WO2010030199A1

WO2010030199A1 - Stem cell culture

Info

Publication number: WO2010030199A1
Application number: PCT/NZ2009/000199
Authority: WO
Inventors: Gorba Thorsten
Original assignee: Neurotrophincell Pty Ltd
Current assignee: Neurotrophincell Pty Ltd
Priority date: 2008-09-15
Filing date: 2009-09-15
Publication date: 2010-03-18
Anticipated expiration: 2011-03-15

Abstract

The invention provides a stem cell culture media composition and methods for culturing stem cells to enhance proliferation, migration and/or differentiation of stem cells in culture. The media composition comprises from 2% to 80% v/v of a choroid plexus (CP) proliferation (CP cells and/or CP conditioned media) in a commercially available stem cell culture medium. The culture media composition of the invention is surprisingly better at enhancing, proliferation and maintaining stem cell viability in culture than the commercially available stem cell media alone. The invention further provides cells cultured by the method of the invention, and the use of the cells in research and therapeutic methods.

Description

STEM CELL CULTURE

FIELD OF THE INVENTION

The present invention relates to the field of stem cell culture in vitro and ex-vivo. The present application is particularly directed to a cell culture medium for maintaining the viability of stem cells in-vivo or ex-vivo. The present invention is also directed to an improved method of stem cell culture to provide factors which enhance the proliferation and viability of stem cells in culture. The cell culture medium and the improved method of stem cell culture provide factors that enhance the motility of migratory stem cells, such as neural stem cells (NSC). The present invention therefore enhances the quality and quantity of stem cells for basic research, drug discovery or therapeutic transplantation.

BACKGROUND OF THE INVENTION

Stem cells can be maintained in culture in vitro and can be differentiated into different cell types. However, most stem cells are notoriously difficult to culture as the cells can be fickle and prone to loss by either cell death or differentiation. They also require numerous factors to be added to a cell culture medium in optimal concentrations. Such factors include buffers, salts, glucose and stem cell signalling factors such as growth factors, survival factors and related molecules necessary for their propagation. While some growth factors are available commercially, they are expensive and have limited stability in storage and are often ineffective at promoting the propagation of stem cells, including neural stem cells (NSC). Thus, currently the propagation of stem cells and in particular of NSC, in the laboratory is difficult, tedious, and uncertain.

Indeed, some studies have identified chemoattractant and chemorepellant factors that appear to influence NSC migration. For example, scatter factor/hepatocyte growth factor (SF/HGF) produced in glioma cell conditioned media was found to be a powerful chemoattractant and induced a five fold increase in NSC migration compared to control media. Stromal-deiϊved factor- 1 (SDF-I) and platelet-derived growth factor (PDGF) have also been shown to have a chemoattractive effect on NSC migration. Slit 2 protein, secreted by the choroid plexus, has been identified as a potent chemorepulsive factor of NSC migration in the developing brain. Potent chemorepulsive guidance molecules (RGM A and RGM B) have also been discovered which are expressed in vivo in mature myelin, choroid plexus and components of developing glial scar tissue.

Current methods used for culturing stem cells, and in particular NSC, vary from laboratory to laboratory and there is currently no standardised method or media proposed for optimising the proliferation and maintaining the viability of NSC in culture. Some researchers use feeder cell systems, supplement cell culture media with serum, include a matrix, such as Matrigel^1M or use cell conditioned media in an attempt to optimise cell growing conditions. No current culture methods have succeeded in increasing proliferation and maintaining the viability of NSC whilst at the same time enhancing their migratory capacity. This may be due to the culture conditions containing the wrong mixture of growth factors and chemoattractant/ chemorepulsive factors .

It is an object of the present invention to go some way towards providing a culture medium and culture method for improving proliferation and maintenance of viability of stem cells in culture; and/or to provide the public with a useful choice.

SUMMARY OF THE INTENTION

The present invention is directed to the use of choroid plexus preparations, particularly choroid plexus (CP) cells and/or CP conditioned media for enhancing the growth, survival and maintenance of viabilityof stem cells grown in long term and short term culture. The CP preparation can also be used to enhance differentiation of stem cells.

The present invention provides a composition comprising a commercially available stem cell culture or differentiation medium supplemented with between 2 and 80% v/v of a CP preparation. The CP preparation comprises CP conditioned media, CP cells or a mixture of both. The composition may comprise a commercially available stem cell culture or differentiation medium supplemented with between 5% and 75% v/v; 7% and 70% v/v; 8% and 65% v/v; 9% and 60% v/v; 10% and 55% v/v; 15% and 50% v/v and 20% and 40% v/v of a CP preparation.

The stem cell culture or differentiation media can be selected from any commercially available stem cell media, for example StemPro®hESC SFM, Knockout™ DMEM, Knockout™ DMEM/F12, StemPro®-34SFM, StemPro®NSCSFM, StemPro®MSC SFM, MesenPRO RS™ medium (all available from Invitrogen, Carlsbad, CA, USA); NeuroCult® media, MesenCult® media, ES-Cult® media, MegaCult® media (all available from Stem Cell Tehcnologies, Vancouver, BC, Canada); Euro med-N (Euroclone SPA, Pero, MI, Italy); Enstem-A® media (MiHipore, USA) etc as would be understood by a sldlled worker.

The stem cell culture media is selected according to the specific stem cell type that is being cultured and/ or differentiated. For example, Euro med-N is particularly useful at culturing neural stem cells. StemPro®hESC SFM is particularly useful for culturing human embryonic stem cells, StemPro® - 34 SFM is particularly useful for culturing human hemapoietic stem cells, while Aggrewell™ is particularly useful for differentiating human embryonic stem cells, and Enstem-A is particularly useful for differentiating mouse and human neural stem cells, as would be appreciated by a sldlled worker.

The compositions of the present invention may further comprise one or more mitogens selected from, but not limited to EGF, bFGF, GDF-15, GDNF, TGFα, IGF-I. IGF-2, NGF, NT-3, NT-4, BDNF, VEGF, PDGF, SDF-I, FGF-2, amphiregulin, SH-HGF, cystatin, α- microglobulin, pleiotrophic factor, LIF and PTHLH. The compositions of the present invention may further comprise one or more other factors selected from, but not limited to neurotrophins, growth factors, vascular endothelial growth factor, trophic factors, cytokines, mitogens, matrix cell support factors, enzymes, proteases capable of degrading toxic protein precipitates, proteins capable of complexing toxic metal ions. Enzymes for use in the methods of the invention include, for example, proteases, alpha-1 antitrypsin, amylase, lipases, sucrase, lactase, and maltase. Protein capable of complexing toxic metal ions for use in the methods of the invention include, for example, transferrin and ceruloplasmin.

The compositions of the present invention are preferably serum free. A composition is "serum-free" when it is prepared without the addition of serum of any type from any source. The compositions of the present invention are surprisingly better than commercially available stem cell culture or differentiation media alone at enhancing growth, survival and maintenance of viability, or differentiation of stem cells grown in long term and short term culture.

A number of stem cells and stem cell lines can be used to practice the invention including embryonic stem cells, neural stem cells, haematopoietic stem cells and mesenchymal stem cells. Examples of such stem cells and stem cell lines are set out in Table 1, below. The invention further provides a method of culturing stem cells comprising providing a composition of the invention and contacting a stem cell population with the composition under conditions to allow for stem cell growth. Conditions to allow for stem cell growth include, for example, appropriate temperature, humidity, CO₂, nutrients etc, as would be understood by a skilled worker.

The invention also provides methods of culturing stem cell including providing a CP preparation; and contacting a stem cell population with the CP preparation under conditions to allow stem cell growth. Conditions to allow for stem cell growth include, for example appropriate temperature, humidity, CO₂, nutrients, etc as would be understood by a skilled worker. In one embodiment, the CP preparation comprises CP conditioned media. In another embodiment the CP preparation comprises conditioned media optionally containing CP cells such that the CP cells are co-cultured with the stem cells. In some embodiments, the CP preparation includes cerebrospinal fluid (CSF). In a preferred embodiment, the method of culturing enhances growth and survival of stem cells relative to control cells not contacted with a CP preparation. The stem cells and/or CP cells may be 'naked' or may be 'encapsulated' in alginate.

In a preferred embodiment, the culture methods of the invention maintain the stem cell population in an undifferentiated, multipotent state for a period longer or for more cell divisions than a stem cell population not contacted with a composition of the invention or with a CP preparation. In some embodiments, where the stem cell population comprises migrating stem cells, the culture methods of the invention maintain or increase the migratory ability or migration of the stem cell population as compared to a stem cell population not contacted with a composition of the invention or with a CP preparation. In some embodiments, the stem cell population is a neural stem cell population. In some embodiments, the stem cell population in culture is further contacted with one or more mitogens. Mitogens include, but are not limited to, EGF, bFGF, TGFb, GDF-15, GDNF, TGFα, IGF-I, IGF-2, NGF, NT-3, NT-4, BDNF, VEGF, PDGF, SDF-I, FGF2, amphiregulin, SH-FIGF, cystatin, α-microglobuHn, pleiotrophic factor, LIF, PTHLH. In some embodiments, the stem cell population is cultured in the presence of one or more other factors including, but not limited to, neuiOtrophins, growth factors, vascular endothelial growth factor, trophic factors, cytokines, mitogens, matrix cell support factors, enzymes, proteases capable of degrading toxic protein precipitates, proteins capable of complexing toxic metal ions. Enzymes for use in the methods of the invention include, for example, proteases, alpha-1 antitrypsin, amylase, lipases, sucrase, lactase, and maltase. Protein capable of complexing toxic metal ions for use in the methods of the invention include, for example, transferrin and ceruloplasmin.

The invention further provides a use of a stem cell population cultured by the methods of the invention. For example, the invention provides methods for prevention and treatment of conditions or disorders, particularly neurological diseases or disorders, by administering to a patient in need thereof, a NSC population cultured using a method of the invention. In a preferred embodiment, a therapeutically effective amount of NSCs are administered to a subject suffering or prone to suffering from a disease or disorder, particularly a neurological disorder. Neurological disorders include, but are not limited to, Alzheimers disease, Parkinsons disease, Atαylo trophic Lateral Sclerosis (ALS), dementia, stroke, multiple sclerosis, anoxia/asphyxia, aging, vascular disease, Huntington's chorea, brain injury due to a physical trauma; and any combination thereof.

The invention provides a number of sources for CP preparations including, but not limited to, a mammalian donor, a primary or secondary CP culture, a CP cell line; and any combination diereof. In some embodiments, the CP cells in culture have been genetically modified. In some embodiments, the CP cells are obtained direcdy from the mammalian donor cerebrospinal fluid. In some embodiments, the CP preparation includes mammalian CSF. In some embodiments, the CP cells comprise single cells, or clusters of CP cells. In some embodiments, the CP cells are free in culture. In other embodiments, CP cells are encapsulated in alginate.

In preferred embodiments of the culture methods of the invention, the stem cells maintain at least one function of a stem cell, e.g., self-renewal, multipotency, migration, or the ability to differentiate.

The invention provides stem cells cultured by the methods of the invention. The invention further provides a cell culture including at least one stem cell in a culture containing a composition of the invention or a CP preparation. The invention further provides a co-culture comprising a population of stem cells and a population of CP cells. In an embodiment, the invention provides a co-culture of a population of neural stem cells and a population of CP cells.

