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WO2017189842A1 - Vésicules extracellulaires provenant de cellules souches ou de sérum de sujets plus jeunes pour thérapies liées à l'âge - Google Patents

Vésicules extracellulaires provenant de cellules souches ou de sérum de sujets plus jeunes pour thérapies liées à l'âge Download PDF

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WO2017189842A1
WO2017189842A1 PCT/US2017/029828 US2017029828W WO2017189842A1 WO 2017189842 A1 WO2017189842 A1 WO 2017189842A1 US 2017029828 W US2017029828 W US 2017029828W WO 2017189842 A1 WO2017189842 A1 WO 2017189842A1
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extracellular vesicles
stem cells
rna
patient
cells
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Paul Robbins
Laura Niedernhofer
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Scripps Research Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • Microvesicles mainly differ from nanovesicles by their size and by their mechanism of generation (Thery, C. et al. Exosomes: composition, biogenesis and function. Nature Rev. Immunol, 2, 569-579 (2002); Andaioussi, S. E. L. et al Extracellular vesicles: biology and emerging therapeutic opportunities. Nature Rev. Drug Discov. 12, 347-357 (2013); Thery, C. et al. Membrane vesicles as conveyors of immune responses. Nature Rev. Immunol. 9, 581— 593 (2009); and Yang, C.
  • Microvesicles are released from the plasma membrane by shedding or budding, they are usually larger than 0.2 ⁇ in size and have been referred to as microparticles or ectosom.es.
  • nanovesicles, including exosomes are between 30-100 nm in diameter, are characterized by an endocytic origin and are formed by the reverse budding of the peripheral membrane of multivesicular bodies (MVBs) or late endosom.es.
  • MVBs multivesicular bodies
  • Certain nanovesicles seem to be derived from the plasma membrane (Booth, A. M. et al. Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J. Cell Biol. 172, 923-935 (2006)).
  • extracellular vesicles mostly reflects that of the parent cells; vesicles are enriched in certain molecules, including adhesion molecules, membrane trafficking molecules, cytoskeleton molecules, heat shock proteins, cytoplasmic enzymes, signal transduction proteins, cytokines, chemokines, proteinases and cell-specific antigens.
  • extracellular vesicles contain mRNAs, non-coding RNAs (ncRNAs), including microRNAs (miRNAs), and even extra-chromosomal DNA, such as amplified MYC6. Almost all cell types release extracellular vesicles that are found in the plasma and other bodily fluids, including breast milk, semen, saliva, urine and sputum.
  • Extracellular vesicles participate in important biological functions, acting as a mode of communication between ceils. This intercellular communication can be conferred by mediators that are expressed on the surface of the extracellular v esicles or that are contained within the extracellular vesicle lumen.
  • Extracellular vesicles that are produced by both immune and non-immune cells have important roles in the regulation of immunity. They can mediate immune stimulation or suppression and they can drive inflammatory, autoimmune and infectious disease pathology (M. Lavasani et al. Muscle-derived stem /progenitor cell dysfunction limits healthspan and lifespan in a murine progeria model, Nature Comm. 3, 608 (2012)).
  • extracellular vesicle types Despite a focus upon use of extracellular vesicles in some therapeutic contexts, the diversity of extracellular vesicle types and their parent cells raises a challenge to discover what, if any, other therapies might be derived from, or modulated by, extracellular vesicles.
  • the present invention provides, in one embodiment, a method for treating a patient suffering from a disease or condition or age- related symptom that is caused by stem, cell dysfunction and/or increased senescence.
  • the method comprises administering to the patient a composition comprising extracellular vesicles obtained from stem cells of a subject that is younger than, and of the same species as, the patient.
  • Another embodiment of the invention is a method for treating a patient suffering from a disease or condition or age-related symptom that is caused by stem cell dysfunction and/or increased senescence.
  • the method comprises administering to the patient a composition comprising extracellular vesicles obtained from the serum, of a subject that is younger or healthier than, and of the same species as, the patient.
  • the stem cells are selected from one or more of muscle-deri ved stem cells, adipose derived stem cells, or mesenchymal stem cells.
  • adipose derived stem cells or mesenchymal stem cells.
