial stem cells.

52. The method of any one of embodiments 48 to 51, wherein the step of administering comprises administering to or causing to be administered to the subject a notch agonist, which is also an HDAC inhibitor.

53. The method of any one of embodiments 48 to 52 wherein the step of administering comprises administering or causing to be administered to the subject a notch agonist comprising a synthetic molecule.

54. The method of any one of embodiments 48 to 53, wherein the step of administering comprises administering or causing to be administered VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the subject (e.g., wherein VPA is a notch agonist and is also an HDAC inhibitor).

55. The method of any one of embodiments 48 to 54, wherein the step of administering comprises administering to or causing to be administered to the subject a Wnt agonist that is also a GSK3- β inhibitor.

56. The method of any one of embodiments 48 to 55, wherein the step of administering comprises administering or causing to be administered to the subject a Wnt agonist comprising a synthetic molecule.

57. The method of any one of embodiments 48-56, wherein the step of administering comprises administering or causing to be administered CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to the subject (e.g., wherein CHIR99021 is a GSK 3-beta inhibitor).

58. The method of any one of embodiments 48-57, wherein the administering step is performed by performing one or more injections into the ear (e.g., trans-tympanic injection into the middle and/or inner ear).

59. The method of any one of embodiments 48 to 58, comprising administering a notch agonist and/or an HDAC inhibitor in a pulsatile manner and administering a GSK3- β inhibitor and/or a Wnt agonist in a sustained manner.

60. A composition comprising the first or second composition of any of the preceding embodiments.

61. A composition, comprising: (a) (ii) a GSK3- β inhibitor or a derivative or a pharmaceutically acceptable salt thereof, and/or a Wnt agonist or a derivative or a pharmaceutically acceptable salt thereof, and (ii) a notch agonist or a derivative or a pharmaceutically acceptable salt thereof, and/or an HDAC inhibitor or a derivative or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

62. A composition, comprising: (a) (ii) a GSK3- β inhibitor or a derivative or a pharmaceutically acceptable salt thereof, and (ii) a notch agonist or a derivative or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

63. A composition, comprising: (a) (ii) a Wnt agonist or a derivative or a pharmaceutically acceptable salt thereof, and (ii) a notch agonist or a derivative or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

64. A composition, comprising: (a) (ii) a GSK3- β inhibitor or a derivative or a pharmaceutically acceptable salt thereof, and (ii) an HDAC inhibitor or a derivative or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

65.A composition, comprising: (a) (ii) a Wnt agonist or a derivative or pharmaceutically acceptable salt thereof, and (ii) an HDAC inhibitor or a derivative or pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

The composition of any one of embodiments 61 to 65, wherein said composition comprises a GSK3- β inhibitor.

The composition of any one of embodiments 61-65, wherein the composition comprises a Wnt agonist.

The composition of any one of embodiments 61 to 65, wherein the composition comprises a notch agonist.

The composition of any one of embodiments 61 to 65, wherein the composition comprises an HDAC inhibitor.

66. A pharmaceutical composition comprising a GSK3- β inhibitor and a notch agonist in lyophilized form.

67. A pharmaceutical composition comprising a hydrated form of a GSK3- β inhibitor and a notch agonist.

68. The pharmaceutical composition of embodiment 66 or 67, wherein the GSK 3-beta inhibitor is CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)).

69. The pharmaceutical composition of embodiment 66 or 68, wherein the notch agonist is VPA (e.g., in a pharmaceutically acceptable form (e.g., a salt)).

70. A method of producing inner ear hair cells, the method comprising:

expanding stem cells in a naive stem cell population (e.g., a stem cell population of a sample/subject in vitro, ex vivo, or in vivo) to produce an expanded stem cell population (e.g., such that the expanded population is at least 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold greater than the naive stem cell population); and

exposing the expanded stem cell population to a GSK3- β inhibitor and/or a Wnt agonist and optionally a notch inhibitor, thereby promoting the production of inner ear hair cells from the expanded stem cell population.

71. A method of producing inner ear hair cells, the method comprising: administering CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., a salt)) to a cell population in the inner ear of the subject, thereby promoting production of inner ear hair cells.

72. A method of producing inner ear hair cells, the method comprising: expanding postnatal LGR5 in a naive stem cell population (e.g., a population of stem cells of a sample/subject in vitro, ex vivo, or in vivo)⁺Cells producing expanded LGR5⁺A population of cells (e.g., such that the expanded population is at least 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold greater than the initial population of stem cells); the amplified LGR5⁺The cell population results in the production of inner ear hair cells.

73. A method of treating a disease or disorder, the method comprising: amplification of postnatal Lgr5 in an initial population of (in vivo) subjects⁺Epithelial cells, resulting in expanded Lgr5⁺Epithelial cell populations (e.g., such that the expanded population is the initial postnatal Lgr5⁺At least 2-fold, 3-fold, 5-fold, 10-fold of the epithelial cell populationOr 20 times).

74. A method of expanding stem cells, the method comprising:

(iii) administering or causing to be administered both (i) and (ii) to a population of stem cells (e.g., a population of stem cells of an in vitro, ex vivo or in vivo sample/subject):

(i) a GSK 3-beta inhibitor and/or a Wnt agonist, and

(ii) a notch agonist and/or an HDAC inhibitor,

thereby expanding the stem cells in the stem cell population and producing an expanded stem cell population; and

inner ear hair cells are generated from the expanded stem cell population.

75. A kit, comprising:

(a) a set of one or more compositions, the set comprising (i) and (ii):

(i) a GSK 3-beta inhibitor and/or a Wnt agonist, and

(ii) a notch agonist and/or an HDAC inhibitor,

each of the one or more compositions is provided in a pharmaceutically acceptable carrier; and

(b) instructions for using the set of one or more compositions to treat an inner ear disorder.

76. The kit of embodiment 75, wherein the set of one or more compositions further comprises a TGF β inhibitor.

77. The kit of embodiment 75 or 76, wherein the set of one or more compositions further comprises a ROS inhibitor.

78. The kit of embodiment 77, wherein the ROS inhibitor is vitamin C or a derivative thereof.

79. The kit of any one of embodiments 75 to 77, wherein a set of one or more compositions is in an injectable form (e.g., by injection via a syringe).

80. The kit of embodiment 79, wherein the set of one or more compositions is in a form injectable into the middle ear.

81. A method of enhancing the stem cell potential of a cochlear support cell population.

82. The method of embodiment 81, wherein the method comprises activating the Wnt pathway in the population of cochlear supporting cells.

83. The method according to any one of the preceding embodiments, wherein the method comprises activating the Wnt pathway upstream of c-myc in the population of cochlear supporting cells.

84. The method of any one of the preceding embodiments, wherein the cochlear support cells are syngeneic to the organ of Corti.

85. The method of any one of the preceding embodiments, wherein the cochlear support cell population comprises Lgr5⁺A population of support cells.

86. The method of any one of the preceding embodiments, wherein the cochlear support cell population comprises Lgr5⁺Supporting cells, and the method further comprises inducing Lgr5⁺Supporting cell division to generate multipotent Lgr5⁺Progeny cells.

87. A method as claimed in any preceding embodiment wherein the parent and/or progeny Lgr5⁺The cells subsequently differentiate into hair cells.

88. The method of any one of the preceding embodiments, wherein Lgr5 expression in a given support cell remains within 25% of its baseline level.

89. The method of any one of the preceding embodiments, wherein Lgr5 is upregulated in support cells to at least 1.25, 1.5, 2, 5, 10, 100, or 1000-fold its baseline expression level.

90. The method of any one of the preceding embodiments, wherein Lgr5 in a population of support cells is upregulated on average at least 1.25, 1.5, 2, 5, 10, 100, or 1000-fold over a baseline expression level of the population, and subsequently supports cell proliferation.

91. The method of any one of the preceding embodiments, wherein Lgr5 in a population of support cells is upregulated on average at least 1.25, 1.5, 2, 5, 10, 100, or 1000-fold over the baseline expression level of the population, and members of the population of support cells divide and differentiate into hair cells.

92. A method of inducing self-renewal of stem/progenitor support cells contained in a cochlear cell population, the method comprising inducing stem/progenitor support cells to proliferate while maintaining the ability to differentiate into hair cells in progeny cells.

93. The method of embodiment 92, wherein said progeny cells are allowed to differentiate into hair cells.

94. The method of embodiment 92, wherein said progeny cells are induced to differentiate into hair cells.

95. The method of embodiment 92, wherein the differentiation of the progeny cells into hair cells is induced by activating the Wnt pathway upstream of the c-myc gene without any genetic modification to the population.

96. The method of embodiment 92, wherein the differentiation of said progeny cells into hair cells is induced by activating the Wnt pathway upstream of the c-myc gene with a small organic molecule that transiently induces such activity.

97. The method of any one of embodiments 92 to 96, wherein said population of support cells comprises LGR5 homologous to Corti apparatus⁺Supporting the cell.

98. A composition capable of inducing self-renewal of a supporting cell population by activating or inhibiting a pathway involved in inducing the properties of stem cells.

99. The composition of embodiment 98, wherein said pathway is selected from the group consisting of Wnt, HDAC, TGF- β, RAR, DKK1, and a combination thereof.

100. The composition of embodiment 98 or 99, wherein the composition comprises a small organic molecule that activates or inhibits the pathway.

101. The composition of embodiment 98, 99, or 100, wherein the composition comprises a biocompatible matrix.

102. The composition of any one of embodiments 98 to 101, wherein a preferred composition, when administered to a support cell population in vitro, induces the population to proliferate to a high degree and with high purity in a stem cell proliferation assay and differentiates the population into a high purity hair cell population in a stem cell differentiation assay.

103. The composition of any one of embodiments 98 to 102, wherein the composition induces and maintains stem cell properties by proliferating a support cell population to produce stem cells that are capable of dividing through many generations and maintaining a high proportion of the ability of the resulting cells to differentiate into hair cells.

104. The composition of any one of embodiments 98 to 102, wherein the proliferating cells express stem cell markers comprising one or more of Lgr5, Sox 5, Opeml, Phex, lin 5, Lgr5, cyclinD 5, Msx 5, Myb, Kit, Gdnf 5, Dppa5, Nanog, Esrrb, Rex 5, Dnmt 35, mtdn 35, Dnmt 35, Tcl 5, Oct 5, Klf 5, Pax 5, Six 5, Otx 5, Bmi 5, CDX 5, STAT 5, Smad 5/3, Smad 5/5, Smad5, and 5.

105. A method of increasing cell density of support cells in a cochlear cell population, the method comprising: activating pathways and mechanisms in the support cells that induce stem cell properties, proliferating the activated support cells while maintaining their pluripotent properties in newly formed progeny cells, and subsequently differentiating or inducing the expanded population into hair cells, thereby forming an expanded cochlear cell population, wherein the cell density of the hair cells in the expanded cochlear cell population is greater than the cell density of the hair cells in the original (unexpanded) cochlear cell population.

106. The method of embodiment 105, wherein the amplified population is differentiated into hair cells.

107. The method of embodiment 105, wherein the induced expansion of the population differentiates into hair cells.

108. The method of any one of embodiments 105-107, wherein the population of support cells is contained in cochlear tissue.

109. The method of any one of embodiments 105 to 108, wherein said support cell population is an in vitro support cell population.

110. The method of any one of embodiments 105 to 108, wherein said support cell population is an in vivo support cell population.

111. The method of any one of embodiments 105 to 110, wherein the proliferation phase is preferably controlled to substantially maintain the natural architecture of the cochlear structure.

112. The method of any one of embodiments 105 to 111, wherein said proliferation is transiently induced by inducing a pathway upstream of c-myc without any genetic modification to said population.

113. The method of any one of embodiments 105 to 112, wherein said proliferation is transiently induced by inducing a pathway upstream of c-myc with a small organic molecule without any genetic modification to said population.

114. The method of any one of embodiments 105 to 113, wherein said support cells comprise LGR5 homologous to Corti apparatus⁺Supporting the cell.

115. Increase Lgr5 in cochlear cell population⁺A method of supporting cell density of a cell, the method comprising: activating Lgr5⁺Supporting induction or maintenance in cellsPathways and mechanisms of stem cell nature, activated Lgr5⁺Supporting cell proliferation while maintaining such stem cell properties, and subsequently differentiating or inducing the expanded population into hair cells, thereby forming an expanded cochlear cell population, wherein the cell density of the hair cells in the expanded cochlear cell population is greater than the cell density of the hair cells in the original (unexpanded) cochlear cell population.

116. The method of embodiment 115, wherein the induced expansion of the population differentiates into hair cells.

117. The method of embodiment 115, wherein the induced expansion of the population differentiates into hair cells.

118. The method of any one of embodiments 115 to 117, wherein said population of support cells is comprised in cochlear tissue.

119. The method of any one of embodiments 115 to 118, wherein said population of support cells is an in vitro population of support cells.

120. The method of any one of embodiments 115 to 118, wherein said support cell population is an in vivo support cell population.

121. The method of any one of embodiments 115 to 120, wherein the proliferation phase is preferably controlled to substantially maintain the natural architecture of the cochlear structure.

122. The method of any one of embodiments 115 to 121, wherein said proliferation is transiently induced by inducing a pathway upstream of c-myc without any genetic modification to said population.

123. The method of any one of embodiments 115 to 122, wherein said proliferation is transiently induced by inducing a pathway upstream of c-myc with a small organic molecule without any genetic modification to said population.

124. The method of any one of embodiments 115 to 123, wherein said population of support cells comprises LGR5 homologous to Corti apparatus⁺A cell.

125. A method of increasing cell density of hair cells in an initial population of cochlear cells, the initial population comprising hair cells, Lgr-supporting cells and Lgr5⁺A support cell, the method comprising: administering to the initial population a stem cell proliferator composition comprising a sternness driver and a differentiation inhibitor, wherein the composition is capable of inducing Lgr5 in the population in a stem cell proliferation assay⁺Supporting the number of cells to expand and allow Lgr5 in the population to differentiate in a stem cell assay⁺Supporting differentiation of the cells into a hair cell population.

126. The method of embodiment 125, wherein the induced expansion of the population differentiates into hair cells.

127. The method of embodiment 125, wherein the induced expansion of the population differentiates into hair cells.

128. The method of any one of embodiments 125-127, wherein the population of support cells is contained in cochlear tissue.

129. The method of any one of embodiments 125 to 128, wherein the population of support cells is an in vitro population of support cells.

130. The method of any one of embodiments 125-128, wherein the population of support cells is a population of support cells in vivo.