DEFINITIONS As used herein the terms "administering", "introducing", and "transplanting" are used interchangeably and refer to the placement of a stem cell population cultured according to the methods of the invention, into a subject by a method or route which results in localization of the stem cells at a desired site. The stem cells can be administered to a subject by any appropriate route which results in delivery of the cells to a desired location in the subject where at least a portion of the cells remain viable. It is preferred that at least about 5%, preferably at least about 10%, more preferably at least about 20%, yet more preferably at least about 30%, still more preferably at least about 40%, and most preferably at least about 50% or more of the cells remain viable after administration into a subject. The period of viability of the cells after adrninistration to a subject can be as short as a few days, to as long as a few weeks to months. Methods of administering, introducing and transplanting cells or compositions for use in the invention are well-known in the art. Cells can be administered in a pharmaceutically acceptable carrier or diluent. Preferably, the stem cells are neural stem cells (NSC). As used herein, an "agent" refers to any protein, recombinant protein, small molecule,

DNA, RNA, or combination thereof, preferably that has or is suspected of having a biological activity.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. Amelioration can require administration of more than one dose of an agent.

As used herein, "choroid plexus" or "CP" is understood as the area on the ventricles of the brain where cerebrospinal fluid (CSF) is produced by modified ependymal cells. Choroid plexus is present in all components of the ventricular system except for the cerebral aqueduct and the occipital and frontal horns of the lateral ventricles. The choroid plexus (CP) consists of many capillaries, separated from the ventricules by choroid epithelial cells. Liquid filters through these cells from blood to become cerebrospinal fluid.

As used herein, a "choroid plexus preparation" or "CP preparation" is understood as fluid useful for maintaining stem cell viability that is prepared by contacting the fluid with a CP cell for sufficient time and under appropriate conditions of temperature, CO₂, nutrients, etc., to allow for the release of factors by the CP cell such that the fluid is useful for the culturing of stem cell, particularly NSCs. A sufficient time is, for example, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more. It is understood that all or part of the fluid on the cells can be changed one or more times during the growth of the cells. Fluids useful for the culturing of stem cells, particularly NSCs, for example, enhance growth (e.g., increase cell division rate or number, or decrease apoptosis), increase the amount of time that stem cells, particularly NSCs can be maintained in culture without differentiating or losing function (e.g., entering a state of senescence), or enhance stem cell, particularly NSC migration. The fluid can be culture media conditioned by growth with a CP cell. The fluid can be CSF. The fluid can include a CP cell population. The preparation can be a mixture of fluids useful for the culturing of stem cells, particularly NSCs obtained from multiple sources. The CP preparation can be diluted, for example, in culture media to produce a composition appropriate for growth of SCs, particularly NSCs. The compositions comprise from about 2% to 80% v/v CP preparation, preferably 5% to 80% v/v in commercially available SC media. The stem cells and/or CP cells can be 'naked' or can be 'encapsulated' in alginate.

A CP preparation can be fractionated using routine protein purification techniques, e.g., selective precipitation, column chromatography, to produce a partially purified fraction, which can be used alone as a CP preparation, or reconstituted with one or more other fractions to produce a partially purified or defined CP preparation.

As used herein, "conditioned media" is cell culture media that has be used for the growth of cells for sufficient time, particularly the growth of a specific cell or tissue type or types that produce desired factors.

As used herein, "conditions to allow growth" in culture and the like is understood as conditions of temperature (typically, optimally around 37 degrees Celsius for mammalian cells), humidity, CO₂ (typically around 5 %), in appropriate media (salt, buffer, serum) etc, such that the cells are able to undergo mitosis or at least maintain viability for at least 24 hours, preferably longer.

By "contacting a cell" is meant providing an agent to a cell in a manner that the agent can have an effect on the cell. For example, in culture, an agent can be added to culture media, with or without the carriers or agents to promote cell entry or uptake (e.g, transfection reagents, liposomes).

By "detecting" or "detection" and the like is meant the process of performing the steps to determine if an analyte, cell, condition, etc. is present. The amount of analyte, number of cells, or indicators of a condition present may be none or below the level of detection of the method.

As used herein, "enhanced" is understood to mean improved, typically increased, and is measured relative to a control sample not contacted ot tested with the agent of interest, e.g., CP preparation, therapeutic agent, or mitogen. For example, enhanced cell growth includes a increase in the frequency or number of cell divisions prior to quiescence and/or differentiation, an increase in the rate of cell divisions (number of divisions per time), or a decrease in the rate or number of apoptosis events, or any combination thereof. Enhanced migration includes an increase in the number of cells that are observed to move from, one location to another, typically through a membrane as in the transwell assay provided herein. Migration can also be detected across any sort of surface, chemoattractant or chemorepulsive field or gradient.

As used herein, the term "isolated" refers to cells which have been separated from their natural environment or other components with which they are naturally associated. This term includes gross physical separation from the natural environment, e.g., removal from the donor animal, and alteration of the cells' relationship with the neighboring cells with which they are in direct contact by, for example, dissociation. For example, an isolated cell can be removed from an animal and placed in a culture dish or another animal. Isolated is not meant as being removed from all other cells. The term "neurological disease" covers any disorder of the central nervous system. It may for example be a global neurodegenerative disease, such as ageing, vascular disease, dementia, Alzheimer's disease (AD), or the more localised Parkinson's disease (PD), or the autoimmune disease multiple sclerosis (MS), it may be a result of an injury, such as a stroke, anoxia/ asphyxia, ot physical injury such as from a blow to the head, it may be a result of exposure to local (eg meningitis) or systemic toxins, and it may be neoplastic. It may be genetically based, such as Huntingtoii's chorea, or a disorder of metabolism such as lysosomal storage disease.

As used herein, "long term culture" includes culture for greater than about 30 days, for example, about 40, 50, 60, 90, or 120 days or more. As used herein, "short term culture" includes culture for any amount of time less than about 30 days, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 days.

As used herein, a "normal cell" is a cell that is derived from tissue that does not include any known mutations or disruptions that predispose the cell to neurodegenerative disease, or a cell having geneotypic or phenotypic properties characteristic of a neurodegenerative disease.

As used herein, "obtaining" is understood as purchase, procure, manufacture, or otherwise come into possession of the desired material.

As used herein, "parenteral administration" is understood as administration of a cell, agent, pharmaceutical composition, and the Mice by any route other than delivery to the digestive tract (e.g., oral or enteral delivery) including injection (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, intra-ocular, intrathecal, intracranial) by bolus or slow infusion using, for example, a needle, catheter, infusion pump or other device; topical administration on skin or mucous membranes. Delivery can be local or to the specific site of injury or disease, or other site within the body as long as the desired therapeutic effect is obtained. It is understood that administration of cells can require the use of special devices and surgical techniques. It is understood that the term coadministration, as used herein, does not require the formulation of multiple components into a single formulation for administration, or even that the components be delivered by the same route of administration. Co-adminsitration refers to the delivery of two or more agents at a dose such that both agents are active in the subject at the same time. This can be readily determined using routine pharmacokinetic and pharmacodynamic analyses. The stem cells will be administered together with a pharmaceutically acceptable carrier.

The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering cells or compounds used in the methods described herein to subjects, e.g., mammals. As used herein, the term "porcine" is used interchangeably with the term "pig" and refers to mammals in the family Suidae. Such mammals include wholly or partially inbred pigs, preferably those members of the Auckland Island pig herd which are described in more detail in applicants co-pending New Zealand specification no. 539491, incorporated herein by reference.

A method for "predicting" or "diagnosing" as used herein refers to a clinical or other assessment of the condition of a subject based on observation, testing, or circumstances.

"Providing," refers to obtaining, by for example, buying or making a cell, compound, conditioned media, or other device or reagent, particularly those required for practicing the methods of the invention, e.g., choroid plexus cells and/or conditioned media from a choroid plexus cell culture, a stem cell, a mitogen or growth factor. As used herein, "selecting" is understood as identifying one or rnone members of a group. For example, a subject prone to a particular disease or condition can be selected for treatment using the methods of the invention.

By "stem cell" is meant a cell characterized by the ability to self-renew through mitotic cell division, i.e., the ability to go through numerous cycles of cell division while maintaining the undifferentiated state; and differentiate into a diverse range of specialized cell types. The two broad types of mammalian stem cells are: embryonic stem cells that are found in blastocysts, and adult stem cells that are found in adult tissues. As used herein, the term "stem cell" includes both pluripotent stem cells that can differentiate into cells derived from any of the three germ layers, and multipotent stem cells can produce only cells of a closely related family of cells. As used herein, a neural stem cell is a stem cell that has the potential to differentiate into a neuron, an astrocyte or oligodendrocyte.. A stem cell can be recognized by one or more markers present on the cell such as those provided in Table 3 below. By "stem cell population" is meant one ot mote stem cells of one or more purified stem cell types.

The term "subject" or "host" or "recipient" as used herein refers to mammals, including laboratory animals such asmice, rats and rabbits as well as pigs, dogs, primates, and humans. In particular embodiments, the subject is suffering from or prone to a disorder and stem cells of the invention of the same or another species are introduced or are to be introduced. A human subject can also be known as a "patient."

A "suitable dosage level" refers to a dosage level of stem cells cultured by the methods of the invention that provides a therapeutically reasonable balance between pharmacological effectiveness and deleterious effects. Preferably the stem cells are encapsulated in alginate capsules of approximately 500-700 microns in diameter and containing approximately 500-3,000 living stem cells per capsule. When CP cells are present, the capsules may contain about 500- 3,000 living CP cells in combination with the stem cells. The number of capsules that are implanted into a patient to give a therapeutic effect can vary depending on the age and weight of the patient as well as the interior dimensions of the implantation site in die body. Typically, when the stem cells are neural stem cells, and the patient is suffering from a neurological disorder, around 1 ,000 to 10,000 capsules/kg would be implanted onto or into the brain of a patient.

Alternatively, similar numbers of naked, or unencapsulated stem cells and CP cells can be implanted into a patient.

As used herein, "susceptible to" or "prone to" a specific disease or condition and the like refers to an individual who based on genetic, environmental, health, and/or other risk factors is more likely to develop a disease or condition than the general population. For example, some mutations in Huntington and ApoE can make a subject prone to Huntington's disease or AD, respectively. Aging, head injury, and some toxins can also make a subject prone to neurological disease.

The language "therapeutically effective amount" or a "therapeutically effective dose" of a compound is the amount necessary to or sufficient to provide a detectable improvement in of at least one sign or symptom associated or caused by the state, disorder or disease being treated. The therapeutically effective amount can be administered as a single dose or in multiple doses over time. As used herein, the terms "effective" and "effectiveness" includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/ or organism level (often referred to as side-effects) resulting from administration of the treatment. On the other hand, the term "ineffective" indicates that a treatment does not provide sufficient pharmacological effect to be therapeutically useful, even in the absence of deleterious effects, at least in the unstratified population. (Such a treatment may be ineffective in a subgroup that can be identified by the expression profile or profiles.) "Less effective" means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects, e.g., greater liver toxicity.

Thus, in connection with the administration of stem cells cultured according to the present invention stem cells which are effective against a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease load, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

The term "treated," "treating" or "treatment" includes the diminishment or alleviation of at least one sign or symptom associated or caused by the state, disorder or disease being treated. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder. Treatment can result in amelioration of a disease. Treatment can require administration of more than one dose. The term "treating" as used herein includes reducing or alleviating at least one adverse effect or symptom of neurological disorders. Examples of adverse effects or symptoms include memory loss, loss of motor skills, loss of cognitive skills, fatigue, muscle cramp, pain, impaired balance and coordination, tremors.