  • mesenchymal stem cells include but are not limited to bone marrow derived mesenchymal stem cells, adipose derived mesenchymal stem cells, and other tissue specific mesenchymal stem, cells.
  • the patient is a mammal.
  • a specific example of a mammal is a human.
  • the disease or condition, driven in part by cellular senescence is one selected from the group consisting of progeroid syndromes, obesity, idiopathic pulmonary fibrosis, and DNA damage.
  • progeroid syndromes include but are not limited to those selected from the group consisting of Werner syndrome, Bloom syndrome, Rothmiind- Thomson syndrome, Cockayne syndrome, xeroderma pigmentosum, trichothiodystrophy, combined xeroderma pigmentosum-Cockayne syndrome, restrictive dermopathy, and Hutchinson-Gilford progeria syndrome .
  • the disease or condition is driven by DNA damage caused by ionizing radiation or chemotherapy that results in an increase in cellular senescence.
  • an increase in oxidative DNA damage with an associated increase in senescence is caused by inflammation, surgical procedures and even obesity.
  • RNA selected from the group consisting of messenger (niRNA), non-coding RNA (ncRNA), and combinations thereof.
  • ncRNA include, but are not limited to, micro-RNA (miRNA), transfer RNA (tRNA), ribosomai RNA (rRNA), small nucleolar RNA (snoRNA), small nuclear RNA
  • RNA small interfering RNA
  • piRNA piwi-interacting RNA
  • IncRNA long noncoding RNA
  • the diameter of the extracellular vesicles is about 40 nm to about 200 nm.
  • the composition described herein can be administered to the patient by injection, pursuant to some embodiments.
  • Another embodiment of the invention is a method for decreasing cellular senescence in a senescent cell from a first host organism.
  • the method comprises contacting the cell with a composition comprising extracellular vesicles obtained from stem cells or serum from a second host organism that is younger or healthier than, and of the same species as, the first host organism.
  • the contacting occurs in vitro. In another embodiment, the contacting occurs in vivo.
  • the method of decreasing cellular senescence is reversing cellular senescence.
  • the invention also provides a method for delaying the onset of cellular senescence in a ceil from a first host organism.
  • the method comprises contacting the cell with a composition comprising extracellular vesicles obtained from stem cells from serum of a second host organism that is younger than, and of the same species as, the first host organism.
  • the ceil from the fi rst host organism expresses senescence-associated beta-galactosidase (SA- -gal).
  • SA- -gal beta-galactosidase
  • the cell from the second host organism does not express SA-p-gal.
  • FIG. 1 shows the time evolution of extracellular vesicles of different weights from muscle- derived stem/progenitor cells (MDSPCs).
  • FIG. 2 shows the time evolution of ectosomes and exosomes in various media.
  • FIG. 3 is an electron microscopy image of extracellular vesicles from murine muscle stem cells (MSCs).
  • FIG. 4 is a plot showing a particle size distribution of extracellular vesicles from murine muscle stem cells.
  • FIG. 5 is plot showing RNA length distribution of extracellular vesicles from murine muscle stem cells.
  • the X axis is the length of the RNA in nucleotides (nu) and the Y axis the intensity of the fluorescent measurement of RNA in arbitrary units (FU).
  • FIG. 6 shows an exemplary process of a determination of senescent muscle stem cells.
  • FIG. 7 shows the effect of various media on senescent muscle stem cells.
  • FIG. 8 compares the effect of extraceliuar vesicles from young and old mice on senescent muscle stem cells.
  • FIG. 9 compares the effect of murine and human extracelluar vesicles on senescent muscle stem cells.
  • FIG. 10 is a plot showing the extended survival of Erccl-/- mice injected with extracellular vesicles derived from young mice.
  • FIG. 11 shows that addition of extracellular vesicles from semm of young mice to senescent murine MSCs reduced the level of senescence.
  • FIG. 12 shows that serum extracellular vesicles from young, but not Erccl-/- mice, reduce senescence of aged MSCs.
  • conditioned media from young, hut not old stem cells can reverse senescence in fibroblasts and aged stem ceils. Furthermore, the positive effect of conditioned media is lost when extracellular vesicles are depleted, and subsequent addition of purified vesicles is as effective as the conditioned media.
  • RNAs in the extracellular vesicles is different than in the young stem cells, thereby indicating a preferential sorting of RNAs into the extracellular vesicles.