131. The method of any one of embodiments 125-130, wherein the proliferation phase is preferably controlled to substantially maintain the natural architecture of the cochlear structure.

132. The method of any one of embodiments 125 to 131, wherein said proliferation is transiently induced by inducing a pathway upstream of c-myc without any genetic modification to said population.

133. The method of any one of embodiments 125 to 132, wherein said proliferation is transiently induced by inducing a pathway upstream of c-myc with a small organic molecule without any genetic modification to said population.

134. The method of any one of embodiments 125 to 133, wherein the population of support cells comprises LGR5 homologous to Corti apparatus⁺A cell.

135. The method of any one of embodiments 125-134, wherein the method produces a stem cell in a stem cell proliferation assay that expresses one or more stem cell markers selected from the group consisting of Sox2, Opeml, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf 1, Dppa 1, nanoppa 1, Nanog, Esrrb, Rex1, Dnmt 31, Tcl1, Oct 1, Klf 1, Pax 1, Six 1, Otx 1, Bmi1, CDX 1, STAT Smad1, smsmad 1, and 1.

136. The method of any one of embodiments 125 to 135, wherein LGR5 in said population is⁺The proportion of cells increases.

137. A method of increasing cell density of hair cells in an initial population of cochlear cells comprising hair cells and support cells, the method comprising:

selectively expanding the number of support cells in the initial population to form an intermediate cochlear cell population, wherein the ratio of the number of support cells to hair cells in the intermediate cochlear cell population is greater than the ratio of the number of support cells to hair cells in the initial cochlear cell population, and

selectively expanding the number of hair cells in the intermediate cochlear cell population to form an expanded cochlear cell population, wherein a ratio of the number of hair cells to support cells in the expanded cochlear cell population is greater than a ratio of the number of hair cells to support cells in the intermediate cochlear cell population.

138. The method of embodiment 137, wherein the intermediate population is induced to differentiate into hair cells.

139. The method of embodiment 137, wherein the intermediate population is induced to differentiate into hair cells.

140. The method of any one of embodiments 137-139, wherein the initial population of cochlear cells is contained in cochlear tissue.

141. The method of any one of embodiments 137 to 139, wherein the initial population of cochlear cells is an in vitro population.

142. The method of any one of embodiments 137 to 139, wherein the initial population of cochlear cells is an in vivo population.

143. The method of any one of embodiments 137 to 142, wherein selective expansion of support cells is controlled to substantially preserve the native architecture of the cochlear structure.

144. The method of any one of embodiments 137 to 143, wherein selective expansion of support cells is transiently induced by inducing a pathway upstream of c-myc without any genetic modification to the population.

145. The method of any one of embodiments 137 to 144, wherein selective expansion of the supporting cells is induced by inducing a pathway upstream of c-myc with a small organic molecule without any genetic modification to the population.

146. The method of any one of embodiments 137 to 145, wherein said population of support cells comprises LGR5 homologous to Corti apparatus⁺A cell.

147. The method of any one of embodiments 137 to 146, wherein the method produces a stem cell in a stem cell proliferation assay that expresses one or more stem cell markers selected from the group consisting of Lgr5, Sox 5, Opeml, Phex, lin 5, Lgr5, cyclin D5, Msx 5, Myb, Kit, Gdnf 5, Dppa5, nanoppa 5, Nanog, Esrrb, Rex 5, Dnmt 35, cdmt 35, Tcl 5, Oct 5, Klf 5, Pax 5, Six 5, Otx 5, Bmi 5, CDX smax 5, STAT 5, klsmd 5, smsmad/5, smsmad 5, and 5.

148. The method of any one of embodiments 137 to 147, whereinLgr5 in middle cluster⁺The proportion of cells is greater than Lgr5 in the initial population⁺The proportion of cells.

149. Increasing Lgr5 in cochlear cell initial population⁺A method of supporting the number of cells or the activity of Lgr5, said initial population comprising hair cells and supporting cells.

150. The method of embodiment 149, wherein an intermediate population is formed, wherein Lgr5⁺The number of supporting cells is expanded relative to the initial population.

151. The method of embodiment 149 or 150, wherein an intermediate population is formed in which the activity of Lgr5 of the supporting cells is increased relative to the initial population.

152. The method of any one of embodiments 149 to 151, wherein Lgr5 is activated by activating Lgr5 in a cell type that normally lacks or has a very low level of Lgr5⁺Expressing in Lgr5⁺The number of cells is increased relative to the initial population of cells.

153. The method of any one of embodiments 149 to 151, wherein an intermediate population is formed, wherein Lgr5 is relative to the initial population of cochlear cells⁺The number of supporting cells was expanded and Lgr5 activity was increased.

154. The method of any one of embodiments 149 to 151, wherein an intermediate population is formed, wherein Lgr5 is relative to the initial population of cochlear cells⁺The number of support cells is expanded and Lgr5 activity is increased, and then the number of hair cells is selectively expanded in the intermediate cochlear cell population to form an expanded cochlear cell population, wherein the ratio of hair cells to support cells in the expanded cochlear cell population is greater than the ratio of hair cells to support cells in the intermediate cochlear cell population.

155. The method of any one of embodiments 81 to 97 and 105 to 154, wherein said method produces Lgr5 in S phase⁺A population of cells.

156. Such as the implementation methodThe method of any one of formulae 81 to 97 and 105 to 154, wherein the method is for adult mammalian cochlear cells and the method produces adult mammalian Lgr5 in S phase⁺A population of cells.

157. The method of any one of embodiments 81-97 and 105-156, wherein the method comprises contacting a cochlear cell population with a stem cell proliferator.

158. The method of any one of embodiments 81-97 and 105-156, wherein the method comprises contacting the cochlear cell population with a stem cell proliferator comprising a sternness driver.

159. The method of any one of embodiments 81-97 and 105-156, wherein the method comprises contacting the cochlear cell population with a differentiation inhibitor.

160. The method of any one of embodiments 81-97 and 105-156, wherein the method comprises contacting a cochlear cell population with a stem cell proliferator comprising a sternness driver and a differentiation inhibitor.

161. The method of any of embodiments 81-97 and 105-160, wherein the dry driver is used to drive Lgr5⁺Proliferation of stem cells.

162. The method of any one of embodiments 81 to 97 and 105 to 161, wherein a sternness driver is used to induce Lgr5 in the absence of an effective differentiation inhibitory concentration of a differentiation inhibitor⁺The cells differentiate into hair cells.

163. The method of any one of embodiments 81 to 97 and 105 to 162, wherein the sternness driver that simultaneously drives proliferation and differentiation comprises a GSK3 β inhibitor and a Wnt agonist.

164. The method of any one of embodiments 81-97 and 105-163, wherein the composition comprises the drydriver and the differentiation inhibitor in formulations that release the drydriver and the differentiation inhibitor at different rates in a release assay.

165. The method of any one of embodiments 81-97 and 105-163, wherein the composition comprises the dry driver and the differentiation inhibitor in a formulation that releases the dry driver and the differentiation inhibitor into the inner ear environment at a constant, sustained, extended, delayed, or pulsed active agent release rate.

166. The method or composition of any one of the preceding embodiments, wherein the differentiation inhibitor may be a Notch agonist or an HDAC inhibitor.

167. A method or composition according to any preceding embodiment, wherein there is a prior proliferation phase of a differentiation inhibitor having an effective sternness driver concentration and an effective differentiation inhibiting concentration, followed by a differentiation phase of a differentiation inhibitor having an effective sternness driver concentration but no effective differentiation inhibiting concentration.

168. The method or composition of any of the preceding embodiments, wherein the sternness driver and differentiation inhibitor are provided to the cochlear cell in formulations that release the sternness driver and differentiation inhibitor at different rates.

169. The method or composition of any of the preceding embodiments, wherein the formulation provides a constant, sustained, prolonged, delayed, or pulsed rate of dry driver and differentiation inhibitor release into the inner ear environment.

170. A method or composition according to any preceding embodiment, wherein the formulation releases the sternness driver and differentiation inhibitor in a manner to provide a pre-proliferative phase having an effective sternness driver concentration and an effective differentiation inhibiting concentration, and a subsequent differentiation phase having an effective sternness driver concentration but no effective differentiation inhibiting concentration of differentiation inhibitor.

171. The method or composition of any preceding embodiment, wherein there is a prior proliferative phase of a Wnt agonist or a GSK3 β inhibitor at an effective dryness driver concentration and a Notch agonist or a HDAC inhibitor at an effective differentiation-inhibiting concentration, followed by a differentiation phase of a Wnt agonist or a GSK3 β inhibitor at an effective dryness driver concentration but no Notch agonist or HDAC inhibitor at an effective differentiation-inhibiting concentration.

172. The method or composition of any preceding embodiment, wherein the formulation provides a constant, sustained, prolonged, delayed or pulsed rate of release of the Wnt agonist or GSK3 β inhibitor and release of the Notch agonist or HDAC inhibitor into the inner ear environment.

173. The method or composition of embodiment 171 or 172, wherein said formulation releases the Wnt agonist or GSK3 β inhibitor and the Notch agonist or HDAC inhibitor in a manner that provides for a pre-proliferative phase having an effective drydriver concentration of the Wnt agonist or GSK3 β inhibitor and an effective differentiation inhibitory concentration of the Notch agonist or HDAC inhibitor followed by a differentiation phase having an effective drydriver concentration of the Wnt agonist or GSK3 β inhibitor but no effective differentiation inhibitory concentration of the Notch agonist or HDAC inhibitor.

174. The method or composition of any preceding embodiment, wherein the differentiation inhibitor is also a sternness driver.

175. The method or composition of any one of the preceding embodiments, wherein the differentiation inhibitor is a Notch agonist and is also a sternness driver.

176. The method or composition of any preceding embodiment, wherein the differentiation inhibitor is valproic acid.

177. The method or composition of any of the preceding embodiments, wherein, if the differentiation inhibitor is also a sternness driver, the concentration of differentiation inhibitor during the differentiation phase is lower than the effective differentiation inhibitory concentration.

178. The method or composition of any of the preceding embodiments, wherein the differentiation inhibitor and the sternness driver are comprised in a sustained release polymer gel.

179. The method or composition of any preceding embodiment, wherein the gel is injected (or adapted to be injected) through a needle and becomes solid within the middle ear space.

180. The method or composition of embodiment 179, wherein the gel comprises a thermoreversible polymer.

181. The method or composition of embodiment 179, wherein the gel comprises poloxamer 407.

182. The method or composition of any one of the preceding embodiments, wherein Notch activity in the support cell retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of Notch activity in a native state of the support cell.

183. A method or composition for producing hair cells, the method comprising: administering or causing the composition comprising (i) and (ii) to be administered to a population of stem cells (e.g., a population of stem cells of an in vitro, volumetric, or in vivo sample/subject): (i) a GSK3 β inhibitor (or derivative or pharmaceutically acceptable salt thereof) and/or a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof), and (ii) a Notch agonist (or derivative or pharmaceutically acceptable salt thereof) and/or a HDAC inhibitor (or derivative or pharmaceutically acceptable salt thereof), thereby proliferating stem cells in the stem cell population and obtaining an expanded stem cell population; and exposing the expanded stem cell population to a GSK3 β inhibitor (or derivative or pharmaceutically acceptable salt thereof) and/or a Wnt agonist (or derivative or pharmaceutically acceptable salt thereof) and optionally a notch inhibitor (or derivative or pharmaceutically acceptable salt thereof), thereby promoting production of inner ear hair cells from the expanded stem cell population.

184. A method of preventing or treating hearing impairment in a subject, the method comprising administering to the subject an effective amount of a composition to treat hearing impairment in the subject, the composition comprising: (a) (ii) an HDAC inhibitor and/or Notch activator and (ii) a GSK3 β inhibitor, a derivative thereof (e.g., a derivative of an HDAC inhibitor, a derivative of a Notch activator, and/or a derivative of a GSK3 β inhibitor), a pharmaceutically acceptable salt thereof (e.g., a pharmaceutically acceptable salt of an HDAC inhibitor, a pharmaceutically acceptable salt of a Notch activator, and/or a pharmaceutically acceptable salt of a GSK3 β inhibitor), or a combination thereof; and (b) a pharmaceutically acceptable carrier or excipient. Thus, for example, the composition may comprise (a) an HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and a GSK3 β inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and (b) a pharmaceutically acceptable carrier or excipient. As another example, the composition may comprise (a) a Notch activator (or a derivative or pharmaceutically acceptable salt thereof) and a GSK3 β inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and (b) a pharmaceutically acceptable carrier or excipient. For another example, the composition may comprise (a) an HDAC inhibitor (or a derivative or pharmaceutically acceptable salt thereof), a Notch activator (or a derivative or pharmaceutically acceptable salt thereof), and a GSK3 β inhibitor (or a derivative or pharmaceutically acceptable salt thereof) and (b) a pharmaceutically acceptable carrier or excipient.

185. A method of identifying an agent that proliferates hair cell progenitors and/or increases the number of hair cells and an agent that protects (e.g., supports the survival of) supporting cells and/or hair cells, and an agent that is toxic or non-toxic to supporting cells or to differentiated progeny, including hair cells.

186. A method of preventing or treating hearing impairment in a subject in need of treatment, the method comprising administering to the subject an effective amount of a composition to treat hearing impairment in the subject, the composition comprising: an HDAC inhibitor and/or Notch activator and a GSK3 β inhibitor or derivative or pharmaceutically acceptable salt thereof, and an acceptable carrier or excipient.

187. A method of inhibiting loss or death of auditory system cells in a subject, comprising administering to the subject an effective amount of a composition described herein or a derivative or pharmaceutically acceptable salt thereof, and an acceptable carrier or excipient, thereby inhibiting loss or death of auditory system cells in the subject.

188. A method of maintaining or promoting growth of cells of the auditory system of a subject, the method comprising administering to the subject an effective amount of a composition comprising an agent described herein or a derivative or pharmaceutically acceptable salt thereof, and an acceptable carrier or excipient, to amplify or initiate endogenous repair, thereby maintaining or promoting growth of cells of the auditory system of the subject.

189. The method of any one of the preceding embodiments, wherein the average in vitro Lgr5 activity in the population of cells at the end of the stem cell proliferation assay is no less than 25%, 50%, 75%, or 90% of the average in vitro Lgr5 activity in the population of cells at the beginning of the stem cell proliferation assay.