Accordingly, the stem cells, preferably NSC, and optionally CP cells and/or CP conditioned media, are transplanted into a patient suffering from or predisposed to a disorder, preferably a neurological disorder, in an amount such that there is at least a partial reduction or alleviation of at least one adverse effect or symptom of the disease, disorder or condition.

In reference to response to a treatment, the term "tolerance" refers to the ability of a patient to accept a treatment, based, e.g., on deleterious effects and/or effects on lifestyle. Frequently, the term principally concerns the patients perceived magnitude of deleterious effects such as nausea, weakness, dizziness, diarrhea, and weight gain, among others. Such experienced effects can, for example, be due to general or cell- specific toxicity, activity on non-target cells, cross-reactivity on non-target cellular constituents (non-mechanism based), and/or side effects of activity on the target cellular substituents (mechanism based), oi: the cause of toxicity may not be understood. The term 'comprising' as used in this specification and claims means 'consisting at least in part of, that is to say when interpreting independent claims including that term, the features prefaced by that term in each claim all need to be present but other features can also be present. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

As used herein, "about" is understood to mean approximately or reasonably close to, and within the tolerances generally accepted in the specific experiment, result, time frame, formulation of a composition, etc. for example within two standard deviations or three standard deviations of the mean of a specific result, or about 1%, 2%, 3%, 5%, 10% variation from the amount within the tolerances of the specific art, experiment, result, formulation, time, or composition. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. For example, any single point mutation or alteration of the wild-type sequence can be combined with any other mutation or alteration of the wild-type sequence.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in more detail by reference to the figures of the accompanying drawings in which:

Figure Ia shows a bar graph, the results demonstrating an increased proliferation of mouse NSC grown in CP conditioned media only; in CP conditioned media in the presence of optimal concentration of EGF and bFGF; or in CP conditioned media in the presence of bFGF only; and shows a clear dose dependency in proliferation response to CP conditioned media only; Fig Ib shows photomicrographs that demonstrate that mouse NSC remain in an undifferentiated state in the presence of CP conditioned proliferation media, evidenced by the maintained ubiquitous expression of the NSC marker vimenti^'n;

Figure 2a shows a photomicrograph of transwell culture plates demonstrating cell migration in control and CP conditioned media; Figure 2b shows a bar graph, the results demonstrating an increased migratory response of mouse NSC in the presence of CP conditioned media; Figure 3 shows in vitro proliferation of mNSC over 5 days of culture in 100% commercially available media (Media A); 20% CP conditioned media (Media B); 50% CP conditioned media (Media C); 80% CP conditioned media (Media D) and 100% CP conditioned media (Media E);

Figure 4 shows a photomicrograph of encapsulated mNSC; Figure 5 shows in vitro proliferation of mNSC in 100% commercially available differentiation media (Media A); 5% CP conditioned media (Media B); 10% CP conditioned media (Media C); 50% CP conditioned media (Media D); 95% CP conditioned media (Media E); 50% CP conditioned media from a different batch (Media I/ 'C);

Figure 6a shows mNSC cultured in 100% commercially available media for 2 days and in plating media for 1 day;

Figure 6b shows mNSC cultured in 100% commercially available media for 2 days and in Enstem-A differentiation media for 1 day;

Figure 6c shows mNSC cultured in 50% CPCM for 2 days and in plating media for 1 day; Figure 6d shows mNSC cultured in 50% CPCM for 2 days and in Enstem-A differentiation media for 1 day; Figure 6e shows plated mNSC cultured in 100% commercially available media (Euro Med-N) for 5 days before being transformed to differentiation media (Enstem A). Cells are stained with Styo21 to show the cell nuclei and with fluorescently labelled Beta tubulin marker;

Figure 6f shows plated mNSC cultured in 100% commercially available differentiation media (Euro Med-N) for 5 days before being transformed to differentiation media (Enstem A). Cells are stained with Styo21 to show cell nuclei and with fluorescently labelled vimentin marker;

Figure 6g shows plated mNSC cultured in Euro Med-N media supplemented with 50% CP conditioned media for 5 days before being transformed to differentiation media (Enstem A). Cells are stained with Styo21 nuclear stain and with fluorescently labelled Beta tubulin marker; and

Figure 6h shows plated mNSC cultured in Euro Med-N media supplemented with 50% CP conditioned media for 5 days before being transformed to differentiation media (Enstem A). Cells are stained with Styo21 nuclear stain and with fluorescently labelled vimentin marker.

DETAILED DESCRIPTION OF THE INVENTION The invention provides compositions and methods for supporting the growth, maintenance, migration, and differentiation of stem cells, particularly neural stem cells, using choroid plexus preparations, and methods of use of stem cells cultured using the methods of the invention, including therapeutic methods for the prevention and treatment of disorders and diseases, particularly neurodegenerative diseases. It is demonstrated herein that a CP preparation, including CP cells and/or CP conditioned media, is useful for enhancing the growth, survival and maintenance of viability of stem cells. In particular, a CP preparation is useful at maintaining stem cells in an undifferentiated state in long term and short term culture, thus maintaining a pure population of undifferentiated stem cells. Moreover, it has been found that CP cells and/or CP conditioned media are useful for enhancing the migratory capacity of migratory stem cells, such as NSC. Supplementation of a CP preparation with the appropriate growth factors and mitogens can promote differentiation of neural stem cells.

The present invention provides a composition comprising a commercially available stem cell culture or differentiation medium supplemented with between 2% and 80% v/v, preferably 5% to 80% v/v of a CP preparation. The CP preparation comprises CP conditioned media, CP cells or a mixture of both. The composition may comprise a commercially available stem cell culture or differentiation medium supplemented between 5% and 75% v/v; 7% and 70% v/v; 8% and 65% v/v; 9% and 60% v/v; 10% and 55% v/v; 15% and 50% v/v and 20% and 40% v/v of a CP preparation. The stem cell culture or differentiation media can be selected from any commercially available stem cell media, for example StemPro®hESC SFM, Knockout™ DMEM, Knockout™ DMEM/F12, StemPro®-34SFM, StemPro®NSCSFM, StemPro®MSC SFM, MesenPRO RS™ medium (all available from Invitrogen, Carlsbad, CA, USA); NeuroCult® media, MesenCult® media, ES-Cult® media, MegaCult® media (all available from Stem Cell Tehcnologies, Vancouver, BC, Canada); Euro med-N (Euroclone SPA, Pero, MI, Italy); Enstem-A® (Millipore, US); etc as would be appreciated by a skilled worker.

The stem cell culture media is selected according to the specific stem cell type that is being cultured and/or differentiated. For example, Euro med-N is particularly useful at culturing neural stem cells, StemPro®hESC SFM is particularly useful for culturing human embryonic stem cells, StemPro® - 34 SFM is particularly useful for culturing human hemapoietic stem cells, while Aggrewell™ is particularly useful for differentiating human embryonic stem cells, and Enstem-A® is particularly useful for differentiating human and mouse neural stem cells as would be appreciated by a skilled worker.

The compositions of the present invention are preferably serum free. The compositions of the present invention are surprisingly better than commercially available stem cell culture or differentiation media alone at enhancing growth, survival and maintenance of viability, or differentiation of stem cells grown in long term and short term culture.

A number of stem cells and stem cell lines can be used to practice the invention including embryonic stem cells, neural stem cells, haematopoietic stem cells, and mesenchymal stem cells. Examples of human embryonic stem cells and stem cell lines are set out in Table 1, below. TABLE l hESC lines Provider

H9 WiCeIl Research Institute

Hl WiCeIl Research Institute

H7 WiCeIl Research Institute

BGOl BresaGen, Inc.

HES-3 ES Cell International

HES-2 ES Cell International

HSF-6 University of California, San Francisco

BG02 BresaGen, Inc.

HES-I ES Cell International

Miz- hESl MizMedi Hospital, Seoul National University

H13 WiCeIl Research Institute

H14 WiCeIl Research Institute

HES-4 ES Cell International

BGOl ES Cell International

SNUhES3 Seoul National University

SA002^c Cellartis AB

AS034^f Cellartis AB

Miz-hES4 MizMedi Hospital, Seoul National University

BGOIV ATCC

Stem cells and stem cell lines can be prepared from any animal species including without limitation ungulates (cows, sheep, goats, pigs etc), horses, dogs, cats, rats, mice, humans and other primates.²⁶'²⁷. For example mouse neural stem cells (mNSC) can be used as described¹⁴, as well as human NSC. The present invention is also directed to the use of CP cells or CP conditioned media, in the manufacture of a CP preparation for enhancing the growth, survival and/or maintenance of viability of stem cells in long term or short term culture, wherein the CP preparation includes one or more factors capable of supporting the proliferation, survival and/ or maintenance of stem cells.

The present invention further provides the use of CP cells or CP conditioned media, in the manufacture of a CP preparation for maintaining stem cells in an undifferentiated state (i.e., suppressing differentiation) in long term or short term culture wherein the CP preparation includes one ot more factors capable of maintaining the stem cells in an undifferentiated state. The present invention also provides the use of CP cells or CP conditioned media in the manufacture of a CP preparation for enhancing differentiation of stem cells in long or short term culture.

The present invention further provides the use of CP cells or CP conditioned media, in the manufacture of a CP preparation for enhancing the migratory capacity of migratory stem cells in long or short term culture, wherein the CP preparation includes one or more factors capable of stimulating the migration of stem cells.

In an embodiment of the invention, the stem cells cultured according to the invention can be used in regenerative medicine, whereby stem cells are transplanted into a patient to replace non-functional or diseased cells, to restore and/or enhance cells function to treat disease. The stem cells can be cultured in a CP preparation according to the invention for a short term prior to transplantation into a recipient. For example, neural stem cells may be cultured for about 1-10 days, 2-7 days, 2-5 days, 3-5 days, 3-7 days, preferably about three days prior to implantation into a recipient for the treatment of neurological disorders. Stem cells of the invention can also be used for the treatment of non-neurological disorders. The stem cells cultured according to the invention can include cells obtained from a different species than the recipient. Thus, the present invention may be useful for enhancing growth, survival, and maintenance of cells that are to be useful in xenotransplantation. Alternatively, the stem cells can be from the same species as the recipient for use in allotransplantation. The stem cells can be 'naked' or encapsulated prior to implantation using encapsulation methods known in the art. In a further embodiment the present invention provides a method for treating neurological disorders in a patient in need thereof comprising administering to the patient's brain, either directly by surgical methods or catheter, or indirectly, by systemic administration, an effective amount of NSC, wherein said NSC has been cultured in a CP preparation of the invention.

Neurological disorders can include, for example, Alzheimer's disease (AD), Parkinson's disease (PD), dementia, Amylotrophic lateral sclerosis (ALS), stroke, multiple sclerosis, anoxia/asphyixia, aging, vascular disease, Huntington's chorea, or brain injury due to a physical trauma, or any combination thereof. For example, AD is known to frequently be accompanied by dementia.

The invention further provides NSCs cultured in a CP preparation of the invention, the NSCs having enhanced migratory capacity.

The invention further provides a use of NSC cultured in a CP preparation of the invention in the manufacture of an implantable therapeutic composition for administration to a subject, for example, for treating neurological disorders in a subject in need thereof.