  • stem, cell derived extracellular vesicles interact with and are taken up preferentially by senescent cells, whereby therapeutic effects are conferred through the targeting and then delivery of non-coding RN As to damaged, senescent ceils.
  • administration such as intraperitoneal (IP) injection, of extracellular vesicles from young mesenchymal stem cells (MSCs) can extend lifespan in an aged mammal.
  • IP intraperitoneal
  • extracellular vesicles isolated from young, but not old stem cells, or isolated from young serum. are used in accordance with the present invention to extend health span, to treat age-related chronic diseases, and to treat diseases associated with an increase in cellular senescence.
  • extracellular vesicles derives from the fact that extracellular vesicles are natural products.
  • extracellular vesicles are readily purified, and they likely work through multiple mechanisms including transfer of small, non-coding RNAs.
  • the fact that they preferentially target damaged, senescent cells moreover enables use of the extracellular vesicles to treat a disease or condition driven by cellular damage and senescence.
  • Extracellular vesicles for use in methods described herein are obtained from a subject or first host organism that is younger or healthier than, and of the same species, as the patient or cells from a second host organism to be treated with the extracellular vesicles.
  • the age of the subject or first host organism ranges from, newborn to adulthood.
  • stem cells likely age in a linear fashion, it may be possible to use extracellular vesicles from a mid-life organism to treat an older organism.
  • one useful demarcation other than chronological age to distinguish patient and subject cells is based on the observation that only senescent cells express the biomarker senescence-associated beta-gaiactosidase (SA-P-gal) and soluble factors such as IL-6, PAH and TNF-a.
  • SA-P-gal biomarker senescence-associated beta-gaiactosidase
  • soluble factors such as IL-6, PAH and TNF-a.
  • Extracellular vesicles in serum for use in the present invention likely are secreted by, and isolated and purified from a variety of cell types. These include, without limitation, reticulocytes; immune ceils, such as T cells, B ceils, macrophages, and dendritic cells;
  • epithelial cells such as intestinal epithelial cells, and stem and progenitor cells.
  • Particularly useful sources of extracellular vesicles are stem cells, such as mesenchymal stem cells (MSCs).
  • MSCs mesenchymal stem cells
  • Useful sources of MSCs include bone marrow and non-marrow tissues such as the placenta, umbilical cord, adipose tissue, and dental pulp.
  • the cells are muscle-derived stem/progenitor cells (MDSPCs).
  • the harvesting of extracellular vesicles is achieved by contacting parent cells from a subject with a suitable medium for a time sufficient to enrich the media in secreted extracellular vesicles.
  • a suitable medium is suitable for this purpose, such as RPMI, DMEM, and AIM V®.
  • the medium is preferably a protein-free medium so as to avoid contamination of extracellular vesicles by media-deri ved proteins.
  • Typical conditioning times range from about 5 hours to about 48 hours. Exemplary conditioning times are about 12 hours, about 16 hours, and about 24 hours.
  • the resulting conditioned media is then separated from the parent cells by conventional means known in the art.
  • a convenient method, among others, includes centrifugation to isolate a fraction of media highly concentrated in extracellular vesicles.
  • extracellular vesicles are isolated from the serum of a young subject or healthy host organism.
  • Extracellular vesicles obtained in this manner are isolated by conventional means. For example, serum from a younger subject or a healthy host organism is centrifuged or ultracentrifuged to separate extracellular vesicles from other serum components.
  • Extracellular vesicles obtained from young subjects or first host organisms exhibit a profile of RNAs that is distinct from extracellular vesicles obtained from old subjects or second host organisms. More specifically, according to some embodiments of the invention, at least a portion of extracellular vesicles for use in the methods described herein contain messenger RNA (mRNA), non-coding RNA (ncRNA), or a combination thereof.
  • mRNA messenger RNA
  • ncRNA non-coding RNA
  • the RNA is non-coding RNA (ncRNA).
  • ncRNA non-coding RNA
  • exemplary types of ncRNAs are micro-RNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA, long noncoding RNA (IncRNA), and combinations thereof.
  • RNA(s) are different than in the young cells from which the extracellular vesicles are secreted, indicating a preferential sorting of RN As in the extracellular vesicles.
