WO2017189842A1

WO2017189842A1 - Extracellular vesicles from young stem cells or serum for age-related therapies

Info

Publication number: WO2017189842A1
Application number: PCT/US2017/029828
Authority: WO
Inventors: Paul Robbins; Laura Niedernhofer
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 2016-04-27
Filing date: 2017-04-27
Publication date: 2017-11-02
Anticipated expiration: 2018-10-27

Abstract

The invention provides methods for treating a patient suffering from a disease or condition or age-related symptom that is caused by stem cell dysfunction or increased senescence. The methods comprise administering to the patient a composition comprising extracellular vesicles obtained from stem cells or serum of a subject that is younger or healthier than, and of the same species as, the patient.

Description

EXTRACELLULAR VESICLES FROM YOUNG STEM CELLS OR SERUM FOR

AGE-RELATED THERAPIES

CLAIM OF PRIORITY This application claims the benefit of priority to U.S. Provisional Application Serial

No. 62/328,196, filed April 27, 2016, which is herein incorporated by reference in its entirety.

BACKGROUND

Almost all cells release different types of membrane extracellular vesicles, such as microvesicles and nanovesicles, which have a variety of important physiological effects. 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. et al. Immunosuppressive exosomes: a new approach for treating arthritis. Int. J. Rheumatol. 2012, 573-528 (2012)). Microvesicles are released from the plasma membrane by shedding or budding, they are usually larger than 0.2 μιη in size and have been referred to as microparticles or ectosom.es. By contrast, 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. 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)).

The protein content of different types of 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. Moreover, 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)).

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.

SUMMARY In meeting this challenge and others, 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. In accordance with this embodiment, 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.

In one embodiment, the stem cells are selected from one or more of muscle-deri ved stem cells, adipose derived stem cells, or mesenchymal stem cells. Illustrative in this regard are mesenchymal stem cells. Examples of 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. n die methods described herein, according to some embodiments, the patient is a mammal. A specific example of a mammal is a human.

In other embodiments of the invention, optionally in combination with any other embodiments, 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. Specific examples of a progeroid syndrome 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 .

In other embodiments, the disease or condition is driven by DNA damage caused by ionizing radiation or chemotherapy that results in an increase in cellular senescence. Also, an increase in oxidative DNA damage with an associated increase in senescence is caused by inflammation, surgical procedures and even obesity. In accordance with some embodiments of the methods described herein , at least a portion of the extracellular vesicles contain RNA selected from the group consisting of messenger (niRNA), non-coding RNA (ncRNA), and combinations thereof. In some embodiments, at least a portion of the extracellular vesicles contain ncRNA. Specific examples of ncRNA include, but are not limited to, micro-RNA (miRNA), transfer RNA (tRNA), ribosomai 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.

In still other embodiments of the methods described herein, 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. According to this embodiment, 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. In one embodiment, the contacting occurs in vitro. In another embodiment, the contacting occurs in vivo.

In accordance with some embodiments, 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. In accordance with this embodiment of the invention, 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. In one embodiment, the method the ceil from the fi rst host organism expresses senescence-associated beta-galactosidase (SA- -gal). In another embodiment, optionally in combination with other embodiments, the cell from the second host organism does not express SA-p-gal.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION Underlying the invention is the discovery that 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. Functional, MSC-derived extracellular vesicles carrying a distinct subset of RNAs, both niRNA and small, non-coding RNAs, that likely are important for their ability to reverse cellular senescence and stem cell dysfunction. We observe that the pattern of RNAs in the extracellular vesicles is different than in the young stem cells, thereby indicating a preferential sorting of RNAs into the extracellular vesicles. Furthermore, as the examples show herein, 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. Moreover, the examples demonstrate that administration, such as intraperitoneal (IP) injection, of extracellular vesicles from young mesenchymal stem cells (MSCs) can extend lifespan in an aged mammal. Thus, 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.

One advantage of using extracellular vesicles derives from the fact that extracellular vesicles are natural products. In addition, 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

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. In general, the age of the subject or first host organism ranges from, newborn to adulthood. However, since 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. According to some embodiments, 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. Hence, an assay of subject cells, as described in more detail below, can be used to identify the suitability of a subject or host organism as a source of extracellular vesicles for use in the inventive methods.

From Ceils

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 according to some embodiments of the invention are stem cells, such as mesenchymal stem cells (MSCs). Useful sources of MSCs include bone marrow and non-marrow tissues such as the placenta, umbilical cord, adipose tissue, and dental pulp. In oilier embodiments, 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. Any appropriate culture 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. An accepted protocol for isolation of exosomes includes ultracentrifugation (Thery et al., Cum Protoc. Cell. Biol, Chapter 3, Unit 3: 22 (2006)), often in combination with sucrose density gradients or sucrose cushions to float the relatively low-density extracellular vesicles.

From Serum Alternatively, according to some embodiments, extracellular vesicles are isolated from the serum of a young subject or healthy host organism. Extracellular vesicles obtained in this manner, analogous to that as described above, 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. RNA in Extracellular Vesicles

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.