The CP cells used in the present invention can be used as feeder cells, grown in contact with the SCs or grown on opposite sides of a membrane having pore sizes large enough to allow for passage of CP preparation components that enhance cell growth, maintenance, and migration, but small enough to prevent the migration of cells across the membrane, or used to produce CP conditioned media, may be obtained directly from a suitable mammalian donor or may be obtained from any primary or secondary CP cell culture, or from a CP cell line including an immortalised CP cell line, or from a combination of any of the above sources. The CP cells in culture may be genetically modified. The CP cells obtained directly from a donor, may comprise cerebro spinal fluid optionally containing one or more CP cells. When CP cells are used to culture stem cells, they may be isolated cells or clusters of cells and may be "naked" or encapsulated, for example, in alginate. When the CP cells are naked, they may be "free" to make direct contact with the stem cells or the CP cells and stem cells may be separated by a biocompatible separation means which allows the diffusion of secreted factors from the CP cells to the stem cells. The encapsulation of CP cells may function as a biocompatible separation means.

Preferably, the stem cells are cultured using CP conditioned media, and more preferably the stem cells are neural stem cells. The present invention provides the use of CP preparations, including CP cells and/or CP conditioned media for enhancing growth, survival and/or viability of stem cells in long term or short term culture.

The CP are tabulated structures comprising a single continuous layer of cells derived from the edendymal layer of the cerebral ventricles. One function of the choroid plexus is the secretion of cerebrospinal fluid (CSF). Cerebrospinal fluid fills the four ventricles of the brain and circulates around the spinal cord and over the convexity of the brain. The CSF is continuous with the brain interstitial (extracellular) fluid, and solutes, including macromolecules, are exchanged freely between CSF and interstitial fluid. In addition to the production of CSF, the choroid plexus has been associated with the formation of the CSF-blood barrier⁸. However, its broader function is the establishment and maintenance of baseline levels if the extracellular milieu throughout the brain and spinal cord, in part by secreting a wide range of growth factors into the CSF. Studies have reported the presence of numerous trophic factors within choroid plexus including TGFb, GDF-15, GDNF, IGF2, NGF, NT-3, NT-4, BDNF, VEGF, PDGF, SDF-I, FGF2 and amphiregulin as well as antioxidant enzymes and detoxifying chaperones ^{■ ■ l > 1}.

The present invention includes the recognition that CP cells are capable of secreting factors such as neurotrophins, growth factors, vascular endothelial growth factors, trophic factors, cytokines, mitogens, matrix cell support factors, enzymes such as proteases, alpha-1 antitrypsin, amylase, lipase, sucrose, lactose or maltase and other proteins which may be useful in enhancing the growth, survival and function of stem cells in long or short term cultures. However, as it was also known that CP cells secrete factors that are inhibitory to stem cell growth, survival and function, especially to the migratory capacity of migratory stem cells such as NSC, it was surprising that CP cells/conditioned media can be used to dramatically improve the proliferation and migration of NSC. Some stem cells have other physiological characteristics, such as motility and again, this can be difficult to replicate or maintain in culture. NSC, for example, are migratory in viiw, migrating over long distances from the subventricular zone to the olfactory bulb. To maintain migratory ability in vitro, the right culture conditions are necessary. It may also be desirable to enhance the migratory activity of NSC in vitro so that, once implanted into a brain in vivo, they will migrate more quickly to a site of injury. The mechanism of NSC migration is not fully understood, however, it is thought that migration of NSC is guided by a number of attractant and/or repulsive chemical signals.

The present invention also provides the use of CP preparations, including CP cells and/or CP conditioned media, for improving the proliferation and survival of stem cells other than NSC in long and short term culture , for example for the culture of stem cells such as mesenchymal stem cells, embryonic stem cells and haematopoietic stem cells. The invention also provides a method to enhance migration or migratory capacity of stem cells using CP preparations. The invention further provides methods for maintaining stem cells in an undifferentiated state in culture medium including a CP preparation. The invention further provides method for enhancing differentiation in c ulture medium including a CP preparation.

The stem cells which are to be cultured using the methods of the invention can be in a viable, but quiescent state, such as freeze dried or frozen (e.g., in growth media, serum, and DMSO or glycerol), before being cultured -with a CP preparation of the invention. Such quiescent stem cells are cultured in a suitable cell medium (e.g., any of a number of commercially available media) under suitable conditions (e.g., temperature, CO₂, humidity, etc.) depending on the cell type as would be understood by a skilled worker. Stem cells for use in the method of the invention can also be obtained fresh from a subject (e.g., bone marrow), including a human. Bone marrow cells can be enriched for stem cells, or stem cells can be isolated using methods known to those of skill in the art. The CP preparation is then added to the stem cells in culture in an amount that enhances the growth, survival and/ or functionality of the stem cells, when compared with such cells to which the CP preparation has not been added.

The CP preparation may comprise a purified population of CP cells, CP conditioned media or a mixture of both. The CP cells can be obtained from any donor mammal, including genetically modified donor animals, including pigs, sheep, cows, goats, rabbits, mice and primates including rhesus monkeys and humans, using methods known in the art, for example as described in WO 00/66188. The CP cells can be obtained directly from die brain of the donor or from cerebrospinal fluid. Alternatively, CP cells may be obtained from a CP cell line including immortalised cell lines such as TR-CSFB cells¹², or Z310 cells¹³.

The CP cells of the CP preparation can be genetically modified to produce one or more desired factors which will enhance the survival and growth of stem cells in long term or short term culture, using genetic modification techniques which are well known in the art. The CP cells can be genetically modified to increase the life span of the cells.

The CP preparation can be freeze dried or frozen for storage prior to use in the present invention as would be appreciated by a skilled worker. Upon use, the frozen or freeze dried CP preparation can be simply reconstituted. The CP preparation can include isolated CP cells or small clusters of such cells which may be "naked", i.e. in their natural state after harvesting from a donor or cell culture, or they may be encapsulated in a biocompatible material such as alginate by methods known in the art (see for example WO 00/66188 and US 6322804). Alternatively, such cells, either naked or encapsulated, may be placed in a confinement means, which acts to separate the CP preparation from the stem cells in culture, such as a tube or other structure made of biocompatible material which will allow the diffusion of growth factors, etc., from the CP preparation to the stem cells in culture.

Alternatively, the CP preparation includes conditioned media from CP cells. Conditioned media is prepared from a CP cell culture in which the ceDs have been cultured for a period of time and under conditions that allow secretion of the growth factors, etc., from the CP cells into tiie media. The conditioned media is then separated from the cells to provide a factor rich, cell- free CP preparation for use in the invention. Media can be fractionated, concentrated, or otherwise treated to increase the stability of the factors present in the media. Biocompatible and/ or pharmacologically acceptable preservatives or agents can be added.

The CP preparation defined herein is useful for culturing stem cells for a short term or a long term. As mentioned above, stem cells can be cultured in the short term prior to transplantation into a recipient. When stem cells are to be cultured over a long period of time, such as for example, in a continuous culture system, the CP preparation can be maintained separately from the stem cell culture and added thereto by way of an infusion device or the like, either manually or automatically, as and when required.

In an embodiment, the CP preparation can be embedded in a polymer or other biocompatible matrix, which may be degradable when in contact with the stem cells in culture, to release the CP cells, or CP conditioned media. Alternatively, the polymer may be non- degradable, but instead may be permeable to the growth factors, etc. secreted OΪ released from the CP preparation.

The invention also contemplates the use of cerebrospinal fluid (CSF) as the CP preparation for use in the present invention. As discussed above, CP cells in vivo secrete growth factors etc into the CSF. Thus, CSF contains CP secreted factors likely capable of enhancing growth, survival and function of stem cells and may be useful in the present invention. The CSF can comprise CP cells.

The CP preparation may be supplemented with optimal concentrations of any combination of growth factors known such as EGF, bFGF, TGFb, GDF-15, GDNF, TGFα, IGF-I, IGF-2, NGF, NT-3, NT-4, BDNF, VEGF, PDGF, SDF-I, FGF2, amphiregulin, SH- HGF, cystatiπ, α-microglobulin, pleiottophic factor, LIF and/or PTHLH to further enhance the stem cell proliferation, survival and/ or function (including migratory capacity), or to enhance the maintenance of stem cells in an undifferentiated state. The invention also provides a method of enhancing the growth, survival and/or maintenance of stem cells in long term or short term culture by incubating stem cells with a CP preparation, the CP preparation including: a) a CP cell population capable of producing one or more factors that support the survival, growth and maintenance of stem cells; and/or b) a CP cell culture capable of producing one or more factors that support the survival, growth and maintenance of stem cells; and/or c) CP conditioned media from a) or b) containing one ot more factors that support the survival, growth and maintenance of stem cells.

The present invention also includes a method of enhancing the migratory capacity of migratory stem cells in long or short term culture by incubating stem cells with a CP preparation, the CP preparation including: a) a CP cell population capable of producing one or more factors that enhance the migratory capacity of stem cells; and/or b) a CP cell culture capable of producing one or more factors that enhance the migratory capacity of stem cells; and/ or c) CP conditioned media from a) or b) containing one or more factors that enhance the migratory capacity of stem cells.

The invention also includes a method of maintaining stem cells in an undifferentiated state in long term or short term culture, comprising incubating stem cells with a CP preparation, the preparation including: a) a CP cell population capable of producing one or more factors that maintain stem cells in an undifferentiated state; and/ or b) a CP cell culture capable of producing one or more factors that maintain stem cells in an undifferentiated state; and/or c) CP conditioned media from a) or b) containing one or more factors that maintain stem cells in an undifferentiated state.

The stem cells maintained in long term or short term culture according to the methods of the invention remain viable and able to differentiate upon incubation in a suitable differentiation media.

In addition, and surprisingly, supplementation of commercially available differentiation media with the CP preparation of the invention enhanced differentiation of stem cells. The CP preparation/composition of the present invention is therefore useful for enhancing both maintenance as well as differentiation of stem cells. Preferably the CP preparation is CP conditioned media and the stem cells are NSC.

In some embodiments, the CP conditioned media can further include one or more of added EGF, bFGF, TGFb, GDF-15, GDNF, TGFα, IGF-I, IGF-2, NGF, NT-3, NT-4, BDNF, VEGF, PDGF, SDF-I, FGF2, amphiregulin, SH-HGF, cystatin, ^-microglobulin, pleio trophic factor, LIF and/ or PTHLH. The present invention further includes a method of preventing or delaying the onset of neurological disorders by administering a therapeutically effective amount NSC cells cultured in a CP preparation of the invention, to a subject, particularly a patient, in need thereof.

According to the invention, NSCs are maintained in an undifferentiated state while cultured in a CP preparation of the invention. It may be desirable to differentiate the NSC prior to implantation. It is known that NSC differentiate into neurons, astrocytes and oligodendrocytes. However, it has been demonstrated that NSCs can differentiate into non- neuronal cell types including hematopoietic cells. To initiate differentiation, the NSC can be transferred into a differentiating media prior to transplantation, for example as described in US 6,103,530. Alternatively, the undifferentiated NSC cultured in the CP preparation of the invention, can be used for transplantation.

The NSCs for use in the therapeutic methods of the invention can be from the same species as the host recipient patient, i.e., allograft, or may be from a different species, i.e. xenograft, from the recipient.

The NSCs for use in the methods of the invention can be genetically modified to produce one or more desired factors which will assist in the treatment of a neurological disorder, using genetic modification techniques that are well known in the art. Similarly, NSCs for use in die method of the invention can be modified to produce growth factors desirable for specific phenotypes, e.g., increased migration, differentiation, etc.