  • composition containing: extracellular vesicles to damaged, senescent cells.
  • the extracellular vesicles are provided in a composition, typically a pharmaceutical composition that comprises an extracellular vesicle composition as disclosed herein and a pharmaceutical vehicle, carrier, or excipient.
  • the pharmaceutical composition is pharmaceutically-acceptable in humans.
  • composition can be formulated as a therapeutic composition for delivery to a subject in some embodiments, so long as the composition maintains intact extracellular vesicles.
  • the pharmaceutical composition includes a pharmaceutical carrier such as aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents.
  • a pharmaceutical carrier such as aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient
  • aqueous and nonaqueous sterile suspensions which can include suspending agents and thickening agents.
  • the pharmaceutical composition can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the formulations can be presented in unit
  • Injectable formulations of the compositions also can contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, poiyols (glycerol, propylene glycol, liquid polyethylene glycol), and the like.
  • various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, poiyols (glycerol, propylene glycol, liquid polyethylene glycol), and the like.
  • water soluble versions of the compositions can be administered by the drip method, whereby a fonnulation including a
  • compositions of the present invention and a physiologically-acceptable excipient are infused.
  • Physiologically-acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
  • Intramuscular preparations can be dissolved and administered in a pharmaceutical excipient such as Water-for-Iiijection, 0.9% saline, or 5%j glucose solution.
  • a suitable insoluble form of the composition can be prepared and administered as a suspension in an aqueous base or a pharmaceutically-acceptable oil base, such as an ester of a long chain fatty acid, ⁇ e.g. , ethyl oleate).
  • the invention provides methods for treating a patient suffering from a disease or condition or age-related symptom that is caused by stem ceil dysfunction or increased senescence.
  • Cellular senescence is defined as the irreversible state of Gi cell cycle arrest in which cells are refractor ⁇ ' to growth factor stimulation.
  • Telomere shortening which occurs each cell division, plays an essential role in driving implicative senescence.
  • SASP senesecence-associated secretory phenotype
  • senescence plays a critical role during embryogenesis and wound healing.
  • Senescent cells accumulate in mammals as they age and are found associated with many age-related degenerative diseases such as atherosclerosis, osteoarthritis, sarcopenia, gastrointestinal ulcers, and Alzheimer's disease.
  • age-related degenerative diseases such as atherosclerosis, osteoarthritis, sarcopenia, gastrointestinal ulcers, and Alzheimer's disease.
  • senolytics new classes of drugs able to induce apoptosis of senescent cells, termed senolytics, are able to enhance cardiovascular function and stem cell function in chronologically aged mice, restore treadmill endurance in radiation-exposed mice, and decrease frailty, neurologic dysfunction and bone loss in progeroid mice.
  • senolytics are able to enhance cardiovascular function and stem cell function in chronologically aged mice, restore treadmill endurance in radiation-exposed mice, and decrease frailty, neurologic dysfunction and bone loss in progeroid mice.
  • cellular senescence is
  • SA-p-gal senescence-associated beta-galactosidase
  • upregulation of pl6 iNK4a and p21 and secretion of certain cytokines, metalloproteinases and growth factors.
  • SA-B-gal is the most-recognized true marker for all types of senescent cells, even though SA-p-gal does not necessarily play a role in driving senescence.
  • a population of cells is assayed, according to one embodiment, by incubating the cells in a culture medium, then contacted with a DNA intercalating dye, such as Hoechst dye.
  • SA-p-gal -positive cells are then quantified by routine methods, such as by sCMOS camera detection technology. The sample population of cells is compared to a control population of SA-p-gal -negative cells to complete the quantitative analysis,
  • Increased senescence thus is expressed as a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%>, at least 100%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold or at least 100-fold increase or more relative to a control level, such as population of SA-p-gal-negative cells.
  • Underlying the disease, condition, or symptom is the biology of ageing, which results from an inability to maintain tissue homeostasis and to repair damaged or patiiological tissues due to injury or disease.
  • the methods according to the invention counteract these mechanisms.
  • a patient suffering from a disease associated with or caused by stem cell dysfunction or damage may not enjoy increased life span, but does enjoy an increased health span, i.e., the prolongation of time during which the patient experiences relatively healthy life, in contrast to a slow deterioration of health over the same lifetime.