190. The method of any one of the preceding embodiments, wherein the average in vitro notch activity in the population of cells at the end of the stem cell proliferation assay is no less than 25%, 50%, 75%, or 90% of the average in vitro notch activity in the population of cells used at the beginning of the stem cell proliferation assay.

191. A method of expanding a population of cochlear stem cells in cochlear tissue comprising a population of parent cells, the parent population comprising supporting cells, the method comprising: contacting cochlear tissue with a stem cell proliferating agent capable of (i) determining Lgr5 in a stem cell proliferation assay in a cell population to form an expanded cell population in cochlear tissue⁺The number of cells increased at least 10-fold, and (ii) in a stem cell differentiation assay from cells comprising Lgr5⁺The cell population of cells forms hair cells.

192. The method of embodiment 191, wherein the at least one stem cell proliferator is capable of measuring stem cell proliferation in a stem cell proliferation assay as Lgr5 in a population of stem cell proliferation assay cells⁺The number of cells increased at least 50-fold.

193. The method as set forth in embodiment 191,wherein the at least one stem cell proliferating agent is capable of proliferating Lgr5 in the stem cell proliferation assay in the population of stem cell proliferation assay cells⁺The number of cells increased at least 100-fold.

194. The method of any one of embodiments 191 to 193, wherein the expanded population of cells comprises more hair cells than the parental population.

195. The method of any one of embodiments 191 to 194, wherein Lgr5 is measured from the beginning to the end of a stem cell proliferation assay⁺The proportion of the total cell number of the cells in the stem cell proliferation assay cell population is increased by at least 2-fold.

196. The method of any one of embodiments 191 to 195, wherein the proportion of total cell number of hair cells in the stem cell differentiation assay cell population is increased by a factor of at least 2 from the beginning to the end of the stem cell differentiation assay.

197. The method of any one of embodiments 191 to 196, wherein the proportion of total cell number of hair cells in the stem cell proliferation assay cell population that decreases by at least 25% from the beginning to the end of the stem cell proliferation assay.

198. The method of any one of embodiments 191 to 197, wherein stem cell proliferation determines an average Lgr5 per cell in the population of cells⁺Activity was increased by at least 10% in the stem cell proliferation assay.

199. The method of any one of embodiments 191 to 198, wherein the average in vitro Lgr5 activity in the population of cells at the end of the stem cell proliferation assay is no less than 25%, 50%, 75%, or 90% of the average in vitro Lgr5 activity in the population of cells at the beginning of the stem cell proliferation assay.

200. The method of any one of embodiments 191 to 199, wherein the average in vitro notch activity in the population of cells at the end of the stem cell proliferation assay is not less than 25%, 50%, 75%, or 90% of the average in vitro notch activity in the population of cells used at the beginning of the stem cell proliferation assay.

201. The method of any one of embodiments 191-200, wherein the cochlear tissue retains a native morphology.

202. The method of any one of embodiments 191-201, wherein the at least one stem cell proliferator is dispersed in a biocompatible matrix.

203. The method of embodiment 202, wherein the biocompatible matrix is a biocompatible gel or foam.

204. The method of any one of embodiments 191 to 203, wherein the composition is a controlled release formulation.

205. The method of any one of embodiments 191-204, wherein the cochlear tissue is cochlear tissue in vivo.

206. The method of any one of embodiments 191-204, wherein the cochlear tissue is ex vivo cochlear tissue.

207. The method of any one of embodiments 191 to 205, wherein said method produces Lgr5 in S phase⁺A population of cells.

208. The method of any one of embodiments 191-207, wherein the at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor.

209. The method of any one of embodiments 191-208, wherein the contacting step provides the cochlear tissue with:

a differentiation inhibitor at an initial stage, at least a dryly driving concentration and at least an effective differentiation inhibiting concentration; and

at least a dryly driving concentration and a differentiation inhibitor at a concentration less than the effective differentiation inhibiting concentration at a subsequent stage.

210. The method of any one of embodiments 191 to 209, wherein the cochlear tissue is in a subject, and the step of contacting the cochlear tissue with the composition is achieved by trans-tympanic administration of the composition to the subject.

211. The method of embodiment 210, wherein the step of contacting the cochlear tissue with the composition improves the subject's auditory function.

212. A composition comprising a biocompatible matrix and at least one stem cell proliferator, wherein the at least one stem cell proliferator is capable of expanding an initial test Lgr5 in a stem cell proliferation assay⁺A cell population to produce an expanded test population, and wherein the Lgr5 of the expanded test population⁺Lgr5 in which the cells are the initial test population⁺At least 10-fold of the cells.

213. The composition of embodiment 212, wherein the at least one stem cell proliferator is capable of measuring stem cell proliferation in a stem cell proliferation assay as Lgr5 in a population of stem cell proliferation assay cells⁺The number of cells increased at least 50-fold.

214. The composition of embodiment 212, wherein the at least one stem cell proliferator is capable of measuring stem cell proliferation in a stem cell proliferation assay as Lgr5 in a population of stem cell proliferation assay cells⁺The number of cells increased at least 100-fold.

215. The composition of any one of embodiments 212-214, wherein the at least one stem cell proliferator is dispersed in a biocompatible matrix.

216. The composition of embodiment 215, wherein the biocompatible matrix is a biocompatible gel or foam.

217. The composition of embodiment 215, wherein the at least one stem cell proliferator is capable of measuring the average per cell Lgr5 in a stem cell proliferation assay for a population of stem cell proliferation assay cells⁺The activity is increased by at least 10%.

218. The composition of any one of embodiments 212-217, wherein the at least one stem cell proliferator comprises at least one of a sternness driver and a differentiation inhibitor.

219. The composition of any one of embodiments 212-218, wherein the at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor.

220. The composition of any one of claims 212-219, wherein the stem cell proliferator comprises a sternness driver at a concentration that is 100 times greater than an effective sternness driver concentration and a differentiation inhibitor at a concentration that is at least 100 times greater than an effective differentiation inhibiting concentration of the differentiation inhibitor.

221. The composition of any one of embodiments 212-220, wherein the composition is a controlled release formulation.

222. The composition of embodiment 221, wherein the controlled release formulation provides immediate release, delayed release, sustained release, extended release, variable release, pulsed release, or bimodal release of the stem cell proliferator when administered to a subject via the tympanic membrane.

223. The composition of embodiment 221 or 222, wherein the controlled release formulation, when administered to a subject, provides: (a) a differentiation inhibitor at an initial stage, at least an effective dryness driver concentration and at least an effective differentiation inhibiting concentration; and (b) at least an effective dryness driver concentration and a differentiation inhibitor at a concentration below the effective differentiation inhibiting concentration at a subsequent stage.

224. The method of any one of embodiments 191 to 211 or the composition of any one of embodiments 212 to 223, wherein the dry driver is: a GSK3 β inhibitor, a GSK3 β inhibitor derivative, a Wnt agonist derivative, or a pharmaceutically acceptable salt of any of them.

225. The method of any one of embodiments 191 to 211 or the composition of any one of embodiments 212 to 223, wherein the differentiation inhibitor is: a notch agonist, a notch agonist derivative, an HDAC inhibitor derivative, or a pharmaceutically acceptable salt of any of them.

226. The method of any one of embodiments 191 to 211 or the composition of any one of embodiments 212 to 223, wherein the dry driver is selected from the group consisting of CHIR99021, LY2090314, lithium, a1070722, BML-284, and SKL 2001.

227. The method of any one of embodiments 191 to 211 or the composition of any one of embodiments 212 to 223, wherein the differentiation inhibitor is a Notch agonist or an HDAC inhibitor selected from the group consisting of valproic acid, SAHA and Tubastatin a.

228. A method of treating a subject having or at risk of developing hearing loss, the method comprising: administering a composition comprising at least one stem cell proliferating agent trans-tympanic to cochlear tissue of the subject.

229. The method of embodiment 228, wherein the at least one stem cell proliferator comprises at least one of a sternness driver and a differentiation inhibitor.

230. The method of embodiment 228 or 229, wherein said at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor. The method of

231. A method of expanding a population of cochlear cells in cochlear tissue comprising a population of parent cells, the method comprising: contacting cochlear tissue with a stem cell proliferating agent to form an expanded cell population in cochlear tissue, wherein the stem cell proliferating agent is capable of forming (i) a proliferation assay final cell population from a proliferation assay initial cell population after undergoing a proliferation assay period in a stem cell proliferation assay, and (ii) a differentiation assay final cell population from a differentiation assay initial cell population after undergoing a differentiation assay period in a stem cell differentiation assay, wherein:

(a) the proliferation assay starting cell population has: (i) proliferation assay initial total number of cells, (ii) proliferation assay initial Lgr5⁺(ii) cell number, (iii) proliferation assay of Primary Hair(iii) cell number, (iv) proliferation assay initial Lgr5⁺Cell fraction equal to the initial Lgr5 of proliferation assay⁺(iv) the ratio of the number of cells to the total number of cells from which the proliferation assay was initiated, and (v) the proliferation assay initiation hair cell ratio, which is equal to the ratio of the proliferation assay initiation hair cell number to the total number of cells from which the proliferation assay was initiated;

(b) the proliferation assay final cell population has: (i) proliferation assay final total cell count, (ii) proliferation assay final Lgr5⁺Cell number, (iii) proliferation assay final hair cell number, (iv) proliferation assay final Lgr5⁺Cell fraction equal to the final Lgr5 of the proliferation assay⁺(iv) the ratio of the number of cells to the final number of cells for the proliferation assay, and (v) the proliferation assay final hair cell ratio, which is equal to the ratio of the proliferation assay final hair cell number to the proliferation assay final cell number;

(c) the differentiation assay initial cell population has: (i) differentiation assay initial total number of cells, (ii) differentiation assay initial Lgr5⁺Cell number, (iii) differentiation assay initial hair cell number, (iv) differentiation assay initial Lgr5⁺Cell proportion equal to the differentiation assay initial Lgr5⁺(iv) the ratio of the number of cells to the total number of cells from which differentiation is determined, and (v) the proportion of hair cells from which differentiation is determined, which is equal to the ratio of the number of hair cells from which differentiation is determined to the total number of cells from which differentiation is determined;

(d) the final cell population of the differentiation assay has: (i) differentiation assay final total cell count, (ii) differentiation assay final Lgr5⁺Cell number, (iii) differentiation assay final hair cell number, (iv) differentiation assay final Lgr5⁺Cell proportion, which is equal to the final Lgr5 of the differentiation assay⁺(iv) the ratio of the number of cells to the total number of cells at the end of the differentiation assay, and (v) the differentiation assay final hair cell ratio, which is equal to the ratio of the number of cells at the end of the differentiation assay to the total number of cells at the end of the differentiation assay;

(e) proliferation assay Final Lgr5⁺Cell number is the initial Lgr5 of proliferation assay⁺At least 10 times the number of cells; and

(f) differentiation assay the final hair cell number was not 0.

232. The method of embodiment 231, wherein the proliferation assay final Lgr5⁺Cell number is the initial Lgr5 of proliferation assay⁺At least 50 times the number of cells.

233. The method of embodiment 231, wherein the proliferation assay final Lgr5⁺Cell number is the initial Lgr5 of proliferation assay⁺At least 100 times the number of cells.

234. The method of any one of embodiments 231 to 233, wherein the expanded population of cells in the cochlear tissue comprises more hair cells than the parent population.

235. The method of any one of embodiments 231 to 234, wherein proliferation assay final Lgr5⁺Cell proportion is the initial Lgr5 of proliferation assay⁺At least 2 times the proportion of cells.

236. The method of any one of embodiments 231 to 235, wherein the differentiation assay final hair cell proportion is at least 2 times greater than the differentiation assay initial hair cell proportion.

237. The method of any one of embodiments 231 to 236, wherein the proliferation assay final hair cell fraction is at least 25% lower than the proliferation assay initial hair cell fraction.

238. The method of any one of embodiments 231 to 237, wherein proliferation assay final Lgr5⁺Cell ratio proliferation assay initial Lgr5⁺The proportion of cells is at least 10% higher.

239. The method of any one of embodiments 231-238, wherein cochlear tissue retains a native morphology.

240. The method of any one of embodiments 231-238, wherein the at least one stem cell proliferator is dispersed in a biocompatible matrix.

241. The method of any one of embodiments 231 to 240, wherein proliferation assay final Lgr5⁺The number of cells is the initial proliferation assayStarting Lgr5⁺At least 100 times the number of cells.

242. The method of any one of embodiments 231 to 240, wherein proliferation assay final Lgr5⁺Cell number is the initial Lgr5 of proliferation assay⁺At least 500 times the number of cells.

243. The method of any one of embodiments 231 to 242, wherein proliferation assay final Lgr5⁺Cell proportion is the initial Lgr5 of proliferation assay⁺At least 2 times the proportion of cells.

244. The method of any one of embodiments 231 to 242, wherein proliferation assay final Lgr5⁺Cell proportion is the initial Lgr5 of proliferation assay⁺At least 4 times the proportion of cells.

245. The method of any one of embodiments 231 to 242, wherein proliferation assay final Lgr5⁺Cell proportion is the initial Lgr5 of proliferation assay⁺At least 8 times the proportion of cells.

246. The method of any one of embodiments 231 to 242, wherein proliferation assay final Lgr5⁺Cell proportion is the initial Lgr5 of proliferation assay⁺At least 16 times the proportion of cells.

247. The method of any one of embodiments 231 to 242, wherein proliferation assay final Lgr5⁺Cell proportion is the initial Lgr5 of proliferation assay⁺At least 32 times the proportion of cells.

248. The method of any one of embodiments 231 to 247, wherein the proliferation assay final hair cell fraction is at least 25% lower than the proliferation assay initial hair cell fraction.

249. The method of any one of embodiments 231 to 247, wherein the proliferation assay final hair cell fraction is at least 50% lower than the proliferation assay initial hair cell fraction.

250. The method of any one of embodiments 231 to 247, wherein the proliferation assay final hair cell fraction is at least 75% lower than the proliferation assay initial hair cell fraction.

251. The method of any one of embodiments 231 to 250, wherein proliferation assay final Lgr5⁺Cell ratio proliferation assay initial Lgr5⁺The proportion of cells is at least 10% higher.

252. The method of any one of embodiments 231 to 250, wherein proliferation assay final Lgr5⁺Cell ratio proliferation assay initial Lgr5⁺The proportion of cells is at least 10% higher.

253. The method of any one of embodiments 231 to 250, wherein proliferation assay final Lgr5⁺Cell ratio proliferation assay initial Lgr5⁺The proportion of cells is at least 20% higher.