The CP cells for use in the methods of the invention can be sourced for the same species as the host recipient and/or the NSCs, or may be from a different species. For clinical use, a safe source of CP cell is porcine choroid plexus in particular from the Auckland Island herd of pigs. These pigs are substantially microorganism free, and in particular have very low PERV copy number, making them highly suitable to culture of stem cells for transplantation into humans. Methods of isolating and culturing CP cells from porcine is described for example in WO 01/52871; WO 02/32437; WO 04/113516, WO 03/027270, WO 00/66188 and NZ 532057. Such pigs can also be an ideal source for CSF for the production of CP preparations.

The present invention further provides a method of treating a neurological disorder by administering a therapeutically effective amount of an implantable composition including NSC to the brain of a patient in need thereof, wherein the NSC have been cultured in a CP preparation of the invention. Neurological disorders for treatment using the compositions and methods of the invention include, for example Alzheimer's disease, Parkinson's disease, dementia, Amylotrophic lateral sclerosis (ALS), stroke, multiple sclerosis, anoxia/ asphyxia, aging, vascular disease, Huntington's chorea or brain injury due to physical trauma.

The amount of NSC, cultured in a CP preparation of the invention, required to be implanted to the brain of a patient in need thereof can be determined by a physician, or skilled person. The actual amount which will be suitable for an individual patient is likely to vary with age, weight, sex, disease, disease stage, and response to the particular patient to be treated.

Preferably the stem cells are encapsulated in alginate capsules of approximately 500-700 microns in diameter and containing approximately 500-3,000 living stem cells per capsule. When CP cells are present, the capsules may contain about 500-3,000 living CP cells in combination with living stem cells. The number of capsules that are implanted into a patient to give a therapeutic effect will vary depending on the age and weight of the patient and the interior dimensions of the implantation site in the body. CP cells and/ or stem cells can be encapsulated using methods known in the art (see for example WO 00/6618 and US 6,322,804). mNSC have been encapsulated according to the method of WO 00/66188 and were viable in long term culture (see Figure 4). Alternatively, equivalent numbers of "naked" or un-encapsulated CP and/or stem cells can be implanted into a patient. Neural stem cells, cultured in conditioned media according to the present invention, can be tested for efficacy in an animal model or in an in vitro model of neurodegenerative disease. For example, NSC cells cultured in CP conditioned media can be transplanted into the brain of rats that have undergone stroke surgery¹⁵'¹⁶ to assess their effect on neuronal function, and in , particular, to assess improvements in motor function produced by the NSC transplant.

Alternatively, cells can be administered prior to stroke surgery to determine if the cells can have a protective or mitigating effect in stroke.

Neural stem cells cultured according to the invention can also be transplanted into the brain of rats which will undergo or have undergone surgery to produce a Huntington's disease model as described in US 2005-0265977 or to produce a Parkinson's disease model, as described in US 2009-0047325. The Huntington's disease model uses quinolinic acid (QA) injection to create a lesion that mimicks the effects of Huntington's disease. It is expected that transplantation of NSC of the invention will alleviate the symptoms of Huntington's disease and improved neurological function. The Parkinson's disease model uses 6-hydroxydopamine (6- OHDA) injection to destroy tyrosine hydroxylase producing cells, followed by injection with amphetamine causing behavioural activity that mimics Parkinsons. It is expected that transplantation of NSC of the invention will alleviate the symptoms of Parkinson's disease and improve neurological function. Other animal models of neurological diseases may be used, for example, as described in US 5602299, US 7393994, WO 01/49107, or in vitro models of neurological disease as described in Sherer et al¹⁷, Miglio et al¹⁸ and Messmer et al¹⁹, for example, as would be understood by a skilled worker.

Mesenchymal stem cells (MSCs) have been used as neural stem cells and to derive neural cells of different types. For example, astrocytes and neurons were generated by injection of MSC into brain of immunocompromised neonatal mice. The cells were detected by the use of cell- surface markers by using antibodies and immunofluorescence (Kopen et al.²⁰. In another study, Eglitis and Mezey²¹ injected MSCs and/or hematopoietic stem cells (HSCs) in mice. Hematopoietic stem cells were found to differentiate into both microglia and macroglia in the brains of adult mice with induced injury to neural tissue. The cells were detected using antibodies to cell-surface proteins. HSCs were used by Mezey et al.²² to generate cells bearing neuronal antigens generated in vivo from bone marrow in a model of induced brain injury. Brazelton et al.²³, similarly demonstrated that bone marrow cells could be used to produce neural cells in a model of brain injury. Transplanted cells were detected by detecting a Y chromosome of the transplanted cells in a female mouse. Bjornson et al²⁴ performed essentially the converse experiment, providing NSCs to an irradiated mouse to reconstitute the hematopoietic system. Transplanted cells were detected using flow cytometry, genetic analysis, and antibody cell surface marker labeling. Neural stem cells were used by Galli et al²⁵ to generate skeletal muscle. Transplanted cells were detected by direct observation of differentiated skeletal muscle cells and analysis of muscle cell-specific proteins and gene expression.

These references demonstrate that it is within the ability of those of skill in the art to use stem cells for transplantation for the repair of damaged tissues. The references further demonstrate the utility of NSCs for regenerative therapies for non-neuronal cells. Provided with an appropriate source of stem cells or neural stem cells, their use in any of a number of applications would be within the ability of those of skill in the art. Moreover, those of skill in the art would know how to test cells for various applications and to identify transplanted cells in animal models of disease or disorder.

These studies demonstrate that stem cells can migrate to the site of injury or disease and need not be implanted specifically at the site of injury. It is understood that the cells cultured by the methods provided herein can be used for any therapeutic intervention including, but not limited to, the treatment of ischemia, e.g., by promoting vascularization, to repair muscle damage, e.g., cardiac damage, to promote bone growth, to provide pancreatic cells for the treatment of diabetes, or any other condition amenable to treatment with stem cells. Without being bound by theory, it is thought that it is the CP secreted proteins of the CP preparation that are effective in enhancing the growth, survival and/or function of stem cells (including migration) and/or in preventing their differentiation. While the exact mechanism is unknown, it is likely a result of the combination of growth factors secreted by the CP such as EGF, bFGF, TGFb, GDF-15, GDNF, TGFα, IGF-I, IGF-2, NGF, NT-3, NT-4, BDNF, VEGF, PDGF, SDF-I, FGF2, amphiregulin, SH-HGF, cystatin, α-microglobuHn, pleiotrophic factor, LIF and/ or PTHLH. Such a combination has not previously been used in stem cell culture. For example, it is known that optimal concentrations of bFGF and EGF are critical for stem cell expansion, especially for NSC expansion, however, culture of NSC in CP conditioned media resulted in a 16-fold increase in proliferation compared to a standard culture medium containing bFGF and EGF.

In addition, a number of factors are secreted by CP, such as Slit 2 and RGM A, which are known to inhibit stem cell migration. It is therefore surprising that a cocktail of CP secreted proteins, including Slit 2 and RGM A enhance the migratory capacity of NSC. It is demonstrated herein, that NSCs cultured in a CP preparation of the invention have a significantly increased migratory capacity. Thus, the NSC of the present invention will be able to migrate more quickly towards the pathology once implanted to a patients' brain. Migration towards pathology is the first critical step in stem cell engagement during regeneration. Culture of NSC in CP cells or CP conditioned media enhances the proliferation and survival of NSC thus providing an abundant, healthy, reproducible source of NSC for research, drug discovery, or therapeutic transplantation.

Culture of NSC in CP cells or CP conditioned media prevents the NSC differentiating and thus prevents loss of NSC from the culture and the maintenance of a pure population of undifferentiated NSC.

This invention can also be said broadly to consist in the parts, elements, and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

The invention consists in the foregoing and also envisages constructions of which the following gives examples only.

EXAMPLE 1 Murine neural stem cell proliferation with choroid plexus (CP) cell conditioned medium

Serum-free media conditioned for 3 days by porcine CP cells were assessed for the capability to enhance proliferation of fetal murine neural stem cells (mNSC).

Materials and Methods

The choroid plexus was removed from a neonatal Auckland Island pig by methods described in WO 00/66188. Clusters of CP cells were grown for 3 days in serum- free full stem cell medium EuroMedN (Euroclone, Italy) supplemented with N2, 2mM L-glutamine, Peniciliα/Streptomycin (Gibco). The media were harvested, filtered and frozen at -80⁰C until use.

Neural stem cell culture

Fetal neural stem cells were derived from El 5 CDl mice forebrain and cultured as neurospheres in the presence of 20 ng/ml EGF and 1 Ong/ml bFGF as described previously¹⁴. Briefly, timed pregnant mice were sacrificed and the embryos removed. Under sterile conditions the brains were removed and the forebrains dissected. The tissue was dissociated by trituration and pelleted by 75g centrifugation for 10 minutes and the cells seeded in non-coated tissue culture flasks in full EuroMedN medium with the recombinant growth factors EGF and bFGF. The neurospheres were passaged by trituration when reaching optimal size.

In vitro proliferation assay

The proliferation of mNSC was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan). CCK-8 is directly added to the cells and the water soluble tetrazolium salt WST-8 is bioreduced by cellular dehydrogenases to an orange formazan product that can be quantified by measurement of absorbance at 450nm in a plate reader. The amount of formazan produced is directly proportional to the number of living cells in the cultures. The supplier provides evidence for this by a correlation of r=0.99 between formazan absorbance and ³H-Thymidine incorporation for several cell lines.

Freshly triturated mNSC were seeded at a density of 10⁴ cells /well in 6 replicate wells per condition into 96 well plates and allowed to proliferate in the incubator for a period of 24 hours in 100 μl full EuroMedN stem cell medium containing no or different percentages of CP CM in presence of both EGF and bFGF, in presence of bFGF only and in the absence of both. After this time had passed, 10 μl of CCK reagent was added to each well and the plates returned into the incubator for colour development for another 18 hours. Subsequently, die optical densities (absorbance 450nm) were read against the control media with CCK reagent (without cells) using an E-Liza MAT 3000 plate reader. In parallel freshly triturated mNSC were plated in laminin- coated wells and cultured in 100% CP conditioned full stem cell medium supplemented with EGF and bFGF for 48 hours. Subsequently, the cultures were incubated for 10 minutes in proliferation medium containing lOOng/ml Syto21 to label the nuclei of viable cells. Then they were fixed with 4% paraformaldehyde and immunostained with a mouse anti-vimentin antibody and a goat anti-mouse Cy3-coupled secondary antibody

Results

Addition of CP CM to fetal mouse forebrain neural stem cell cultures resulted in significantly increased proliferation in the presence of optimal concentrations of both standard mitogens for neural stem cell expansion EGF and bFGF, in the presence of bFGF only and in the absence of both mitogens (Figure Ia). These result were confirmed in two independent experiments and are statistically significant as shown by Student's t-test, p<0.001 and One-way ANOVA followed by Dunnet's post-hoc test, p<0.0001/p<0.001. I_n the presence of 80% CP CM the proliferation rate of mNSC increased by 33%, 178% and 536% compared to EGF and bFGF only, bFGF only and absence of both mitogens, respectively. A clear dose-dependency for the concentration of CP CM on the stimulation of proliferation was observed. After exposure to 100% CP conditioned full stem cell medium for 48 hours the mNSC remained in an undifferentiated state, demonstrated by ubiquitous staining with the NSC marker vimentin (Fig Ib, left microphotograph) in all cells, shown by double-labelling for nuclei of viable cells (Fig Ib, right photomicrograph)

Summary

These experiments indicate that CP conditioned media contains factors that enhance the proliferation of neural stem cells, which can partly substitute for the addition of the recombinant mitogens EGF and bFGF. Interestingly, there was a synergistic effect of CP CM when both EGF and bFGF were present in optimal concentrations, indicating that the conditioned medium contains other proliferation factors than these two standard mitogens for NSC expansion.