  • the methods comprise the administration of an extracellular vesicle composition as described herein to the patient.
  • the extracellular vesicles are obtained from stem cells of a young or healthier subject.
  • the extracellular vesicles are obtained from the serum of a young subject.
  • the extracellular vesicles as described as herein are useful in treating a patient who suffers from a disease or condition or age-related symptom that is caused by stem ceil dysfunction or increased senescence.
  • the methods therefore are useful in treating disease or condition or age-related symptoms such as progeroid syndromes, obesity, idiopathic pulmonary fibrosis, and DNA damage.
  • the term "treating” encompasses the arrest of an otherwise progressive disease.
  • the term also contemplates reversal or amelioration of the disease or condition.
  • the disease or condition is a progeroid syndrome, such as Werner syndrome. Bloom syndrome, Rothmund-Thomson syndrome, Cockayne syndrome, xeroderma pigmentosum, trichothiodystrophy, combined xeroderma pigmentosum-Cockayne syndrome, restrictive dermopathy, or Hutchinson -Gilford progeria syndrome.
  • DNA damage such as damage caused by ionizing radiation.
  • DNA damage also can arise from chemotherapy regimens.
  • the methods of the invention are useful in treating these examples of DNA damage.
  • the invention also contemplates methods for decreasing or delaying the onset of cellular senescence in a cell by contacting the cell with an extracellular vesicle composition as described herein.
  • extracellular vesicles can be administered to cells by addition of the extracellular vesicles to cell cultures in. vitro, injection of these extracellular vesicles intravenously, or by any other route, in vivo as is known in the art.
  • Extracellular vesicles can be targeted to any cell in the body, including cells in the cardiovascular system, skeletal muscle cells, joint cells, neural cells, gut cells, lung cells, liver cells or kidney cells, or cells in the immune system, or to any type of cell with any function or dysfunction in the body of humans or animals, including malignant cells.
  • the features of the present invention will be more clearly understood by reference to the following examples and embodiments, which are not to be construed as limiting the invention.
  • Example 1 Rejuvenation of ERCC1 -deficient MDSPCs by extracellular vesicles from young MDSPCs
  • MDSPCs Erccl -deficient Muscle derived stem/progenitor cells
  • An imaging system Automated Cell Technologies, Inc. equipped with a 20 ⁇ objective was used to acquire images at 10-minute intervals over a period of 72 hrs. Forty images at each time point were randomly selected and analyzed from each treatment group. Conditioned media was isolated from MDSPCs from juvenile mice.
  • conditioned media was then passed through Centricon filters of different sizes, resulting in conditioned media containing components under 1000, 300, 100, 50 or 30 kD.
  • Microvesicles are removed by the 300 kD cutoff and exosomes by the 100 kD cutoff ( Figure 1).
  • Exosomes or microvesicles (ectosomes) were isolated by differential centrifugation of the conditioned media (10,000 X g for ectosomes and 100,000 X g for exosomes). The vesicles were then added back to non-conditioned media and the effect on cell growth determined, in addition, conditioned media depleted of ectosomes or ectosomes and exosomes was also tested ( Figure 2).
  • Exosomes were isolated from, conditioned media from young, murine MSCs by differential centrifugation.
  • the vesicles present in the 1000,000 x g pellet had a size similar to exosomes, as demonstrated by electron microscopy ( Figure 3) and Nanosight analysis ( Figure 4).
  • the vesicles also contained protein markers of exosomes (Hsp70 and CD63) as well as were enriched for small RNA between 20 and 100 nucleotides in length (Figure 5),
  • Example 3 Conditioned media from young MSCs rescues senescence in aged MSCs.
  • the ratio of conditioned media to non-condiiioned media was 1 : 1.
  • 48 hours after addition of the conditioned media the percent of SA-B-gal positive cells was determined by X-gal staining.
  • conditioned media depleted of exosomes by centrifugation at 1 0,000 x g was also tested (Figure 7).
  • Example 4 Exosomes from young but not old MSCs recues senescence in aged MSCs. Exosomes were isolated from, conditioned media from murine MSCs derived from young (8 weeks) and old (2 years) mice by differential centrifugation. The number and size of the vesicles present in the 1000,000 x g pellet was determined by Nanosight analysis. Addition of 3 10 9 and, in particular 3 10 10 exosomal particles to senescent MSCs from old mice reduced the level of senescence at a 48 hour time point ( Figure 8).