254. The method of any one of embodiments 231 to 250, wherein proliferation assay final Lgr5⁺Cell ratio proliferation assay initial Lgr5⁺The proportion of cells is at least 30% higher.

255. The method of any one of embodiments 231 to 250, wherein proliferation assay final Lgr5⁺Cell ratio proliferation assay initial Lgr5⁺The proportion of cells is at least 50% higher.

Other embodiments include:

1. amplification of a nucleic acid molecule having LGR5⁺LGR5 in cochlear tissue of cell population⁺A method of cell population, the method comprising:

contacting the cochlear tissue with a composition comprising at least one stem cell proliferating agent.

2. The method of claim 1, wherein the at least one stem cell proliferator is capable of expanding an initial test Lgr5 in a stem cell proliferation assay⁺A cell population to produce an expanded test population, and wherein the Lgr5 of the expanded test population⁺Lgr5 in which the cells are the initial test population⁺At least 10-fold of the cells.

3. Such asThe method of claim 2, wherein said expanded test population has an ability to differentiate Lgr5⁺Cells are the primary test population of Lgr5 capable of differentiation⁺At least 2-fold of the cells.

4. The method of claim 2, wherein said expanded test population has an ability to differentiate Lgr5⁺Cells are the primary test population of Lgr5 capable of differentiation⁺At least 10-fold of the cells.

5. The method of any one of claims 1 to 4, wherein one of a plurality of morphological features of cochlear tissue is preserved.

6. The method of any one of claims 1 to 5, wherein the at least one stem cell proliferator is dispersed in a biocompatible matrix.

7. The method of claim 6, wherein the biocompatible matrix is a biocompatible gel or foam.

8. The composition according to any one of claims 1 to 7, wherein the composition is a controlled release formulation.

9. The method of any one of claims 1 to 8, wherein the cochlear tissue is an in vivo cochlear tissue.

10. The method of any one of claims 1 to 8, wherein the cochlear tissue is ex vivo cochlear tissue.

11. The method of any one of claims 1 to 10, wherein the at least one stem cell proliferator comprises at least one of a sternness driver and a differentiation inhibitor.

12. The method of claim 11, wherein the at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor.

13. The method of claim 12, wherein the contacting step provides:

at an initial stage, an effective proliferation concentration of at least a sternness driver and an effective differentiation inhibiting concentration of at least a differentiation inhibitor; and

at a later stage, at least an effective proliferation concentration of a desiccating driver and a differentiation inhibitor below an effective differentiation-inhibiting concentration.

14. The method of any one of claims 1 to 9 or 11 to 13, wherein the cochlear tissue is in a subject, and the step of contacting the cochlear tissue with the composition is achieved by administering the composition trans-tympanic to the subject.

15. The method of claim 14, wherein the step of contacting the cochlear tissue with the composition improves the subject's auditory function.

16. A composition, comprising:

at least one stem cell proliferator, wherein the at least one stem cell proliferator is capable of expanding the initial test Lgr5 in a stem cell proliferation assay⁺A cell population to produce an expanded test population, and wherein the Lgr5 of the expanded test population⁺Lgr5 in which the cells are the initial test population⁺At least 10-fold of the cells.

17. The composition of claim 16, wherein said expanded test population has an ability to differentiate Lgr5⁺Cells are the primary test population of Lgr5 capable of differentiation⁺At least 2-fold of the cells. Please confirm that this is a suitable multiple.]

18. The composition of claim 16, wherein said expanded test population has an ability to differentiate Lgr5⁺Cells are the primary test population of Lgr5 capable of differentiation⁺At least 10-fold of the cells.

19. The composition of claim 16 or 18, wherein the at least one stem cell proliferator is dispersed in a biocompatible matrix.

20. The composition of claim 19, wherein the biocompatible matrix is a biocompatible gel or foam.

21. A composition, comprising:

at least one stem cell proliferation agent dispersed in a biocompatible matrix suitable for cochlear administration.

22. The composition of any one of claims 16 to 21, wherein the at least one stem cell proliferator comprises at least one of a sternness driver and a differentiation inhibitor.

23. The composition of any one of claims 16 to 22, wherein the at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor.

24. The composition of any one of claims 16 to 23, wherein the composition is a controlled release formulation.

25. The composition of claim 24, wherein the controlled release formulation provides immediate release, delayed release, sustained release, extended release, variable release, pulsed release, or bimodal release of the stem cell proliferator when administered to a subject via the tympanic membrane.

26. A composition according to claim 24 or 25, wherein the controlled release formulation provides, when administered to a subject: (a) at an initial stage, an effective proliferation concentration of at least a sternness driver and an effective differentiation inhibiting concentration of at least a differentiation inhibitor; and (b) at a later stage, at least an effective proliferation concentration of a sternness driver and a less than effective differentiation inhibitory concentration of a differentiation inhibitor.

27. The method of any one of claims 11 to 15 or the composition of any one of claims 16 to 26, wherein the dry driver is: a GSK3 β inhibitor, a GSK3 β inhibitor derivative, a Wnt agonist derivative, or a pharmaceutically acceptable salt of any of them.

28. The method of any one of claims 11 to 15 or 27 or the composition of any one of claims 16 to 26 or 27, wherein the differentiation inhibitor is: a notch agonist, a notch agonist derivative, an HDAC inhibitor derivative, or a pharmaceutically acceptable salt of any of them.

29. The method of claim 27 or 28 or the composition of claim 27 or 28, wherein the dry driver is selected from the group consisting of CHIR99021, LY2090314, lithium, a1070722, BML-284 and SKL 2001.

30. The method of any one of claims 27 to 29 or the composition of any one of claims 27 to 29, wherein the differentiation inhibitor is a Notch agonist or an HDAC inhibitor selected from the group consisting of valproic acid, SAHA and Tubastatin a.

31. A method of treating a subject having or at risk of developing hearing loss, the method comprising:

administering a composition comprising at least one stem cell proliferating agent trans-tympanic to cochlear tissue of the subject.

32. The method of claim 31, wherein the at least one stem cell proliferator comprises at least one of a sternness driver and a differentiation inhibitor.

33. The method of claim 31 or 32, wherein the at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor.

34. A population of post-natal mammalian cochlear cells of proliferative Lgr5 that expresses at least one of the following compared to a non-proliferative native state: reduced histone deacetylase, more Notch, optionally wherein: the cell is a support cell; the supporting cell is Lgr5⁺The notch agonist is an HDAC inhibitor and further comprises a Wnt agonist, the Wnt agonist is a GSK3 β inhibitor, and/or the GSK3 β inhibitor is CHIR 99021.

35. A method of administering a composition comprising VPA to the ear of a subject.

36. A method of administering a composition comprising CHIR99021 to the ear of a subject.

37. A method of administering a composition comprising VPA and CHIR99021 to the ear of a subject.

38. A method of increasing the ratio of hair cells to Lgr5 supporting cells by providing an agonist of the Wnt pathway and an agonist of the notch pathway or a histone deacetylase antagonist, optionally wherein: WNT activation is achieved by providing one or more GSK3b antagonists and/or histone deacetylase antagonists; or by providing VPA to effect Notch activation.

39. A method of expanding Lgr5 cells at least 3-fold and/or up-regulating Lgr5 expression at least 3-fold by providing one or more Wnt agonists and one or more histone deacetylase antagonists or notch agonists.

40. A method of expanding inner ear precursor cells by contacting the cells with one or more Notch agonists or histone deacetylase antagonists and Wnt agonists to proliferate the cells, optionally wherein: exposing the cells to additional growth factors; and/or additional factors including one or more Notch agonists or histone deacetylase antagonists and Wnt agonists, administered in vivo to achieve a transient proliferative response.

41. An in vitro differentiated hair cell population obtained from culture expanded Lgr5 cells using one or more Notch agonists and/or histone deacetylase antagonists and/or one or more wnt agonists.

42. A method of differentiating Lgr5 precursor cells, the method comprising: contacting the cell with an amount of a Notch inhibitor and CHIR99021, optionally wherein it is differentiated into a hair cell.

43. A method of differentiating Lgr5 precursor cells into hair cells, the method comprising: contacting a cell expressing Lgr5 with CHIR99021 and a Notch antagonist.

44. Any of the compositions herein, with an ROS antagonist added to enhance Lgr5 expression and/or cell number expansion.

45. Any of the compositions herein, wherein the Notch agonist is an HDAC inhibitor.

46. Any composition herein, wherein the HDAC inhibitor is VPA.

47. Any of the compositions herein, wherein the Wnt agonist is CHIR 99021.

Delivery of CHIR to the ear to amplify and/or increase Lgr5⁺Supporting the expression of the cells.

Delivery of CHIR to the ear to increase the number of hair cells.

Delivery of HDAC inhibitors (e.g. VPA) to the ear to amplify and/or increase Lgr5⁺Supporting the expression of the cells.

Delivery of HDAC inhibitors (e.g. VPA) and CHIR99021 to the ear to increase Lgr5⁺Supporting the expression of the cells.

52. Any composition herein delivered to the middle and/or inner ear.

53. Any of the compositions herein, wherein the Notch agonist or histone deacetylase antagonist is delivered in a pulsed manner and the Wnt agonist is delivered in a sustained manner.

54. A method of treating or preventing hearing loss by administering CHIR99021 in a pharmaceutically relevant vehicle, optionally wherein the vehicle used herein is saline.

55. Any composition herein administered to the middle and/or inner ear.

56. A method of anesthetizing the tympanic membrane and/or surrounding tissue, placing a needle in the middle ear, and injecting an agent as described herein.

57. A method of enhancing the delivery of the agents described herein in the ear by penetration enhancers, ultrasound, electroporation, and other methods known to those skilled in the art.

58. Any of the methods described herein, used in combination with an agent capable of increasing survival of a support cell and/or hair cell.

59. Any suitable agent described herein that is delivered to a patient to enable delivery of a higher concentration of drug associated with dose-limiting ototoxicity.

60. A method wherein cells produced in vitro by a medicament or method described herein are used for research purposes and/or high throughput screening.

61. Method or composition, wherein screening is used to identify promoted Lgr5⁺An agent that supports cell proliferation, and/or testing the toxicity of the drug to the supporting cell and/or its progeny, and/or testing the ability of the agent to improve hair cell survival.

62. Any composition herein further comprising at least one protective drug (which protective drug is capable of enhancing survival or preventing death of cells in the inner ear, including but not limited to hair cells).

63. A kit, comprising: an inner ear supportive cell proliferation inducing amount of a Notch activator and/or an HDAC inhibitor and a GSK3 β inhibitor in a pharmaceutically acceptable carrier; and instructions for using the container contents to treat an inner ear disorder.

64. A method of treating or preventing hearing loss by administering an inhibitor of GSK3 β in a pharmaceutically acceptable carrier.

65.A method of producing Atoh-1+ in inner ear cells by treatment with CHIR99021, optionally wherein the inner ear cells are Lgr5⁺And differentiating in the presence of CHIR99021 to produce Atoh-1+ cells, and/or wherein the Atoh-1+ cells are hair cells.

66. A method of identifying a candidate agent that promotes proliferation of epithelial stem cells capable of differentiating to express atoh-1.

67. A method of identifying a candidate agent that promotes differentiation of epithelial stem cells capable of expressing atoh-1.

68. A method of identifying a candidate agent that promotes survival of epithelial stem cells or cells that express atoh-1.

Additional comments regarding the method:

general Experimental methods

One skilled in the art will recognize that there are many ways to apply, test and treat with the agents described herein. Some non-limiting examples are described below.

Animal(s) production

For experiments using The inner ear ball, C57BL/6(The Jackson Laboratory) or Atoh1-nGFP reporter mice (Lumpkin et al, 2003) (Jane Johnson gift, university of Texas) were used for both sexes. To generate Corti device explants with reduced hair cells, we crossed Pou4f3-Cre mice (Sage et al, 2005) (Douglas Vetter gift from Tufts university) with Mos-iCsp3 mice (line 17) (Fujioka et al, 2011). For all in vivo experiments we used the Cre reporter strain mT/mG (the Jackson laboratory) to cross 4 weeks old Sox2CreER mice (Arnold et al, 2011) (Konrad Hochedlinger, Massachusetts general Hospital gift). After genotyping, lineage tracing was performed using double transgenic animals. We used young adult wild type littermates of mT/mG Sox2-CreER mice to prevent breed effects in response to noise, which are known to vary by background (Harding et al, 2005; Wang et al, 2002). Mice were genotyped using PCR. All procedures were approved by the public animal protection and use committee of the eye-ear medical institution, massachusetts, or the ethical committee of the joint laboratory animal medicine, Keio university, and met public health service policies regarding human care and use of laboratory animals.

Separation of inner ear ball

Dissecting the oval sacs and cochlea of 1-3 days old postnatal mice of two sexes, carefully removing nerve trunk and mesenchymal tissues, and carrying out trypsin digestion and dispersion on the nerve trunk and the mesenchymal tissues. The dispersed cells were centrifuged and the pellet was resuspended in DMEM/F12 medium containing N2/B27 supplement, EGF (20ng/ml), IGF1(50ng/ml), bFGF (10ng/ml) and heparan sulfate (50ng/ml) (Sigma) and filtered through a 70 μm cell filter (BD Biosciences discovery laboratory). Single cells were cultured in non-adherent culture dishes (Greiner Bio-One) to initiate clonal growth of spheres (Martinez-Monedero et al, 2008). And (4) carrying out subculture once every 4-6 days on the spheres formed after 2-3 days of culture. The spheres were centrifuged and the pellet was mechanically dispersed with a pipette tip and resuspended in culture medium. The experiments described herein were performed using spheres from passage 3 to 4. Before differentiation begins, these cells are negative for hair cell markers (Oshima et al, 2007). For differentiation, floating spheres were transferred to fibronectin-coated 4-well plates (Greiner Bio-One) previously described (Martinez-Monedero et al, 2008; Oshima et al, 2007). Attached pellets were allowed to differentiate for 5-7 days in DMEM/F12 medium with N2/B27 supplements but without growth factors. Several concentrations of gamma-secretase inhibitor, DAPT, L-685458, MDL28170(Sigma), and LY411575(Santa Cruz) were added the next day after cell attachment.