EXAMPLE 2:

Murine neutal stem cell migration with chot oid plexus cell conditioned medium

Serum-free CP conditioned media was assessed for the capability to enhance migration of fetal murine neural stem cells (mNSC).

MATERIALS AND METHODS Generation of CP cell CM and culture of mNSC were done as described in Example 1.

In vitro migration assay

The migratory response of mNSC to CP cell conditioned medium was assessed with a haptotactic transwell migration assay over a period of 24 hours using Migration plates with 8 μm pore size membrane inserts (Costar). The principle of the assay is that a chemoattractant or repellent is added to the medium in the bottom compartment of the plate, but not into the medium within the insert, generating a concentration gradient which affects the chemotactic migratory response of cells seeded into the insert on top of the porous membrane. At the end of the set migration period, cells that remain at the top of the membrane are removed with a cotton swab, the inserts fixed and cells that crossed through the pores to the underside of the membrane stained and counted as migrated cells under the microscope. For the control condition six wells per plate were filled with 500 μl EuroMedN full medium to which 10 mg/ml BSA as a protein control ^■was added and the other six with 500 μl of 100% CP-condtioned EuroMedN. The inserts were coated with 50 μg/rnl Poly-L-lysine (Sigma) for 30 minutes and rinsed once with water. 3x10⁴ freshly triturated mNSC were seeded in 100 μl EuroMedN with 10 mg/ml BSA into every insert and the plate placed in the incubator for 24 hours. After this time the inserts were fixed with 4% PFA for one hour, washed with Hanks balanced salt solution (HBSS) and then stained with 1 uM Syto 21 fluorescent nucleic acid stain (Invitrogen) in HBBS for 20 minutes in the dark. Then cells that had migrated to the lower membrane surface in each insert were counted by their nuclear staining with an Olympus microscope at 20Ox magnification under fluorescent light.

Results The in vitro migration assay demonstrated that molecules secreted from CP cultures possess potent chemoattractive effects on murine neural stem cells. The average number of cells migrated in the control condition was set to 1.0 and the average number of cells migrated in the presence of CP CM calculated as fold of controls. 100% CP CM caused 2.95x as many mNSC to migrate to the bottom compartment of the transwell culture plates compared to controls with BSA only (Figure 2a). The bar graph (Figure 2b) represents the combined results of two independent experiments done in six replicates per condition each. The observed increase in migratory response of mNSC was statistically significant (Student's t-test, p<0.0001).

Summary

These data indicate that CP conditioned media contains factors that stimulate the migration of neural stem and progenitor cells. Interestingly, the net effect is a positive chemoattraction even though the CP is known to secrete high concentrations of slit proteins, which are repellent to neural progenitors⁶. Positive chemoattractants contained in the CP CM, like for example stromal-deiϊved factor-1 (SDF-I) and platelet-derived growth factor (PDGF) appear to have a dominant action on the migratory behaviour of neural stem cells. EXAMPLE 3:

Murine neural stem cell survival and neurogenesis with choroid plexus cell conditioned medium under differentiation-inducing conditions

Serum-free media conditioned for 3 days by CP cells were assessed for the capability to enhance survival and neurogenesis under differentiation-inducing conditions of fetal murine neural stem cells (mNSC). Materials and methods

Clusters of CP cells were grown for 3 days in serum-free full neuronal differentiation medium as described in example 1. The media were harvested, filtered and frozen at -80⁰C until use. In vitto differentiation assay

When reaching a sufficient size, neurospheres were dissociated by trituration and plated at a density of 200,000 cells per well on laminin-coated 13mm diameter cover slips placed in 24 well plates. The plating medium was a 1:1 mixture of DMEM/F12 supplemented with N2 and Neurobasal supplemented with B27 and 2 mM glutamine. After 24h the medium was replaced with a neuronal differentiation-promoting medium (1:3 mixture of DMEM/F12 supplemented with N2 and Neurobasal supplemented with B27 and 2 mM glutamine). Conditions were full differentiation media only (Control) or full differentiation media mixed 1:1 with full differentiation media conditioned by CP clusters for 3 days (CP CM). The medium was exchanged every other day. Seven days aftei plating the differentiating cells were incubated for 10 minutes in differentiation medium containing 100ng/ml Syto21 to label the nuclei of viable cells. Subsequently, they were fixed with 4% paraformaldehyde and immunostained with a mouse anti- BIII tubulin antibody and a goat anti-mouse Cy3-coupled secondary antibody

After staining the cover slips were removed from the tissue culture plate and mounted on slides, using Immunofluore fluorescent mounting medium. To analyse the percentage of neurons of total cells, with an Olympus microscope, equipped with analySIS LE software, at 20Ox magnification images of two random fields per well were taken in the red (tubukn stained neurons) and green (Syto21 stained nuclei) fluorescent channels. Neurons and nuclei per field were counted and the neuronal percentage of total cell number determined. Further, the numbei of neurons and number of total cells per mm² was determined. (See Table 2 below). Results

The in vitro differentiation assay demonstrated that molecules secreted from CP cultures confer potent survival effects on murine neural stem cells under differentiation inducing conditions and do not inhibit neuronal differentiation, but to the contrary slightly enhance it. The normally observed massive cell death upon seeding of dissociated neurospheies under differentiating conditions and withdrawal of mitogens was largely suppressed by addition of 50% CP CM. While the percentage of differentiated neurons of total cells increased only by 15%, the overall improved survival resulted in a 63% increase in the yield of neuronal cells in the cultures (Table 2). The table represents the combined results of two independent experiments done in four replicates per condition each.

Summary

The data indicate that CP conditioned media contains factors that enhance the survival of mNSC under differentiation-inducing conditions and thereby increase the yield of differentiated neurons in the cultures.

Table 2

Media conditions Neurons %/total cells Neurons/mm2 Cells/mm2

Full differentiation medium

20.9 196 942

(Control)

Full differentiation medium

24.1 312 1306

+50% CP CM

% increase with CP CM 15 63 39

EXAMPLE 4

Murine neural stem cell proliferation response and maintenance with varying concentrations of CP conditioned medium for up to 8 days

Serum-free media conditioned for 48 hrs by porcine CP cells was also assessed for its capability of proliferation and maintenance at varying concentrations over an 8 day period.

Materials and Methods

The murine neural stem cells were dissociated by routine enzyme and trituration methods to get a uniform single cell suspension. The cell suspension was pelleted by 75g centrifugation for 10 minutes and the cells seeded in non-coated tissue culture flasks in full Euro Med-N medium with glutamax, N2, antibiotics and recombinant growth factors EGF and bFGF. The neurospheres were passaged with enzyme and trituration when reaching optimal size using techniques known in the art. Generation of CP cell CM and culture of mNSC were carried out as described in Example 1. Five culture media compositions containing different concentrations of CP conditioned media (CPCM) were prepared as follows: Media A 100% Euro med- N, Glutamax, N2, antibiotic and mitogens (EGF,bFGF)

Media B 80% Media A, 20% CPCM

Media C 50% Media A, 50% CPCM

Media D 20% Media A, 80% CPCM Media E 100% CPCM

In vitro Proliferation Assay

Freshly triturated mNSC were seeded at a density of 5000 cells/well in 6 replicate 96 well plates and allowed to proliferate in the incubator for 24, 48, 72, 96, and 192 hrs for each of the five different media types A-E. The total volume of the media added per well was 100 μl. Blank control media (without cells) in duplicate were also included. Media A, with the abovementioned composition, was used for sub culturing of the neurospheres to provide maximum proliferation potential.

After the specified incubation time had passed, 10 ul of CCK i.e Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was added to the plate and the plate was incubated at 37°C for colour development . The water soluble tetrazolium salt WST-8 is bioreduced by cellular dehydrogenases to an orange formazan product. The amount of formazan produced is directly proportional to the number of living cells in the culture.The plate was read at 450nm and 630nm after 4 hours and 24 hours to allow for colour development and quantified by measurement of absorbance using an E-Liza MAT 3000 plate reader. Results

CP CM addition to neural stem cell cultures resulted in significantly increased proliferation in the presence of optimal concentrations of both standard mitogens EGF and bFGF for neural stem cell expansion. A clear dose-dependency for the concentration of CP CM on the stimulation of proliferation was observed (see Figure 3). Addition of 20%, 50% or 80% CPCM to a commercially available stem cell media suitable for maintenance of mNSC (Euro med- N) (Media B, C and D) resulted in a significant increase in mNSC proliferation at days 3, 4 and 5 compared to Euro med- N alone (Media A). TIxLs significant increase in proliferation of mNSC appeared to be synergistic at days 4 and 5, see figure 3. Media B (comprising 20% CPCM) continued to be synergistic compared to control Media A at day 8 and allowed the proliferation, and maintenance of the neurospheres, while Media D (80% CPCM) and control Media A had about the same rate of proliferation at day 8 (data not shown)

Interestingly, Media E (100% CPCM) did not increase the rate of mNSC proliferation above that achieved with the control Media A and from day 4, resulted in a decreased rate of proliferation of mNSC compared to the control media.

Summary

This experiment indicates that CPCM contains factors that enhance the proliferation of neural stem cells however CPCM alone does not contain all of the proliferation factors necessary to enhance NSC growth and maintenance. Addition of 20%, 50% or 80% of CPCM to a commercially available NSC media (Euro med-N) enhanced proliferation synergistically for up to 5 days over the Euro med-N media alone, indicating that CPCM contains other important proliferation factors. The synergistic effect continued for the 20% and 50% CPCM up to 8 days. These experiments indicate that CP conditioned media contains factors that enhance the proliferation of neural stem cells, which can partly substitute for the addition of the recombinant mitogens EGF and bFGF. Interestingly, there was a synergistic effect of CPCM even when both EGF and bFGF were present in optimal concentrations, indicating that the conditioned medium contains other proliferation factors than these two standard mitogens for NSC expansion.

EXAMPLE 5 Murine neural stem cell proliferation with varying concentration of CP conditioned media for up to 6 days.

Varying concentration of serum-free media conditioned for 3 days by porcine CP cells were assessed for the capability to enhance proliferation of mNSC.

Materials and Methods Generation of CP cell CM and culture of mNSC were carried out as described in example

1. Five culture media compositions contain different concentrations of CP conditioned media (CPCM) were prepared as follows:

Media A 100% Euro med-N, Glutamax, N2, antibiotic and mitogens (EGF, bFGF)

Media B 95% Media A, 5% CPCM Media C 90% Media A, 10% CPCM Media D 50% Media A, 50% CPCM

Media E 5% Media A, 95% CPCM

Media I/C 50% Media A, 50% CPCM from a different batch. In vitro Prolifer ation Assay The proliferation was assessed as described in example 4, above.