  • Example 5 Exosomes from human adipose derived MSCs reduces senescence in murine aged MSCs.
  • Exosomes were isolated by differential centrifugation from conditioned media from young murine MSCs and from human MSCs isolated from adipose tissue of human donors. The number and size of the vesicles present in the 1000,000 x g pellet was determined by Nanosight analysis. Addition of 3 1() 9 and/or 3 1() !0 exosomal particles from murine and human MSCs to senescent MSG from old mice reduced the level of senescence ( Figure 9).
  • Example 6 Exosomes from young MSCs extends lifespan senescence in Erccl-deficient mice, Exosomes were isolated from conditioned media from murine MSCs derived from young (8 weeks) mice by differential centrifugation. The number and size of the vesicles present in the 1000,000 x g pellet was determined by Nanosight analysis. Littermate pairs of Erccl ⁇ ! ⁇ mice were injected intraperitoneally (IP) with 2 * 10'" exosomes particles at day 10 and 17 or with vehicle and the effect on lifespan measured ( Figure 10).
  • Example 7 Mouse serum derived exosomes reduce senescence of aged MSCs.
  • Exosomes were isolated from serum from young, wildtype mice (8 weeks) derived from by differential centrifugation. Here, the serum was diluted 1 :20 in PBS prior to centrifugation. The number and size of the vesicles present in the 1 00,000 x g pellet was determined by Nanosight analysis. Addition of exosomal particles from serum of young mice to senescent murine MSCs reduced the level of senescence ( Figure 11).
  • Example 8 Serum derived exosomes from young, but not Erect 1' mice reduce senescence of aged MSCs
  • Exosomes were isolated from conditioned media from murine MSCs derived from young (8 weeks) mice by differential centrifugation. The number and size of the vesicles present in the 1000,000 x g pellet was determined by Nanosight analysis. Addition of exosomal particles from serum of young, but not E eel '1 ' ' mice to senescent murine MSCs reduced the level of senescence (Figure 12).

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Abstract

L'invention concerne des méthodes destinées à traiter un patient souffrant d'une maladie, d'une affection ou d'un symptôme lié à l'âge qui est provoqué par un dysfonctionnement des cellules souches ou une sénescence accrue. Les méthodes comprennent l'administration au patient d'une composition comprenant des vésicules extracellulaires obtenues à partir des cellules souches ou du sérum d'un sujet plus jeune ou en meilleure santé, et de la même espèce, que le patient.
PCT/US2017/029828 2016-04-27 2017-04-27 Vésicules extracellulaires provenant de cellules souches ou de sérum de sujets plus jeunes pour thérapies liées à l'âge Ceased WO2017189842A1 (fr)

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CN108103206A (zh) * 2017-12-18 2018-06-01 中国农业科学院北京畜牧兽医研究所 一种肌内脂肪相关的lncRNA及其应用
CN111494417A (zh) * 2020-02-10 2020-08-07 寇晓星 诱导性细胞外囊泡在制备治疗肿瘤药物中的应用
WO2020249567A1 (fr) 2019-06-10 2020-12-17 Unicyte Ev Ag Vésicules extracellulaires provenant de cellules souches hépatiques humaines (hlsc-evs) pour réduire la sénescence cellulaire
CN114748504A (zh) * 2020-12-29 2022-07-15 南京优智源医药科技有限公司 年轻人血浆外泌体在制备治疗衰老引发的退行性疾病的药物中的应用
CN115044543A (zh) * 2022-08-17 2022-09-13 山东卓东生物科技有限公司 一种提高衰老人体来源肌肉干细胞活性的方法
WO2024108263A1 (fr) * 2022-11-23 2024-05-30 Geoffrey Keipert Procédé de prévention ou de traitement d'états liés à l'âge à l'aide de vésicules extracellulaires dérivées d'un jeune mammifère

Citations (4)

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CN111494417B (zh) * 2020-02-10 2024-04-09 寇晓星 诱导性细胞外囊泡在制备治疗肿瘤药物中的应用
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