Neonatal cochlear explant

Both sexes of C57BL/6 or Mos-iCsp3 after 3 days of birth were dissected in Hank's solution (Invitrogen); pou4f3-Cre cochlea of a double transgenic mouse. To obtain a flat cochlear surface preparation, we removed the spiral ganglion, Reissner's membrane and the most basal cochlear segment. Explants were plated onto 4-well plates (Greiner Bio-One) coated with poly-L-ornithine (0.01%, Sigma) and laminin (50 μ g/ml, Becton Dickinson). Cochlear explants were cultured in dmem (invitrogen) with 10% fetal bovine serum. All cultures were maintained at 5% CO₂/20％O₂In a humidified incubator (Forma Scientific).

Excessive acoustic exposure

In a small reverberation chamber, 4 week old mice were exposed to an open sound fieldExposure stimulation was generated by a custom white noise source, filtered (Brickwall filter with 60 dB/octave slope), amplified (Crown Power Amplifier), and output through an exponential curved horn fixed in the reverberant box top hole (JBL compression driver). 0.25 inch Br ü el and Br ü el were used, and a JBL compression driverThe condenser microphone measures the sound exposure level at four locations in each cage: it was found that the sound pressure differed between these measurement positions<0.5dB。

Systemic or round window administration of a target agent (e.g., LY411575)

4-week-old mice weighing 12-16 g were used. Before surgery, animals were anesthetized with ketamine (20mg/kg, intraperitoneally [ i.p. ]) and xylazine (100mg/kg, i.p.), and after topical administration of lidocaine (1%), incisions were made behind the auricles near the outer ear canal. The ear canal (otic bulla) is opened to access the round window niche. The end of a length of PE 10 tubing (Becton Dickinson) was pulled to a thin tip in a flame and gently inserted into the round window niche. LY411575 was dissolved in DMSO and diluted 10-fold in polyethylene glycol 400(Sigma) to a final concentration of 4 mM. This solution (total volume 1 μ l) was injected into the round window niche of the left ear. Polyethylene glycol 400 with 10% DMSO was injected into the right ear as a control. The solution was applied for 2 minutes. This approach is widely used clinically and has the advantage of not occupying the inner ear but still delivering the drug into the inner ear using the topical route provided by the round window membrane (Mikulec et al, 2008). Gelatin is placed over the niche to maintain the solution and the wound is closed.

For systemic administration, LY411575(50mg/kg) dissolved in 0.5% (w/v) methylcellulose (WAKO) was injected orally once daily for 5 consecutive days. Hearing was measured by ABR 1 day before, 2 days, 1 week, 2 weeks, and 1,2, and 3 months after noise exposure.

qRT-PCR

Corti instruments were dissected in HBSS (Invitrogen) and stored in RNAlater (Ambion) at-80 ℃ until further use. Total RNA was extracted using RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. For reverse transcription, SuperScript II (Invitrogen) with random hexamers was used. The reverse transcription conditions were: 10 minutes at 25 ℃ and then 60 minutes at 37 ℃. The reaction was terminated at 95 ℃ for 5 minutes. The cDNA was mixed with Taqman Gene Expression Mastermix (Applied Biosystems) and Hes5, Atoh1 or 18S primers (Applied Biosystems) according to the manufacturer' S instructions. Samples were analyzed in triplicate in 96 wells by qPCR (Applied Biosystems 7900HT), PCR thermal cycling conditions were as follows: initial denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 15 sec, annealing and extension at 60 ℃ for one min, 45 cycles. The conditions for each primer remained unchanged. Each PCR reaction was performed in triplicate. Relative gene expression was analyzed using the Δ Δ CT method. Gene expression was calculated relative to 18S RNA and the amount of cDNA used was adjusted so that the Ct value of 18S RNA was between 8 and 11.

Immunohistochemistry

For spheres, cells were fixed with 4% paraformaldehyde in PBS for 10 min. Immunostaining was initiated by blocking with 0.1% Triton X-100 in PBS supplemented with 1% BSA and 5% goat serum (PBT1) for 1 hour. The fixed and permeabilized cells were incubated overnight with polyclonal antibodies to myosin VIIa (protein Biosciences) in PBT 1. Samples were washed 3 times with PBS for 20 minutes. Primary antibodies were detected with secondary antibodies (Molecular Probes) conjugated with Alexa488 and only secondary antibody was used as negative control. The samples were counterstained with DAPI (vector laboratories) or Hoechst 33258(Invitrogen) for 10 minutes and observed with an epifluorescence microscope (Axioskop 2 Mot Axiocam, Zeiss).

For explants, Corti was fixed with 4% paraformaldehyde in PBS for 15 minutes. Immunostaining was initiated by blocking the tissue for 1 hour with 0.1% Triton X-100 in PBS supplemented with 5% donkey serum (PBT 1). The fixed and permeabilized block was incubated overnight in PBT1 with antibodies to myosin VIIa (Proteus Biosciences), Sox2(Santa Cruz), GFP (Invitrogen), prestin (Santa Cruz), neurofilament H (Chemicon), and CtBP2(BD Biosciences). Samples were washed 3 times with PBS for 20 minutes. Primary antibodies were detected with secondary antibodies (molecular probes) conjugated with Alexa488 and 647. The samples were stained with rhodamine phalloidin (Invitrogen) for 15 min and observed with a confocal fluorescence microscope (TCS SP5, Leica).

To collect mature cochlea, deeply anesthetized mice were perfused transapically with 0.01M phosphate buffer (pH 7.4) containing 8.6% sucrose, followed by a fixative consisting of freshly disaggregated 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). After decapitation, the temporal bone was removed and immediately placed in the same fixative at 4 ℃. A small opening is formed at the round window, the oval window and the cochlea tip. After soaking in the fixative overnight at 4 ℃, the temporal bone was decalcified in 0.1M EDTA (pH 7.4) containing 5% sucrose with stirring at 4 ℃ for 2 days. After decalcification, the cochlea was microdissected into four pieces for integral preparation. Immunostaining was initiated by blocking the tissue for 1 hour with 0.1% Triton X-100 in PBS supplemented with 5% donkey serum (PBT 1). The fixed and permeabilized block was incubated overnight in PBT1 with antibodies to myosin viia (proteus biosciences), Sox2(santa cruz) and gfp (invitrogen). Samples were washed 3 times with PBS for 20 minutes. Primary antibodies were detected with secondary antibodies (Molecular Probes) conjugated with Alexa488, 568 and 647 and visualized with a confocal fluorescence microscope (TCS SP5, Leica). Cochlear length was obtained in each case and cochlear frequency maps were computed to pinpoint inner hair cells from 5.6, 8.0, 11.3, 16.0, 22.6, 32 and 45.2kHz regions. For cross-sections, the fixed temporal bone was immersed in 30% sucrose in PBS at 4 ℃, incubated for 1 hour in OCT at room temperature, and frozen in liquid nitrogen. The staining protocol was the same as above except for counterstaining with DAPI (vector laboratories). The samples were observed with an epifluorescence microscope (Axioskop 2 MotAxiocam, Zeiss).

Number of cells

The spheres were cell counted using MetaMorph software. Cell numbers were determined by DAPI or Hoechst positive nuclei. Duplicate cell counts resulted in test differences of < 1%. For explants, the number of supporting cells in the inner, outer and outer hair cell regions is counted across the cochlea. Hair cells were identified with either myosin VIIa antibody or endogenous GFP in Atoh1-nGFP mice. High power images of full length cochlear or cochlear explant cultures were integrated and analyzed in PhotoShop CS4 (Adobe). The total length of the cochlea in its entirety and the length of the single counted segments were measured using ImageJ software (NIH). The total number of supporting cells in the inner hair cells, outer hair cells and outer hair cell area is counted in each of four cochlear segments (apical, mid-basal and basal segments) of 1,200-1,400 μm. The density of each segment (per 100 micron of cells) was then calculated. For mature cochlea, high power images of frequency specific regions (5.6, 8.0, 11.3, and 16.0kHz) according to the calculated frequency map were integrated and analyzed. The number of supporting cells in the inner hair cell, outer hair cell and outer hair cell regions within 100 μm was counted in each of the four frequency specific regions of the cochlea. The number of Sox2 lineage positive cells identified by GFP was counted in the same way.

ABR measurement

Auditory brainstem responses were measured at 7 log-spaced frequencies (half-octave steps of 5.6 to 45.2 kHz) 1 day before and 1 day after noise exposure, and 1 week, 1 month and 3 months post-surgery for each animal (Kujawa and Liberman, 1997; Maison et al, 2003). Mice were anesthetized with ketamine (100mg/kg, i.p.) and xylazine (20mg/kg, i.p.). Needle electrodes are inserted at the vertex and pinna and grounded near the tail. ABR was excited with a short 5ms tone (0.5ms rise and fall, cos2 start envelope sent at 35/s). Responses were amplified, filtered, and averaged in a LabVIEW-driven data acquisition system. The sound level is increased in 5dB steps from ≧ 10dB below the threshold to <80dB SPL. At each sound level, an average of 1,024 responses (with alternating stimulation polarities) is taken, and an "artifact reject" is used, whereby the response waveform is discarded when the peak-to-peak response amplitude exceeds 15 μ V. When visually inspecting the stacked waveforms, the "ABR threshold" is defined as the lowest SPL sound level at which any wave can be detected, which generally corresponds to a sound level below the sound level at which the peak-to-peak response amplitude is significantly higher than the noise floor (about 0.25 μ V). When no response is observed at the highest level available, the threshold is assigned 5dB higher than that level so that statistical tests can be completed. For the amplitude-to-sound level function, the bove I peak is determined by visual inspection at each sound level, and the peak-to-peak amplitude is calculated.

Quantitative and statistical analysis

The two-tailed Mann-Whitney U test was used to compare differences between treatment groups. The same animal was analyzed for changes before and after treatment using the two-tailed Wilcoxon t test. Time-dependent differences between groups were compared using repeated measures analysis of variance. Data are presented as mean ± SEM in text and image form. p values less than 0.05 are considered significant.

Genotyping primer

We used the following genotyping primers: LacZ F: 5'-ttcactggccgtcgttttacaacgtcgtga-3' and LacZ R: 5'-atgtgagcgagtaacaacccgtcggattct-3' for Mos-iCsp3 mice; cre F: 5'-tgggcggcatggtgcaagtt-3' and Cre R: 5'-cggtgctaaccagcgttttc-3' for Pou4F3Cre and Sox2creER mice; and oIMR7318 wild type F: 5'-ctctgctgcctcctggcttct-3', oIMR7319 wild type R: 5'-cgaggcggatcacaagcaata-3', and oIMR7320 mutant R: 5'-tcaatgggcgggggtcgtt-3' for mT/mG mice.

Reference to the literature

1.Balak KJ,Corwin JT,Jones JE.Regenerated hair cells can originatefrom supporting cell progeny:evidence from phototoxicity and laser ablationexperiments in the lateral line system.The Journal of neuroscience:theofficial journal of the Society for Neuroscience 1990；10:2502-12.

2.Stone JS,Cotanche DA.Identification of the timingof S phase andthe patterns of cell proliferation during hair cell regeneration in the chickcochlea.The Journal of comparative neurology 1994；341:50-67.

3.Stone JS,Cotanche DA.Identification of the timing of S phase andthe patterns of cell proliferation during hair cell regeneration in the chickcochlea.The Journal of comparative neurology 2000；417:1-16.

4.Warchol ME,Corwin JT.Regenerative proliferation in organ culturesof the avian cochlea:identification of the initial progenitors anddetermination of the latency of the proliferative response.The Journal ofneuroscience:the official journal of the Society for Neuroscience 1996；16:5466-77.

Mizutari K, Fujioka M, Hosoya M, etc., Notch inhibition indexes cochlearhair cell regeneration and recovery of heart after acid train 2013; 77:58-69.

6.Ryals BM,Dent ML,Dooling RJ.Return of function after hair cellregeneration.Hearing research 2013；297:113-20.

7.Raphael Y.Evidence for supporting cell mitosis in response toacoustic trauma in the avian inner ear.Journal of neurocytology 1992；21:663-71.

8.Salt AN,Hartsock J,Plontke S,LeBel C,Piu F.Distribution ofdexamethasone and preservation of inner ear function following intratympanicdelivery of a gel-based formulation.Audiology&neuro-otology 2011；16:323-35.

9.Salt AN,Gill RM,Plontke SK.Dependence of hearing changes on thedose of intratympanically applied gentamicin:a meta-analysis usingmathematical simulations of clinical drug delivery protocols.The Laryngoscope2008；118:1793-800.

10.Plontke SK,Biegner T,Kammerer B,Delabar U,Salt AN.Dexamethasoneconcentration gradients along scala tympani after application to the roundwindow membrane.Otology&neurotology:official publication of the AmericanOtological Society,American Neurotology Society[and]European Academy ofOtology and Neurotology 2008；29:401-6.

11.Plontke SK,Salt AN.Simulation of application strategies for localdrug delivery to the inner ear.ORL；journal for oto-rhino-laryngology and itsrelated specialties 2006；68:386-92.

Plontke SK, Lowenheim H, Mertens J et al, Randomized, double blind, planar controlled tertiary on the safety and effectiveness of a connected interactive synthetic alkaline delayed via a round window detector for a section to a section of a robust synthetic resinous sensitive adhesive after failure of a system of a Largoscope 2009; 119:359-69.

13.Van Tomme SR,Storm G,Hennink WE.In situ gelling hydrogels forpharmaceutical and biomedical applications.International journal ofpharmaceutics 2008；355:1-18.

14.Mundada AS,Avari JG.In situ gelling polymers in ocular drugdelivery systems:a review.Critical reviews in therapeutic drug carriersystems 2009；26:85-118.

15.Almeida H,Amaral MH,Lobao P,Lobo JM.In situ gelling systems:astrategy to improve the bioavailability of ophthalmic pharmaceuticalformulations.Drug discovery today 2014；19:400-12.

16.Shoichet MS,Tator CH,Poon P,Kang C,Baumann MD.Intrathecal drugdelivery strategy is safe and efficacious for localized delivery to thespinal cord.Progress in brain research 2007；161:385-92.

17.Gupta D,Tator CH,Shoichet MS.Fast-gelling injectable blend ofhyaluronan and methylcellulose for intrathecal,localized delivery to theinjured spinal cord.Biomaterials 2006；27:2370-9.

18.Wise AK,Gillespie LN.Drug delivery to the inner ear.Journal ofneural engineering 2012；9:065002.

19.Swan EE,Mescher MJ,Sewell WF,Tao SL,Borenstein JT.Inner ear drugdelivery for auditory applications.Advanced drug delivery reviews 2008；60:1583-99.