Results

Addition of 5%, 10% and 50% CPCM to a commercially available stem cell culture medium suitable for maintenance of mNSC ((Euro med-N) (Media B, C and D) resulted in significant increases in mNSC proliferation at day 5 and 6. In particular, the lower concentrations of CPCM favoured a synergistic effect compared to higher concentrations of the same media (see figure 5). The 95% CPCM addition (Media E) decreased the rate of mNSC proliferation at days 5 and 6.

Media I/C in figure 5 is an internal control media from a different batch of CPCM preparation and shows that the culture conditions and mNSC were optimal. Summary

This experiment indicates that CPCM at a concentration of as little as 5% v/v (Media B) is sufficient to enhance proliferation of mNSC over control media alone (Media A). This experiment also confirms that the previous results are reproducible, and that a 95% CPCM v/v media (Media E) had an inhibitory effect. The results of examples 4 and 5 show that CPCM from 5-80% v/v is effective at enhancing mNSC proliferation in vivo.

EXAMPLE 6

Murine neural stem cell differentiation and neurogenesis when cultured in choroid plexus cell conditioned medium for 5 days under differentiation-inducing conditions Fetal murine neural stem cells (mNSC) cultured and proliferated in CP media combinations were assessed for the capability to enhance survival and neurogenesis under differentiation-inducing conditions. Materials and methods

Clusters of CP cells were grown for 2 days in serum-free medium as described in example 1. The media were harvested, filtered and frozen at -80⁰C until use.

In vitto differentiation assay mNSC were grown in ether control media (Euro Med-N) or CP media (50:50 CPCM: Euro Med-N) for 5 days. When reaching a sufficient size, neurospheres were dissociated by trituration and plated at a density of 50,000 and 5,000 cells per well on laminin-coated 24 well plates and chamber slides respectively. The plating medium was a 1:1 mixture of Euro med-N: Neurobasal supplemented with N2, B27 and 2 mM Glutamax. After 24h the medium was replaced with one of two neuronal differentiation-promoting media: 1. (1:3 mixture of Euro med- N: Neurobasal supplemented with N2, B27 and 2 mM Glutamax) or 2. (Enstem A supplemented with glutamax and N2).

The medium was exchanged every other day. Three days after plating bright field images of the cells were taken. The differentiating cells were incubated for 10 minutes in differentiation medium containing lOOng/ml Syto21 to label the nuclei of viable cells. Subsequently, they were fixed with 4% paraformaldehyde, blocked and immunostained with a mouse anti-BIII tubulin antibody and a goat anti-mouse Cy3-coupled secondary antibody. The other wells were stained with vimentin to analyse the non differentiated cells but still having the neuronal markers on them so as to confirm that have not yielded to any other cell type in the inducing condition.

After staining the cells were mounted on slides using Immunofluore fluorescent mounting medium. The percentage of neurons that had differentiated in each of the two differentiation media as compared to the total cells, was measured with an Olympus microscope, equipped with analySIS LE software, at 20Ox magnification, images of two random fields per well were taken in the red (tubulin stained neurons) and green (Syto21 stained nuclei) fluorescent channels. The same analysis was carried out for wells stained with vimentin.

Results

The in vitro differentiation assay demonstrated that neurospheres pre-cultured in media containing 50% v/v CP conditioned media differentiated into neurons, oligodendrocytes and/or astrocytes when induced to differentiate (as shown in figures 6a to 6h). Preculture in 50% CPCM enhanced proliferation and differentiation of NSC. Summary

Preculture of mNSC in CPCM preserved the viability of mNSC and their ability to differentiate into neurons, oligodendrocytes and/or astrocytes.

EXAMPLE 7 In vitro culture of human embryonic stem cells using CP conditioned media.

Varying concentrations of serum-free media conditioned for 3 days by porcine CP cells will be assessed for the capability to enhance proliferation of human embryonic stem cells.

Materials and Methods

Generation of CP cell CM will be carried out as described in example 1. Human embryonic stem cells will be purchased from a suitable supplier, for example, BGOlV cells can be obtained from ATCC; H9 cells from WiCeIl Research Institute (See Table 1) or any other human embryonic stem cell source can be obtained that is commercially available.

Different compositions containing different concentrations of CP conditioned media (CPCM) will be prepared containing from 2% to 80% v/v CPCM in a commercially available human embryonic stem cell media, such as StemPro®hESC SFM (Invitrogen). Other commercially available media can be used as would be appreciated by a skilled worker. Growth factors /mitogens may be added to the commercial media to make sure the media is optimal for hESC growth.

In Vitro Proliferation Assay The proliferation of hESC will be assessed as for mNSC as described in example 4, above.

Results

We expect that addition of from 2% to 80% v/v CPCM to a commercially available stem cell culture medium suitable for maintenance of hESC (e.g. StemPro®hESC SFM) will result in significant increases in hESC proliferation compared to the commercially available stem cell culture medium alone. EXAMPLE 8

In vivo implantation of mNSC into rat models of stroke, Huntington's Chorea and Parkinsons Disease.

Three models of neurological disorders will be used to assess the effect of mNSC that have been cultured according to the methods of the invention on neurological function.

Materials and Methods mNSC will be cultured as described in the examples above in from 2% to 80% v/v CPCM or in a CP preparation that comprises CP cells. The cultured mNSC (and CP cells when present) can be encapsulated as described in the specification or can be 'free'. The free or encapsulated CP and/or mNSC will be implanted into the brains of rats that have undergone surgery to produce a model of stroke, Huntington's chorea or Parkinson's disease as described in US 2009-0047325.

Results

It is expected that the implanted mNSC will demonstrate neuroprotective effects in the rat models of various neurological disorders. The implantation of CP cells in combination with mNSC is expected to result in an enhanced neuroprotective effect.

INDUSTRIAL APPLICABILITY

The present invention is particularly useful for the in vitro culture of stem cells, to maintain viable stem cells in an undifferentiated state in long term or short term culture; to enhance proliferation or differentiation of stem cells in culture; and/or to produce viable stem cells for use in regenerative medicine. In particular, the invention is useful for culturing neural stem cells for implantation to the brain of patients to treat neurological disorders.

It will be appreciated that it is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variants are possible without departing from the scope of the invention as set out in the accompanying claims. REFERENCES

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All references, patents, patent publications, and Accession Numbers are hereby incorporated herein by reference as if each were incorporated by reference individually.

Table 3. Stem cell markers from The National Institutes of Health website http://stemcells.nih.gov/info/sckeport/ appendixe.asp#eii

Blood Vessel

Fetal liver kinase-1 (Flkl) Endothelial Cell-surface receptor protein that identifies endothelial cell progenitor; marker of cell-cell contacts

Smooth muscle cell-specific myosin heavy chain Smooth muscle Identifies smooth muscle cells in the wall of blood vessels

Vascular endothelial cell cadheiϊn Smooth muscle Identifies smooth muscle cells in the wall of blood vessels Bone

Bone-specific alkaline phosphatase (BAP) Osteoblast Enzyme expressed in osteoblast; activity indicates bone formation

Hydroxyapatite Osteoblast Minerϋzed bone matrix that provides structural integrity; market of bone formation Osteocalcin (OC) Osteoblast Mineral-binding protein uniquely synthesized by osteoblast; marker of bone formation

Bone Marrow and Blood

Bone morphogenetic protein receptor (BMPR) Mesenchymal stem and progenitor cells Important for the differentiation of committed mesenchymal cell types from mesenchymal stem and progenitor cells; BMPR identifies early mesenchymal lineages (stem and progenitor cells)

CD4 and CD8 White blood cell (WBC) Cell-surface protein markers specific for mature T lymphocyte (WBC subtype)

CD34 Hematopoietic stem cell (HSC), satellite, endothelial progenitor Cell-surface protein on bone marrow cell, indicative of a HSC and endothelial progenitor; CD34 also identifies muscle satellite, a muscle stem cell

CD34+Scal+ Lin- profile Mesencyhmal stem cell (MSC) Identifies MSCs, which can differentiate into adipocyte, osteocyte, chondrocyte, and myocyte

CD38 Absent on HSC Present on WBC lineages Cell-surface molecule that identifies WBC lineages. Selection of CD34+/CD38- cells allows for purification of HSC populations

CD44 Mesenchymal A type of cell-adhesion molecule used to identify specific types of mesenchymal cells c-Kit HSC, MSC Cell-surface receptor on BM cell types that identifies HSC and MSC; binding by fetal calf serum (FCS) enhances proliferation of ES cells, HSCs, MSCs, and hematopoietic progenitor cells

Colony-forming unit (CFU) HSC, MSC progenitor CFU assay detects the ability of a single stem cell or progenitor cell to give rise to one or more cell lineages, such as red blood cell (RBC) and/ or white blood cell (WBC) lineages

Fibroblast colony-forming unit (CFU-F) Bone marrow fibroblast An individual bone marrow cell that has given rise to a colony of multipotent fibroblastic cells; such identified cells are precursors of differentiated mesenchymal lineages

Hoechst dye Absent on HSC Fluorescent dye that binds DNA; HSC extrudes the dye and stains lightly compared with other cell types

Leukocyte common antigen (CD45) WBC Cell-surface protein on WBC progenitor- Lineage surface antigen (Lin) HSC, MSC Differentiated RBC and WBC lineages

Thirteen to 14 different cell-surface proteins that are markers of mature blood cell lineages; detection of Lin-negative cells assists in the purification of HSC and hematopoietic progenitor populations

Mac-1 WBC Cell-surface protein specific for mature granulocyte and macrophage (WBC subtypes)

Muc-18 (CDl 46) Bone marrow fibroblasts, endothelial Cell-surface protein (immunoglobulin superfamily) found on bone marrow fibroblasts, which may be important in hematopoiesis; a subpopulation of Muc-18+ cells are mesenchymal precursors

Stem cell antigen (Sca-1) HSC, MSC Cell-surface protein on bone marrow (BM) cell, indicative of HSC and MSC Bone Marrow and Blood cont.

Stto-1 antigen Stromal (mesenchymal) precursor cells, hematopoietic cells Cell-surface glycoprotein on subsets of bone marrow stromal (mesenchymal) cells; selection of Stro-1+ cells assists in isolating mesenchymal precursor cells, which are multipotent cells that give rise to adipocytes, osteocytes, smooth myocytes, fibroblasts, chondrocytes, and blood cells

Thy-1 HSC, MSC Cell-surface protein; negative or low detection is suggestive of HSC Cartilage Collagen types II and IV Chondrocyte Structural proteins produced specifically by chondrocyte Keratin Keratinocyte Principal protein of skin; identifies differentiated keratinocyte

Sulfated proteoglycan Chondrocyte Molecule found in connective tissues; synthesized by chondrocyte

Fat Adipocyte lipid-binding protein (ALBP) Adipocyte Lipid-binding protein located specifically in adipocyte

Fatty acid transporter (FAT) Adipocyte Transport molecule located specifically in adipocyte

Adipocyte lipid-binding protein (ALBP) Adipocyte Lipid-binding protein located specifically in adipocyte General

Y chromosome Male cells Male-specific chromosome used in labeling and detecting donor cells in female transplant recipients

Karyotype Most cell types Analysis of chromosome structure and number in a cell Liver Albumin Hepatocyte Principal protein produced by the liver; indicates functioning of maturing and fully differentiated hepatocytes

B-I integrin Hepatocyte Cell-adhesion molecule important in cell-cell interactions; marker expressed during development of liver

Nervous System CDl 33 Neural stem cell, HSC Cell-surface protein that identifies neural stem cells, which give rise to neurons and glial cells

Glial fibrillary acidic protein (GFAP) Astrocyte Protein specifically produced by astrocyte Microtubule-associated protein-2 (MAP-2) Neuron Dendrite-specific MAP; protein found specifically in dendritic branching of neuron

Myelin basic protein (MPB) Oligodendrocyte Protein produced by mature oligodendrocytes; located in the myelin sheath surrounding neuronal structures Nestin Neural progenitor Intermediate filament structural protein expressed in primitive neural tissue

Neural tubulin Neuron Important structural protein for neuron; identifies differentiated neuron

Neurofilament (NF) Neuron Important structural protein for neuron; identifies differentiated neuron Neurosphere Embryoid body (EB), ES Cluster of primitive neural cells in culture of differentiating ES cells; indicates presence of early neurons and glia

Noggin Neuron A neuron-specific gene expressed during the development of neurons O4 Oligodendrocyte Cell-surface marker on immature, developing oligodendrocyte Ol Oligodendrocyte Cell-surface marker that characterizes mature oligodendrocyte Synaptophysin Neuron Neuronal protein located in synapses; indicates connections between neurons

Tau Neuron Type of MAP; helps maintain structure of the axon Pancreas

Cytokeratin 19 (CKl 9) Pancreatic epithelium CKl 9 identifies specific pancreatic epithelial cells that are progenitors for islet cells and ductal cells

Glucagon Pancreatic islet Expressed by alpha-islet cell of pancreas Insulin Pancreatic islet Expressed by beta-islet cell of pancreas Pancreas

Insulin-promoting factor-1 (PDX-I) Pancreatic islet Transcription factor expressed by beta-islet cell of pancreas Nestin Pancreatic progenitor Structural filament protein indicative of progenitor cell lines including pancreatic

Pancreatic polypeptide Pancreatic islet Expressed by gamma-islet cell of pancreas

Somatostatin Pancreatic islet Expressed by delta-islet cell of pancreas Pluripotent Stem Cells

Alkaline phosphatase Embryonic stem (ES), embryonal carcinoma (EC) Elevated expression of this enzyme is associated with undifferentiated pluripotent stem cell (PSC)

Alpha- fetoprotein (AFP) Endoderm Protein expressed during development of primitive endoderm; reflects endodermal differentiation Pluripotent Stem Cells

Bone morphogenetic protein-4 Mesoderm Growth and differentiation factor expressed during early mesoderm formation and differentiation

Brachyury Mesoderm Transcription factor important in the earliest phases of mesoderm formation and differentiation; used as the earliest indicator of mesoderm formation Cluster designation 30 (CD30) ES, EC Surface receptor molecule found specifically on PSC

Cϊipto (TDGF-I) ES, cardiomyocyte Gene for growth factor expressed by ES cells, primitive ectoderm, and developing cardiomyocyte

GATA-4 gene Endoderm Expression increases as ES differentiates into endoderm

GCTM-2 ES, EC Antibody to a specific extracellular-matrix molecule that is synthesized by undifferentiated PSCs

Genesis ES, EC Transcription factor uniquely expressed by ES cells either in or during the undifferentiated state of PSCs

Germ cell nuclear factor ES, EC Transcription factor expressed by PSCs

Hepatocyte nuclear factor-4 (HNF-4) Endoderm Transcription factor expressed early in endoderm formation

Nestin Ectoderm, neural and pancreatic progenitor Intermediate filaments within cells; characteristic of primitive neuroectoderm formation

Neuronal cell-adhesion molecule (N-CAM) Ectoderm Cell-surface molecule that promotes cell- cell interaction; indicates primitive neuroectoderm formation Oct-4 ES, EC Transcription factor unique to PSCs; essential for establishment and maintenance of undifferentiated PSCs

Pax6 Ectoderm Transcription factor expressed as ES cell differentiates into neuroepithelium

Stage-specific embryonic antigen-3 (SSEA-3) ES, EC Glycoprotein specifically expressed in early embryonic development and by undifferentiated PSCs Stage-specific embryonic antigen-4 (SSEA-4) ES, EC Glycoprotein specifically expressed in early embryonic development and by undifferentiated PSCs

Stem cell factor (SCF or c-Kit ligand) ES, EC, HSC, MSC Membrane protein that enhances proliferation of ES and EC cells, hematopoietic stem cell (HSCs), and mesenchymal stem cells (MSCs); binds the receptor c-Kit

Telomerase ES, EC An enzyme uniquely associated with immortal cell lines; useful for identifying undifferentiated PSCs

TRA-1-60 ES, EC Antibody to a specific extracellular matrix molecule is synthesized by undifferentiated PSCs TRA-I -81 ES, EC Antibody to a specific extracellular matrix molecule normally synthesized by undifferentiated PSCs

Vimentin Ectoderm, neural and pancreatic progenitor Intermediate filaments within cells; characteristic of primitive neuroectoderm formation

Skeletal Muscle/Cardiac/Smooth Muscle MyoD and Pax7 Myoblast, myocyte Transcription factors that direct differentiation of myoblasts into mature myocytes

Myogenin and MR4 Skeletal myocyte Secondary transcription factors required for differentiation of myoblasts from muscle stem cells

Myosin heavy chain Cardiomyocyte A component of structural and contractile protein found in cardiomyocyte

Myosin light chain Skeletal myocyte A component of structural and contractile protein found in skeletal myocyte

Claims

WE CLAIM:

1. A method of culturing stem cells comprising: providing a choroid plexus (CP) preparation; and contacting the stem cells with the CP preparation under conditions to allow stem cell growth, thereby culturing the stem cells.

2. The method of claim 1, wherein a CP preparation comprises CP conditioned media.

3. The method of claim 1 or 2, wherein the CP preparation comprises CP cells.

4. The method of any one of claims 1 to 3, wherein the CP preparation comprises between 2% and 80% CP conditioned media and/or CP cells in a commercially available stem cell media.

5. The method of claim 3 or 4, wherein the CP cells are encapsulated in alginate.

6. The method of any one of claims 1 to 5, wherein the method of culturing enhances growth or survival of stem cells relative to stem cells not contacted with a CP preparation.

7. The method of any one of claims 1 to 6, wherein the stem cells are neural stem cells.

8. The method of any one of claims 1 to 7, wherein the stem cells are maintained in an undifferentiated state.

9. The method of any one of claims 1 to 8, further comprising contacting the stem cells with an added mitogen.

10. The method of claim 9, wherein the added mitogen is selected from the group consisting of EGF, bFGF, TGFb, GDF-15, GDNF, TGFα, IGF-I, IGF-2, NGF, NT-3, NT-4,

BDNF, VEGF, PDGF, SDF-I, FGF2, amphiregulin, SH-HGF, cystatin, α-microglobulin, pleiotrophic factor, LIF, PTHLH; and any combination thereof.

11. A method of enhancing migratory capacity of stem cells in culture comprising: providing a choroid plexus (CP) preparation; and contacting the stem cells with the CP preparation under conditions to allow stem cell growth, thereby enhancing the migratory capacity of the stem cells as compared to stem cells not contacted with a CP preparation.

12. The method of claim 11, wherein a CP preparation comprises CP conditioned media.

13. The method of claim 11 or 12, wherein the CP preparation comprises CP cells.

14. The method of any one of claims 11 to 13, wherein the CP preparation comprises between 2% and 80% CP conditioned media and/or CP cells in a commercially available stem cell media.

15. The method of any one of claims 11 to 14, wherein the stem cells are neural stem cells.

16. The method of any one of claims 11 to 15, furdier comprising contacting the stem cells with an added mitogen.

17. The method of claim 16, wherein the added mitogen is selected from the group consisting of EGF, bFGF, TGFb₃ GDF-15, GDNF, TGFα, IGF-I, IGF-2, NGF, NT-3, NT-4, BDNF, VEGF, PDGF, SDF-I , FGF2, amphiregulin, SH-HGF, cystatin, oc- microglobulin, pleiotrophic factor, LIF, PTHLH; and any combination thereof.

18. The method of any one of claims 1-17, further comprising contacting the stem cells with a factor selected from the group consisting of neurotrophins, growth factors, vascular endothelial growth factor, trophic factors, cytokines, mitogens, matrix cell support factors, enzymes, proteases capable of degrading toxic protein precipitates, proteins capable of complexing toxic metal ions; and any combination thereof.

19. The method of claim 18, wherein the enzymes are selected from the group consisting of proteases, alpha-1 antitrypsin, amylase, lipases, sucrase, lactase, and maltase.

20. The method of claim 18, wherein the proteins capable of complexing toxic metal ions are selected from the group consisting of transferrin and ceruloplasmin.

21. The method of any one of the previous claims, wherein the CP cells used to prepare a CP preparation are provided from a source selected from a mammalian donor, a primary or secondary CP culture, a CP cell line; and any combination thereof.

22. The method of claim 21, wherein the CP cells have been genetically modified.

23. The method of any one of the previous claims, wherein the CP cell comprises single CP cells, or clusters of CP cells.

24. The method of any of claims 1 to 23wherein the method maintains at least one function of a stem cell.

25. Stem cells cultured according to the method of any one of claims 1-24.

26. Stem cells as claimed in claim 25, comprising NSC.

27. Stem cells as claimed in claims 25 or 26 comprising encapsulated stem cells.

28. A stem cell culture composition comprising a commercially available stem cell culture or differentiation medium supplemented with between 2% and 80% v/v of a CP preparation.

29. A stem cell culture composition as claimed in claim 28, wherein the CP preparation comprises CP conditioned media.

30. A stem cell culture composition as claimed in claims 28 or 29, wherein the CP preparation comprises CP cells.

31. A stem cell culture composition as claimed in any one of claims 28-30, selected from a composition comprising between 5% and 75% v/v, 7% and 70% v/v, 8% and 65% v/v, 9% and 60% v/v, 10% and 55% v/v, 15% and 50% v/v, and 20% and 40% v/v of a CP preparation.

32. A stem cell culture composition as claimed in any one of claims 28-31, comprising 5% v/v of a CP preparation.

33. A stem cell culture composition as claimed in any one of claims 28-32, comprising 10% v/v of a CP preparation.

34. A stem cell culture composition as claimed in any one of claims 28-33, comprising 15% v/v of a CP preparation.

35. A stem cell culture composition as claimed in any one of claims 28-34, comprising 20% v/v of a CP preparation.

36. A method of culturing stem cells comprising contacting a stem cell culture composition of any one of claims 28-35 with stem cells under conditions to allow stem cell growth, thereby culturing the stem cells.

37. Stem cells cultured by the method of claim 36.

38. A cell culture comprising stem cells in media comprising a CP preparation.

39. A co-culture comprising stem cells and CP cells.

40. The co-culture of claim 39, wherein the stem cells are neural stem cells.

41. A cell culture comprising stem cells in a stem cell culture composition as claimed in any one of claims 28-35.

42. A cell culture as claimed in claim 41, wherein the stem cells are neural stem cells and the stem cell culture composition comprises Euro med-N: CP preparation in a ratio of 98:2% v/v to 20:80% v/v.

43. A use of the stem cells of any one of claims 25-27 and 37 in the manufacture of an implant for treating a neurological disease or disorder in a mammal in need thereof.

44. A use as claimed in claim 43, wherein the neurological disease or disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Amylotrophic Lateral Sclerosis (ALS), dementia, stroke, multiple, sclerosis, anoxia/ asphyxia, aging, vascular disease, Huntington's chorea, brain injury due to a physical trauma; and any combination thereof.