Surovtseva EV, Johnston AH, Zhang W et al, presting binding peptides for targeted polymeric medium mediated delivery to outer hair cells in the inner ear. 424:121-7.

21.Staecker H,Brough DE,Praetorius M,Baker K.Drug delivery to theinner ear using gene therapy.Otolaryngologic clinics of North America 2004；37:1091-108.

22.Seidman MD.Glutamate Antagonists,Steroids,and Antioxidants asTherapeutic Options for Hearing Loss and Tinnitus and the Use of an Inner EarDrug Delivery System.The international tinnitus journal 1998；4:148-54.

23.Salt AN,Plontke SK.Principles of local drug delivery to the innerear.Audiology&neuro-otology 2009；14:350-60.

24.Salt AN,Plontke SK.Local inner-ear drug delivery andpharmacokinetics.Drug discovery today 2005；10:1299-306.

25.Salt A.Guest editorial:drug delivery for treatment of inner eardisease:current state of knowledge.Ear and hearing 2010；31:155.

Sakamoto T, Nakagawa T, Horie RT et al, Inner ear drug delivery system from the clinical point of view. acta oto-laryngologica supplement 2010:101-4.

Roy S, Johnston AH, Newman TA, etc., Cell-specific targeting in the said mobile discovery of nanoparticles conjugated with a neural-derived peptide and a functional tool for the drug delivery. International journal of pharmaceuticals 2010; 390:214-24.

Roy S, Glueckert R, Johnston AH et al, Strategies for drug delivery to the human inner ear by multifunctional nanoparticles 2012; 7:55-63.

29.Rivera T,Sanz L,Camarero G,Varela-Nieto I.Drug delivery to theinner ear:strategies and their therapeutic implications for sensorineuralhearing loss.Current drug delivery 2012；9:231-42.

30.Richardson RT,Wise AK,Andrew JK,O'Leary SJ.Novel drug deliverysystems for inner ear protection and regeneration after hearing loss.Expertopinion on drug delivery 2008；5:1059-76.

31.Plontke SK,Zimmermann R,Zenner HP,Lowenheim H.Technical note onmicrocatheter implantation for local inner ear drug delivery:surgicaltechnique and safety aspects.Otology&neurotology:official publication of theAmerican Otological Society,American Neurotology Society[and]European Academyof Otology and Neurotology 2006；27:912-7.

32.Plontke SK,Siedow N,Wegener R,Zenner HP,Salt AN.Cochlearpharmacokinetics with local inner ear drug delivery using a three-dimensionalfinite-element computer model.Audiology&neuro-otology 2007；12:37-48.

33.Plontke SK,Plinkert PK,Plinkert B,Koitschev A,Zenner HP,LowenheimH.Transtympanic endoscopy for drug delivery to the inner ear using a newmicroendoscope.Advances in oto-rhino-laryngology 2002；59:149-55.

34.Plontke SK.Evaluation of the round window niche before local drugdelivery to the inner ear using a new mini-otoscope.Otology&neurotology:official publication of the American Otological Society,American NeurotologySociety[and]European Academyof Otology and Neurotology 2011；32:183-5.

35.Plontke S,Zenner HP.Pharmacokinetic considerations inintratympanic drug delivery to the inner ear.Acta oto-rhino-laryngologicaBelgica 2002；56:369-70.

36.Plontke S,Siedow N,Hahn H,Wegener R,Zenner HP,Salt AN.[1D-and 3D-computer simulation for experimental planning and interpretation ofpharmacokinetic studies in the inner ear after local drug delivery].Altex2004；21Suppl 3:77-85.

37.Paulson DP,Abuzeid W,Jiang H,Oe T,O'Malley BW,Li D.A novelcontrolled local drug delivery system for inner ear disease.The Laryngoscope2008；118:706-11.

Pararas EE, Chen Z, Firing J, etc., kinetic of recording delivery to the inner ear. journal of Controlled Release of special journal of Controlled Release social 2011; 152:270-7.

39.Pararas EE,Borkholder DA,Borenstein JT.Microsystems technologiesfor drug delivery to the inner ear.Advanced drug delivery reviews 2012；64:1650-60.

40.Paasche G,Gibson P,Averbeck T,Becker H,Lenarz T,Stover T.Technicalreport:modification of a cochlear implant electrode for drug delivery to theinner ear.Otology&neurotology:official publication of the American OtologicalSociety,American Neurotology Society[and]European Academy of Otology andNeurotology 2003；24:222-7.

41.Nakagawa T,Ito J.Local drug delivery to the inner ear usingbiodegradable materials.Therapeutic delivery 2011；2:807-14.

42.Mimura T,Funatsu H,Usui T,Yamagami S,Noma H,Amano S.Topical oculardrug delivery to inner ear disease and sinusitis.Southern medical journal2006；99:1287-9.

43.McCall AA,Swan EE,Borenstein JT,Sewell WF,Kujawa SG,McKennaMJ.Drug delivery for treatment of inner ear disease:current state ofknowledge.Ear and hearing 2010；31:156-65.

Li ML, Lee LC, Cheng YR et al, A novel aerosol-mediated drug delivery system for inner therapy: integrated microbiological aerosol metallic transducer. IEEE transactions on bio-physical engineering 2013; 60:2450-60.

45.Lehner R,Brugger H,Maassen MM,Zenner HP.A totally implantable drugdelivery system for local therapy of the middle and inner ear.Ear,nose,&throat journal 1997；76:567-70.

46, Lajud SA, Han Z, Chi FL, etc., A regulated delivery system for inner area application. journal of controlled Release of special journal of the controlled Release facility 2013; 166:268-76.

Drive delivery 2014, a drive of a drive delivery system for the inner apparatus using poly (amino acid).

(iii) Kanzaki S, Fujioka M, Yasuda A et al, Novel in video imaging analysis of an inner air address delivery system in micro, composition of inner air address over time transfer and system information. plos 2012 one; e48480 at 7.

49.Herraiz C,Miguel Aparicio J,Plaza G.[Intratympanic drug deliveryfor the treatment of inner ear diseases].Acta otorrinolaringologica espanola2010；61:225-32.

50.Engleder E,Honeder C,Klobasa J,Wirth M,Arnoldner C,GaborF.Preclinical evaluation of thermoreversible triamcinolone acetonidehydrogels for drug delivery to the inner ear.International journal ofpharmaceutics 2014；471:297-302.

Chen Z, Kujawa SG, McKenna MJ et al, Innerear drug delivery vision imaging perfusion system in the guineae pig. journal of control drag extract of the Controlled Release Society 2005; 110:1-19.

52.Chen G,Hou SX,Hu P,Jin MZ,Liu J.[Preliminary study on brain-targeted drug delivery via inner ear].Yao xue xue bao＝Acta pharmaceuticaSinica 2007；42:1102-6.

53.Bohl A, Rohm HW, Ceschi P, etc., Development of a specific tissue loss system for the prediction of fibrous impurities within the inner apparatus.journal of materials science material media 2012; 23:2151-62.

54.Hoskison E,Daniel M,Al-Zahid S,Shakesheff KM,Bayston R,BirchallJP.Drug delivery to the ear.Therapeutic delivery 2013；4:115-24.

55.Staecker H,Rodgers B.Developments in delivery of medications forinner ear disease.Expert opinion on drug delivery 2013；10:639-50.

56.Borenstein JT.Intracochlear drug delivery systems.Expert opinionon drug delivery 2011；8:1161-74.

57.Pritz CO,Dudas J,Rask-Andersen H,Schrott-Fischer A,GlueckertR.Nanomedicine strategies for drug delivery to the ear.Nanomedicine 2013；8:1155-72.

58.Peer D,Karp JM,Hong S,Farokhzad OC,Margalit R,LangerR.Nanocarriers as an emerging platform for cancer therapy.Naturenanotechnology 2007；2:751-60.

59.Zahnert T.The differential diagnosis of hearing loss.DeutschesArzteblatt international 2011；108:433-43；quiz 44.

60.Sataloff RT,Sataloff J.Differential diagnosis of occupationalhearing loss.Occupational health&safety 2001；70:126-9.

61.Mills DM.Determining the cause of hearing loss:differentialdiagnosis using a comparison of audiometric and otoacoustic emissionresponses.Ear and hearing 2006；27:508-25.

62.Isaacson JE,Vora NM.Differential diagnosis and treatment ofhearing loss.American family physician 2003；68:1125-32.

63.Lasak JM,Allen P,McVay T,Lewis D.Hearing loss:diagnosis andmanagement.Primary care 2014；41:19-31.

64.Brazilian Association of O,Brazilian College of R.Sensorineuralhearing loss:radiologic diagnosis.Revista da Associacao Medica Brasileira2012；58:519-29.

65.Alford RL, Arnos KS, Fox M, et al, American College of Medical Genetics and Genetics guidelines for the clinical evaluation and ethical diagnosis of diagnosis in medicine of clinical journal of the American College of Medical Genetics 2014; 16:347-55.

66.Shi F,Cheng YF,Wang XL,Edge AS.Beta-catenin up-regulatesAtoh1expression in neural progenitor cells by interaction with an Atoh1 3'enhancer.The Journal of biological chemistry 2010；285:392-400.

67.Jeon SJ,Fujioka M,Kim SC,Edge AS.Notch signaling alters sensory orneuronal cell fate specification of inner ear stem cells.The Journal ofneuroscience:the official journal of the Society for Neuroscience 2011；31:8351-8.

68.Bramhall NF,Shi F,Arnold K,Hochedlinger K,Edge AS.Lgr5-positivesupporting cells generate new hair cells in the postnatal cochlea.Stem cellreports 2014；2:311-22.

69.Purow B.Notch inhibition as a promising new approach to cancertherapy.Advances in experimental medicine and biology 2012；727:305-19.

70.Wu L,Griffin JD.Modulation of Notch signaling by mastermind-like(MAML)transcriptional co-activators and their involvement intumorigenesis.Seminars in cancer biology 2004；14:348-56.

71.Shih Ie M,Wang TL.Notch signaling,gamma-secretase inhibitors,andcancer therapy.Cancer research 2007；67:1879-82.

72.Olsauskas-Kuprys R,Zlobin A,Osipo C.Gamma secretase inhibitors ofNotch signaling.OncoTargets and therapy 2013；6:943-55.

73.Meng RD, Shelton CC, Li YM and the like, gamma-Secretase inhibitors of amino acids-induced activation of the Notch-1signaling pathway in colloidal cells quenching in enhanced chemical activity. cancer research 2009; 69:573-82.

Examples

Example 1

Cell culture: hybrid Lgr5-EGFP-IRES-CreERT2 mice were obtained from Jackson Labs and neonatal P2-P5 mice were used for cell isolation. The Corti apparatus was isolated from Lgr5-GFP mice and further dissociated into single cells using trypsin. The cells were then cultured as described previously (Yin et al, 2014). Briefly, cells were embedded in matrigel and plated in the center of wells of a 24-well plate. After matrigel polymerization, 500 μ L of medium (Advanced DMEM/F12 with N2 and B27) containing growth factors including EGF (50ng/ml) (epidermal growth factor), bFGF (20ng/ml) (fibroblast growth factor) and IGF1(50ng/ml) (insulin-like growth factor 1), and small molecules including CHIR99021(5 μ M), valproic acid (1mM), 2-phospho-L-ascorbic acid (280 μ M) and 616452(2 μ M) were added. Y-27632 (10. mu.M) was added on the first 2 days. In some experiments, 50ng/ml bFGF was used.

To isolate single cells, the Corti apparatus was subsequently treated with a cell recovery solution (Corning) for 1 hour to separate the cochlear epithelium from the underlying stroma. The epithelium was then collected and treated with trypsin (Life Technologies) for 15-20 minutes at 37 ℃. The single cells obtained by mechanical milling were filtered with a cell filter (40 μm) and washed with cell culture medium. For FACS sorting of high and low Lgr5 and 5 cells, FACS ARIA (BD) was used and Lgr5-GFP was used as an indicator of Lgr5 levels. Sorted single cells were cultured in matrigel in inner ear cell culture medium with supplemented growth factors and small molecules. Y-27632 was added on the first 2 days.

FACS analysis: the cell culture medium was removed and trypsin was added. After incubation at 37 ℃ for 10-20 minutes, the colonies were dispersed into single cells. Viable cell numbers were counted using a hemocytometer and trypan blue staining. The cells were then stained with Propidium Iodide (PI) and analyzed by flow cytometry. The number of GFP positive cells was calculated by multiplying the total number of cells by the percentage of GFP positive cells.

Results

Lgr5 cells are present within a subset of supporting cells within the cochlea epithelium. Using the Lgr5-GFP mouse strain, we tested a strain used to amplify a single Lgr5⁺It has been shown that inner ear epithelial cells can be cultured as neurospheres in the presence of growth factors including epidermal growth factor (EGF or E), basic fibroblast growth factor (bFGF or F) and insulin-like growth factor 1(IGF-1 or I) (Li et al, 2003.) however, under this condition no growth of Lgr5-GFP cells was observed (fig. 1A.) we initially tested a combination of small molecules including glycogen synthase kinase 3 β (GSK3 β) inhibitor, CHIR99021(CHIR or C) and Histone Deacetylase (HDAC) inhibitor, valproic acid (VPA or V) with a mixture of growth factors⁺Cell culture conditions (Yin et al, 2014) similar phenotypes were observed when inner ear Lgr5 cells were cultured, but to a lesser extent (FIG. 2B). Number of cellsThe quantification of (a) shows that the addition of CHIR and VPA significantly increased cell proliferation and cellular Lgr5-GFP expression, resulting in a 500-fold increase in Lgr5-GFP cells (fig. 2C).

Unlike intestinal colonies, cells from the inner ear epithelium lose proliferative capacity after passage. We concluded that other factors are needed for long-term cell culture and that screening was performed to identify additional factors. Addition of 2-phospho-L-ascorbic acid (pVc, Sigma), a stable form of vitamin C, further led to Lgr5⁺Increased cell expansion to 2-3 fold (fig. 3 and 8b) and further inclusion of increased bFGF concentration (from 20ng/ml to 50ng/ml) in culture medium (fig. 4), so that addition of TTNPB (an RAR agonist) to 50ng/ml of bFGF used in subsequent experiments slightly increased cell proliferation but not GFP expression (fig. 5), so that further screening of small molecule modulators of the primary signaling pathway, which did not contain TTNPB in culture conditions for novacells, demonstrated that these signaling pathway modulators did not increase Lgr5-GFP expression (fig. 6) interestingly, we found that addition of BMP inhibitors, different from the effect of inhibition in small intestine stem cells necessary to promote expression of Lgr5, greatly reduced expression of Lgr5-gf (fig. 6), in addition to the effect of inhibition in small intestine stem cells, which addition of BMP inhibitors was necessary to promote expression of Lgr5, was able to increase expression of Lgr5-gf (fig. 6), in addition to verify that addition of GFP receptor inhibitors, which had the effect of increased growth of GFP (cvgrp 3-5631) over the whole growth cycle, which was observed under conditions of large passage 3-3, which increased GFP (fig. 11) and which increased growth of GFP 3, which increased the addition of GFP receptor 3-365631, showed that addition of GFP (fig. 3) and which increased the addition of GFP-365631, showed that addition of GFP-365631, increased the addition of cgf-GFP-3, increased the total under the growth of cgf-36567, showed that the addition of cgf-3, which increased the growth of cgf-36567, increased the cells had the growth of cgf-3, which had the growth of cgf-3⁺Increase in cell number>2000 times (fig. 8 and 9).

To examine the relative importance of individual factors in our culture system (no passage), we removed each factor individually from the medium and quantified the cell proliferation and Lgr5 expression of the inner ear epithelial cells after 10 days of culture (fig. 8 b). Removal of CHIR or bFGF on proliferationThe effect was maximal, whereas removal of CHIR had the greatest effect on expression of Lgr 5. Removal of EGF or 616452 resulted in a significant reduction in proliferation, while removal of VPA or pVc greatly reduced Lgr5 expression. The presence of IGF1 had minimal beneficial effects on cell proliferation and Lgr5 expression. Treatment with the combination of reagents (EFICVP6) yielded the highest total cell number, Lgr5, after 10 days of culture⁺Cell number and Lgr5⁺Percentage of cells. These results indicate that bFGF and CHIR are most critical, while other factors promote maximal proliferation and Lgr5 expression. Direct visualization of GFP expression and cell growth gave similar results (fig. 8 c).

We further investigated the potential functions of the individual factors. The effect of CHIR in promoting cell proliferation and expression of Lgr5 could be partially replicated using Wnt3a in binding to R-spondin1 (fig. 9a), suggesting a role for CHIR in activating the Wnt pathway. Using the Atoh1-nGFP mouse strain, we found that VPA inhibited the spontaneous differentiation of hair cells (FIG. 9d), consistent with the role of VPA in maintaining Notch activation in intestinal stem cells (Yin et al, 2014).

To demonstrate that single sorted Lgr5-GFP cells were able to grow into GFP + colonies under our expansion conditions (EFICVP6), we sorted single Lgr5-GFP cells from the inner ear epithelium into a high GFP fraction and a low GFP fraction. After 14 days of expansion under EFICVP6, cultures initiated from single high GFP cells contained highly pure colonies highly expressing Lgr 5-GFP. Whereas cultures initiated with low GFP cells contained both high and low GFP colonies as well as GFP negative colonies (FIG. 10). This experiment demonstrated that single sorted Lgr5-GFP cells were able to expand under EFICVP6 conditions.

Example 2: differentiation of expanded Lgr5 positive cells into hair cells

Materials and methods

Differentiation protocol: to differentiate expanded Lgr5 positive cells, after 10 days of culture under cell expansion conditions (EFICVP6), cell colonies were transferred to fresh matrigel and further cultured in differentiation medium. Differentiation media contained Notch pathway inhibitors (e.g., DAPT, D, 5 μ M or LY411575, LY, 5 μ M), with or without a Gsk3 β inhibitor (e.g., CHIR 99021). The medium was changed every other day. After an additional 6-10 days of culture in differentiation medium, colonies were collected for qPCR analysis or fixed with 4% PFA and immunostained with hair cell markers Myo7a and Prestin.

RNA extraction and quantitative real-time pcr (qpcr): RNA was isolated from the cultured cells according to the manufacturer's protocol (RNeasy Mini kit; Qiagen). Quantitative real-time PCR was performed using the QuantiTect Probe PCR kit (Qiagen) using commercially available primers and TaqMan probes (Myo7a and Hprt, Life Technologies).

Immunocytochemistry staining: colonies were fixed in 4% paraformaldehyde/PBS for 15-20 min at room temperature and then washed twice with PBS containing 0.1% BSA. Cells were then permeabilized with 0.25% Triton X-100 in PBS for 30 minutes at 4 ℃. After washing 2 times with PBS containing 0.1% BSA, cells were incubated with blocking solution (Power Block, biogenet) for 1 hour. The diluted primary antibody (in Power Block solution) was applied at room temperature for 4 hours or at 4 ℃ overnight. The primary antibodies used were myosin VIIA (1:500, rabbit polyclonal antibody from Proteus Biosciences) and Prestin (1:400, goat polyclonal antibody from Santa Cruz). After 3 washes (5 minutes each), secondary antibodies (Alexafluor 594 and 647 conjugated secondary antibodies; Invitrogen) were added at a dilution of 1:500 and incubated for 30 minutes at room temperature. Nuclei were visualized with 4, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Staining was visualized with an inverted fluorescence microscope (EVOS; Advanced Microcopy Group).

Results

As important functional evidence that expanded Lgr5 positive cells are stem cells, we tested the ability of expanded cells to differentiate in vitro. Notch inhibition was shown to promote in vivo differentiation of Lgr 5-supportive cells into hair cells (Jeon et al, 2011). Furthermore, Wnt pathway activation through β -catenin expression has also been shown to promote Atoh1 expression and hair cell differentiation (Shi et al, 2013). Therefore, we tested these conditions in terms of inducing hair cell differentiation of expanded Lgr5 cells.

We first tested multiple conditions with different combinations of growth factors (EGF, bFGF, IGF1) or small molecules (CHIR, VPA, 616452, pVc) and Notch inhibitors (e.g., LY411575) for expression of the hair cell marker Myo7 a. Conditions without growth factors or small molecules but with CHIR and Notch inhibitors produced the highest increase in Myo7a expression (fig. 11), indicating that growth factors (EGF, bFGF, IGF1) or small molecules (VPA, 616452 and pVc) inhibit hair cell differentiation and should be removed in differentiation medium.

We treated Lgr5-GFP cells expanded by the above procedure with DAPT, a gamma-secretase inhibitor and CHIR (GSK3 beta inhibitor). After 10 days of differentiation, hair cell production was visualized by staining with hair cell markers including myosin VIIA and Prestin. The combination of DAPT and CHIR induced hair cell production as indicated by myosin VIIA and Prestin positive colonies (outer hair cells) (fig. 12a, top panel) and myosin VIIA positive but Prestin negative colonies (inner hair cells) (fig. 12a, bottom panel). Little hair cell production was observed either when Wnt signaling was not activated with GSK3 β inhibitors, or when Wnt signaling was inhibited with a small molecule Wnt pathway inhibitor (IWP-2,2 μ M) (fig. 12 b).

Example 3: expansion and hair cell differentiation of Lgr5 expressing cells from adult inner ear tissue

Materials and methods

For adult tissues, striated blood vessels are removed, but the epithelium is not removed from the underlying stroma due to the limited number of intact cochlea that can be extracted. For adult cells, additional small molecule TTNPB (2 μ M, Tocris) was added, and the complete medium contained EGF, bFGF, IGF-1, CHIR99021, VPA, pVc, 616452 and TTNPB.

Results

We found that although condition eficgp 6 supported survival and growth of Lgr5 inner ear cells from adult mice (fig. 13), proliferation was very slow. Therefore, we performed additive screening of an additive factor capable of promoting the proliferation of adult mouse Lgr5 cells. We found that small molecule RA signaling pathway agonist TTNPB significantly promoted proliferation of cultured cells (fig. 14). Therefore, it was further added to the amplification medium of inner ear cells of Lgr 5.

We followed cell colony formation of cells isolated from 6-week-old adult mice. After 5 days of culture under EFICVP6+ TTNPB conditions, single cells formed large GFP + colonies, confirming proliferation of adult Lgr5 cells under these conditions (FIG. 15). A large number of GFP + colonies could be amplified from a single cochlea (fig. 14).

Discussion of examples 1-3

The experiments shown in FIGS. 1-14 show the following:

FIGS. 1 and 2

The mixture containing growth factors and small molecules (including CHIR and VPA) promoted proliferation and GFP expression of Lgr5 inner ear progenitor cells in vitro and allowed these cells to expand.

Fig. 3.

Addition of pVc (2-phospho-L-ascorbic acid) increased cell proliferation of Lgr5 inner ear progenitor cells.

Fig. 4.

Increasing bFGF concentration promoted proliferation of Lgr5 inner ear progenitor cells.

Fig. 5-7.

The additional small molecule (616452) promotes proliferation of Lgr5 inner ear progenitor cells.

Fig. 8.

1. The mixture containing growth factors and small molecules maintains Lgr5 in vitro⁺Inner ear progenitor cells。

2. The mixture containing all the factors (EGF, bFGF, IGF1, CHIR99021, VPA, pVc, 616452(EFICVP6)) showed the best results in supporting cell proliferation and GFP expression, with 4.7 × 10⁵Total number of cells, 58% GFP + cells and 2.7 × 10⁵GFP + cells.

3. Most of the factors in the mixture are important:

CHIR is important for promoting cell proliferation and GFP expression:

removal of CHIR resulted in an approximately 100-fold reduction in total cell number (4.7 × 10)⁵v.s.5.0×10³) And about 50-fold reduction in GFP + cells (58% v.s.1.3%). about 4000-fold reduction in GFP + cells (2.7 × 10)⁵v.s. about 65). bFGF is important for promoting cell proliferation and for GFP expression.removal of bFGF results in a 10-fold reduction in cell number (4.2 × 10)⁴Total number of cells) and an approximately 2-fold decrease in percentage of GFP + (58% v.s.32%) and a 20-fold decrease in the number of GFP + cells (2.7 × 10)⁵v.s.1.4×10⁴Individual GFP + cells).

EGF, 616452, is important for promoting cell proliferation removal results in total cell number (2.2 × 10)⁵) And GFP + cell number (1.1 × 10)⁵) About 2-fold reduction removal 616452 resulted in total cell number (1.7 × 10)⁵) And GFP + cell number (9.8 × 10)⁴) About 3 times less.

VPA removal and pVc resulted in a percentage of GFP of 28% and a GFP + cell count (1.1 × 10)⁵) pVc removal resulted in a percentage of GFP of 25% and a GFP + cell count (1.1 × 10)⁵) A 2-fold reduction.

IGF-1 for promoting cell proliferation (4.1X 10)⁵Total cells) or GFP retention (58% v.s.53% GFP percentage).

Fig. 9.

CHIR functions via the Wnt pathway.

2. In combination with R-spondin1, Wnt3a could replace CHIR, but was not as effective.

VPA acts by inhibiting differentiation (possibly by activating/maintaining Notch activation).

4. HDAC6 inhibitors did not work when HDAC6 inhibitors were used instead of VPA (data not shown, Tubastatin a, ACY1215, and CAY10603 were attempted).

pVc promote GFP expression.

6. Laminin 511 promotes GFP expression.

An inhibitor 616452 of the Tgf β type I receptor (Tgf β R1, ALK5) is capable of extending cell culture.

CHIR is able to promote differentiation into Atoh-1+ cells.

Fig. 10.

Single sorted Lgr5-GFP cells can be expanded in a mixture containing growth factors and small molecules.

Fig. 11.

The presence of the Wnt pathway/CHIR increases the differentiation efficiency of Notch inhibitors.

2. Cultured Lgr5 progenitor cells are capable of producing inner and outer hair cells.

In some in vivo embodiments, no growth factors are required.

FIG. 13.

1.LGR5⁺The cell expansion protocol was applicable to adult cochlea from mice, and cells could be passaged without loss of Lgr5 expression.

FIGS. 14-15.

Small molecule TTNPB promotes the proliferation of adult Lgr5 inner ear progenitor cells.

Example 4: poloxamer 407 hydrogels for delivery of small molecules to the inner ear for hair cell regeneration

1. Method of producing a composite material

1.1 preparation of the formulations

Poloxamer 407 hydrogel was prepared using the "cold method". Briefly, a weighed amount of poloxamer 407 was added to 40ml of cold ultrapure water or cold PBS (pH 7.4) and stirred overnight at 4 ℃ on a magnetic stir plate to achieve complete dissolution. Solutions of poloxamer 407 were prepared at various concentrations ranging from 18% (w/w) to 25% (w/w). Hydrophilic drugs, including valproic acid (VPA) and phosphorylated ascorbic acid (PAC), were added to 5ml of poloxamer 407 solution and dissolved on a magnetic stir plate at 4 ℃. The weight ratio of poloxamer 407 to drug was varied to understand the effect of the drug on the gelling properties of poloxamer 407 and to determine the optimal formulation for gelling at 37 ℃ with the maximum possible loading of hydrophilic drug. The gelation temperature of the formulation was determined by "visual tube inversion method". Briefly, a glass vial containing a solution of poloxamer 407 with or without a hydrophilic drug was placed in a water bath. The temperature was slowly increased and the temperature at which the flow of the solution stopped when the glass vial was tilted was recorded as the gelation temperature.

To encapsulate hydrophobic drugs, including CHIR99021 (CHIR), Repsox and TTNPB, an appropriate volume from its DMSO stock solution was added to the hydrophilic drug-containing poloxamer 407 solution and mixed with suction at 4 ℃. The maximum DMSO concentration to which the hydrophobic drug is added is limited to 5-6% (v/v) of the total volume of the gel. Higher concentrations of DMSO lower the gelation temperature of the gel. The gelation temperature of the formulation was determined by "visual tube inversion" method as described previously.

1.2 in vitro drug Release

To understand the kinetics of release of the encapsulated drug from the poloxamer 407 hydrogel, in vitro release studies were performed using the dialysis bag method at pH 7.4 and 37 ℃. Briefly, a sealed dialysis bag (3.5-5 kDa cut-off) containing 30. mu.L of gel suspended in 1mL of PBS was placed in 10mL of release medium (PBS). The release medium was stirred at 100rpm to prevent the formation of a stagnant layer at the interface of the membrane and the external solution. At predetermined intervals, 1mL aliquots were removed from the medium and analyzed for drug using HPLC and replaced with an equal volume of fresh medium.

Results

2.1 formulation and gelation: the thermogelling behavior of the different formulations was studied to determine the optimal formulation that would provide a fast and reproducible liquid-gel transition between room temperature and body temperature when loaded with the full drug. For the poloxamer 407 solution, the gelation temperature decreased with increasing poloxamer 407 concentration in the absence of drug. The addition of hydrophilic drugs, including VPA and pVc at concentrations greater than 88mg/ml and 14mg/ml, respectively, inhibited gelation of the poloxamer 407 solution. Thus, gels were prepared using 18% (w/w) poloxamer solutions with VPA and pVc concentrations equal to or less than 88mg/ml and 14mg/ml, respectively. An appropriate volume of hydrophobic drug (including CHIR, Repsox, and TTNPB) from its DMSO stock solution was added to the poloxamer 407 solution containing the hydrophilic drug and mixed with suction at 4 ℃. The concentrations of drugs CHIR, Repsox and TTNPB in the stock solutions were maintained at 55.6-69.5mg/ml, 23-28.75mg/ml and 35mg/ml, respectively, to ensure that the total DMSO concentration in the final formulation was less than 5-6%. Higher concentrations of DMSO significantly reduced the gelation temperature of the formulation. The final formulation was a viscous liquid at storage temperature (4 ℃) and formed a semi-solid gel above its liquid-gel transition temperature (37 ℃).

2.2 in vitro drug release: the in vitro release of encapsulated drugs from hydrogel formulations was studied using the dialysis bag method. CHIR and VPA showed initial burst release of 10% and 17% within 2 hours and 0.5 hours, respectively, followed by a sustained release over 48 hours. Furthermore, the release kinetics of VPA were found to be significantly faster compared to CHIR. Quantitatively, 33% of CHIR cumulative release was observed over 48 hours and 98% of VPA cumulative release was observed over 48 hours. The detailed release profile is shown in figures 16 and 17.

Example 5: hearing recovery in mice

A17% (w/w) stock solution of poloxamer 407 gel (Sigma-Aldrich) was prepared by slowly adding it to cold 1X phosphate buffered saline pH 7.4. The solution is liquid at refrigeration or at room temperature, but solidifies at body temperature. The gel was stained with evans blue dye (50ppm) for visualization during application.

Formulations ("VPA/CHIR") were prepared as described in example 4, using 527mM VPA and 2.975mM mchir in 17% poloxamer 407 with 5% DMSO. For comparison, 4mM Notch inhibitor LY411575 ("LY 411575") was prepared in 17% poloxamer 407 containing 5% DMSO. In addition, 17% poloxamer 407 containing 5% DMSO was prepared as a vehicle only control ("control").

In the noise room, CBA/CaJ mice were deaf by exposure to the 8-16kHz octave band noise band for 2 hours at 120dB SPL. Hearing was assessed 24 hours after noise exposure by measuring Auditory Brainstem Response (ABR). The minimum Sound Pressure Level (SPL) required for visual detection of the ABR wave I was determined at 5, 10, 20, 28.3 and 40 kHz. Following ABR measurements, trans-tympanic injections of a poloxamer gel drug mixture (referred to above as "VPA/CHIR", "LY 411575" or "control") were delivered to fill the middle ear cavity. After 30 days, ABR was evaluated and the improvement in hearing threshold was determined from 24 hours to 30 days post noise exposure. The results are shown in FIG. 18.

For a given sound, a10 dB increase in threshold produces double loudness and is considered clinically significant. The "VPA/CHIR" formulation achieved a10 dB recovery. Statistical significance improvement is shown by stars (. sup.p < 0.05).

It will be apparent from the foregoing that various changes and modifications may be made to the invention described herein to adapt it to various usages and conditions. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. Such embodiments are also within the scope of the following claims. Recitation of a list of elements recited herein in any definition of a variable includes the definition of the variable as any single element or combination (or sub-combination) of the listed elements. Recitation of embodiments herein includes embodiments taken as any single embodiment or in combination with any other embodiments or portions thereof. The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. While the present invention has been particularly shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (40)

1. A method of expanding a population of cochlear cells in cochlear tissue comprising a population of parent cells, the method comprising: contacting cochlear tissue with a stem cell proliferating agent to form an expanded cell population in cochlear tissue, wherein the stem cell proliferating agent is capable of (i) determining Lgr5 in a stem cell proliferation assay⁺The number of cells increased at least 10-fold, and (ii) in a stem cell differentiation assay from cells comprising Lgr5⁺The cell population of cells forms hair cells.

2. A method of expanding a population of cochlear cells in cochlear tissue comprising a population of parent cells, the method comprising: contacting cochlear tissue with a stem cell proliferating agent to form an expanded cell population in cochlear tissue, wherein:

the stem cell proliferating agent is capable of (i) forming a proliferation assay final cell population from a proliferation assay initial cell population after undergoing a proliferation assay time period in a stem cell proliferation assay, and (ii) forming a differentiation assay final cell population from a differentiation assay initial cell population after undergoing a differentiation assay time period in a stem cell differentiation assay, wherein:

(a) the proliferation assay starting cell population has: (i) proliferation assay initial total number of cells, (ii) proliferation assay initial Lgr5⁺Cell number, (iii) proliferation assay initial hair cell number, (iv) proliferation assay initial Lgr5⁺Cell fraction equal to the initial Lgr5 of proliferation assay⁺(iv) the ratio of the number of cells to the total number of cells from which the proliferation assay was initiated, and (v) the proliferation assay initiation hair cell ratio, which is equal to the ratio of the proliferation assay initiation hair cell number to the total number of cells from which the proliferation assay was initiated;

(b) the proliferation assay final cell population has: (i) proliferation assay final total cell count, (ii) proliferation assay final Lgr5⁺Cell number, (iii) proliferation assay final hair cell number, (iv) proliferation assay final Lgr5⁺Cell fraction equal to the final Lgr5 of the proliferation assay⁺(iv) the ratio of the number of cells to the final number of cells for the proliferation assay, and (v) the proliferation assay final hair cell ratio, which is equal to the ratio of the proliferation assay final hair cell number to the proliferation assay final cell number;

(c) the differentiation assay initial cell population has: (i) differentiation assay initial total number of cells, (ii) differentiation assay initial Lgr5⁺Cell number, (iii) differentiation assay initial hair cell number, (iv) differentiation assay initial Lgr5⁺Cell proportion equal to the differentiation assay initial Lgr5⁺(iv) the ratio of the number of cells to the total number of cells from which differentiation is determined, and (v) the proportion of hair cells from which differentiation is determined, which is equal to the ratio of the number of hair cells from which differentiation is determined to the total number of cells from which differentiation is determined;

(d) the final cell population of the differentiation assay has: (i) final fine of differentiation assayTotal number of cells, (ii) differentiation assay final Lgr5⁺Cell number, (iii) differentiation assay final hair cell number, (iv) differentiation assay final Lgr5⁺Cell proportion, which is equal to the final Lgr5 of the differentiation assay⁺(iv) the ratio of the number of cells to the total number of cells at the end of the differentiation assay, and (v) the differentiation assay final hair cell ratio, which is equal to the ratio of the number of cells at the end of the differentiation assay to the total number of cells at the end of the differentiation assay;

(e) the proliferation assay Final Lgr5⁺Cell number is the initial Lgr5 of the proliferation assay⁺At least 10 times the number of cells; and

(f) the differentiation assay final hair cell number is not 0.

3. The method of claim 2, wherein the proliferation assay final Lgr5⁺Cell number is the initial Lgr5 of the proliferation assay⁺At least 50 times the number of cells.

4. The method of claim 2, wherein the proliferation assay final Lgr5⁺Cell number is the initial Lgr5 of the proliferation assay⁺At least 100 times the number of cells.

5. The method of any one of claims 2 to 4, wherein the expanded population of cells in the cochlear tissue comprises more hair cells than the parent population.

6. The method of any one of claims 2 to 5, wherein the proliferation assay final Lgr5⁺Cell proportion is the initial Lgr5 of the proliferation assay⁺At least 2 times the proportion of cells.

7. The method of any one of claims 2 to 6, wherein the differentiation assay final hair cell proportion is at least 2 times greater than the differentiation assay initial hair cell proportion.

8. The method of any one of claims 2 to 7, wherein the proliferation assay final hair cell fraction is at least 25% lower than the proliferation assay initial hair cell fraction.

9. The method of any one of claims 2 to 8, wherein the proliferation assay final Lgr5⁺Cell ratio initial Lgr5 of the proliferation assay⁺The proportion of cells is at least 10% higher.

10. The method of any one of claims 2 to 9, wherein the cochlear tissue retains a native morphology.

11. The method of any one of claims 2 to 10, wherein the at least one stem cell proliferator is dispersed in a biocompatible matrix.

12. The method of claim 11, wherein the biocompatible matrix is a biocompatible gel or foam.

13. The method of any one of claims 2 to 12, wherein the stem cell proliferator is a component of a controlled release formulation.

14. The method of any one of claims 2 to 13, wherein the cochlear tissue is an in vivo cochlear tissue.

15. The method of any one of claims 2 to 13, wherein the cochlear tissue is ex vivo cochlear tissue.

16. A method as claimed in any one of claims 2 to 15, wherein the method produces Lgr5 in S phase⁺A population of cells.

17. The method of any one of claims 2 to 16, wherein the at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor.

18. The method of claim 16, wherein the contacting step provides to the cochlear tissue:

at an initial stage, an effective proliferation concentration of at least a sternness driver and an effective differentiation inhibiting concentration of at least a differentiation inhibitor; and

at a later stage, at least an effective proliferation concentration of a desiccating driver and a differentiation inhibitor below an effective differentiation-inhibiting concentration.

19. The method of any one of claims 2 to 14 or 16 to 18, wherein the cochlear tissue is in a subject, and the step of contacting the cochlear tissue with the composition is achieved by trans-tympanic administration of the composition to the subject.

20. The method of claim 19, wherein the step of contacting the cochlear tissue with the composition improves the subject's auditory function.

21. A composition, comprising:

a biocompatible matrix and at least one stem cell proliferating agent, wherein the at least one stem cell proliferating agent is capable of expanding an initial test Lgr5 in a stem cell proliferation assay⁺A cell population to produce an expanded test population, and wherein the Lgr5 of the expanded test population⁺Lgr5 in which the cells are the initial test population⁺At least 10-fold of the cells.

22. A composition, comprising:

a biocompatible matrix and at least one stem cell proliferating agent, wherein the at least one stem cell proliferating agent is capable of expanding a cell population comprising Lgr5 in a stem cell proliferation assay⁺An initial population of cells to produce a final population of cells, and wherein the final population of cells is Lgr5⁺Lgr5 in which the cells are the primary cell population⁺At least 10-fold of cells; and wherein:

(a) the initial population of cells has: (i) initial total number of cells, (ii) initial Lgr5⁺Cell number, (iii) initial hair cell number, (iv) initial Lgr5⁺Cell fraction equal to the initial Lgr5 of proliferation assay⁺(iv) the ratio of the number of cells to the total number of initial cells of the proliferation assay, and (v) the ratio of initial hair cells, which is equal to the ratio of the number of initial hair cells to the total number of initial cells; and

(b) the final population of cells has: (i) final total number of cells, (ii) final Lgr5⁺Cell number, (iii) final hair cell number, (iv) final Lgr5⁺Cell fraction equal to final Lgr5⁺(iv) the ratio of the number of cells to the final total number of cells, and (v) the final hair cell ratio, which is equal to the ratio of the final number of hair cells to the final total number of cells.

23. The composition of claim 22, wherein said final Lgr5⁺The number of cells is the initial Lgr5⁺At least 50 times the number of cells.

24. The composition of claim 22, wherein said final Lgr5⁺The number of cells is the initial Lgr5⁺At least 100 times the number of cells.

25. The composition of claim 22 or 24, wherein the at least one stem cell proliferator is dispersed in a biocompatible matrix.

26. The composition of claim 25, wherein the biocompatible matrix is a biocompatible gel or foam.

27. The composition of claim 16, wherein the proliferation assay final Lgr5⁺Cell ratio proliferation assay Final Lgr5⁺The proportion of cells is at least 10% higher.

28. The composition of any one of claims 22-27, wherein the at least one stem cell proliferator comprises at least one of a sternness driver and a differentiation inhibitor.

29. The composition of any one of claims 22 to 28, wherein the at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor.

30. The composition of any one of claims 22-28, wherein the stem cell proliferator comprises a sternness driver at a concentration that is 100 times the effective proliferation concentration of the sternness driver and a differentiation inhibitor at a concentration that is at least 100 times the effective differentiation inhibitory concentration of the differentiation inhibitor.

31. The composition of any one of claims 22 to 29, wherein the composition is a controlled release formulation.

32. The composition of claim 29, wherein the controlled release formulation provides immediate release, delayed release, sustained release, extended release, variable release, pulsed release, or bimodal release of the stem cell proliferator when administered to a subject via the tympanic membrane.

33. A composition according to claim 29 or 32, wherein the controlled release formulation provides, when administered to a subject: (a) at an initial stage, an effective proliferation concentration of at least a sternness driver and an effective differentiation inhibiting concentration of at least a differentiation inhibitor; and (b) at a later stage, at least an effective proliferation concentration of a sternness driver and a less than effective differentiation inhibitory concentration of a differentiation inhibitor.

34. The method of any one of claims 16 to 20 or the composition of any one of claims 22 to 33, wherein the dry driver is a GSK- β inhibitor, a BSK- β inhibitor derivative, a wnt agonist derivative, or a pharmaceutically acceptable salt of any one thereof.

35. The method of any one of claims 16 to 20 or 34 or the composition of any one of claims 22 to 33 or 34, wherein the differentiation inhibitor is a notch agonist, a notch agonist derivative, an HDAC inhibitor derivative, or a pharmaceutically acceptable salt of any one thereof.

36. The method of claim 34 or 35 or the composition of claim 34 or 35, wherein the dry driver is selected from the group consisting of CHIR99021, LY2090314, lithium, a1070722, BML-284 and SKL 2001.

37. The method of any one of claims 34 to 36 or the composition of any one of claims 34 to 36, wherein the differentiation inhibitor is a Notch agonist or an HDAC inhibitor selected from the group consisting of valproic acid, SAHA and Tubastatin a.

38. A method of treating a subject having or at risk of developing hearing loss, the method comprising:

administering a composition comprising at least one stem cell proliferating agent trans-tympanic to cochlear tissue of the subject.

39. The method of claim 38, wherein the at least one stem cell proliferator comprises at least one of a sternness driver and a differentiation inhibitor.

40. The method of claim 38 or 39, wherein the at least one stem cell proliferator comprises a sternness driver and a differentiation inhibitor.