In accordance with some embodiments, the RNA is non-coding RNA (ncRNA). 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. Without wishing to bind or otherwise limit the invention by any particular theor ', the inventors observed that patterns of the foregoing RNAs in the extracellular vesicles is different than in the young cells from which the extracellular vesicles are secreted, indicating a preferential sorting of RN As in the extracellular vesicles. Some embodiments of the invention therefore contemplate the transfer of RNA(s) from

extracellular vesicles to damaged, senescent cells. Composition

In some embodiments, 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. In some embodiments, the pharmaceutical composition is pharmaceutically-acceptable in humans. The

pharmaceutical 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.

In some embodiments, 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. 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. Additionally, the formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials.

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. For intravenous injections, water soluble versions of the compositions can be administered by the drip method, whereby a fonnulation including a

pharmaceutical composition of the present invention and a physiologically-acceptable excipient is 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). Methods

In accordance with some embodiments, 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.

In addition, mitogenic signaling and cellular damage, including oxidative and genotoxic stress, have been demonstrated to induce senescence. Notably, oncogene-induced senescence (OIS) is an important tumor suppressive mechanism, preventing uncontrolled growth and replication of impaired genome. Senescent cells secrete numerous factors including pro-inflammatory cytokines, growth factors and matrix metalloproteinases, termed the senesecence-associated secretory phenotype (SASP), which impact tissue homeostasis and regeneration. The secreted factors also function to attract immune cells that can clear the damaged senescent cells.

Furthermore, by facilitating tissue remodeling, 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. Recent studies in transgenic mouse models of natural and accelerated aging demonstrated that removal of a percentage of senescent cells attenuated multiple age-related symptoms. Moreover, 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. Thus cellular senescence is a physiologically important process in cancer, aging and diseases and represents an excellent therapeutic target for improving human health.

There are several markers of cellular senescence, including senescence-associated beta-galactosidase (SA-p-gal), 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. Hence, 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. Thus, 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. In one embodiment, the extracellular vesicles are obtained from stem cells of a young or healthier subject. In another embodiment, 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. As used herein, the term "treating" encompasses the arrest of an otherwise progressive disease. The term also contemplates reversal or amelioration of the disease or condition. In particular, according to some embodiments, 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. Stem cell dysfunction and other age-related symptoms also can arise from 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. To administer the extracellular vesicle composition to recipient cells or tissues, 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.

EXAMPLES

Example 1 : Rejuvenation of ERCC1 -deficient MDSPCs by extracellular vesicles from young MDSPCs

Erccl -deficient Muscle derived stem/progenitor cells (MDSPCs) were plated at a density of 2.000 cells/well in a collagen type I-coated 12, -well plate. An imaging system (Automated Cell Technologies, Inc.) equipped with a 20^χ 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.

The 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).

Example 2: Characterization of exosomes from MSCs

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.

Conditioned media from young MSCs grown at 3% oxygen, or grown at 3% oxygen and then subjected briefly (48 hours) to oxidative stress at 20% oxygen (primed), was to senescent aged MSCs (Figure 6). 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. In addition, 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 3 10⁹ and, in particular 3 10¹⁰ 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).

Claims

CLAIMS WE CLAIM:

1. A method for treating a patient suffering from a disease or condition or age-related symptom that is caused by stem cell dysfunction or increased senescence, the method comprising administering to the patient a composition comprising extracellular vesicles obtained from stem cells of a subject that is younger or healthier than, and of the same species as, the patient.

2. A method for treating a patient suffering from a disease or condition or age-related symptom that is caused by stem cell dysfunction or increased senescence, the method comprising administering to the patient a composition comprising extracellular vesicles obtained from the serum of a subject that younger or healthier than, and of the same species as, the patient,

3. The method according to claim 1, wherein the stem cells are selected from one or more of muscle-derived stem cells and mesenchymal stem cells.

4. The method according to claim 2, wherein the stem cells are mesenchymal stem cells.

5. The method according to claim 4, wherein the mesenchymal stem cells are bone marrow derived mesenchymal stem, cells, adipose derived mesenchymal stem cells, or muscle derived mesenchymal stem cells.

6. The method according to claim 1 or 2, wherein the patient is a human.

7. The method according to claim 1 or 2, wherein the disease or condition is one selected from the group consisting of progeroid syndromes, obesity, idiopathic pulmonary fibrosis, and cellular senescence associated with DNA damage.

8. The method according to claim 7, wherein the disease or condition is a progeroid syndrome selected from the group consisting of Werner syndrome, Bloom syndrome,

Rothmund-Thomson syndrome, Cockayne syndrome, xeroderma pigmentosum,

trichothiodystrophy, combined xeroderma pigmentosum-Cockayne syndrome, restrictive dermopathy, and Hutchinson -Gil ford progeria syndrome.

9. The method according to claim 7, wherein the disease or condition is DNA damage caused by ionizing radiation or chemotherapy.

10. The method according to claim 1 or 2, wherein at least a portion of the extracellular vesicles contain RNA selected from the group consisting of messenger (mRN A), non-coding RN A (ncRNA), and combinations thereof,

11. The method according to claim 10, wherein at least a portion of the extracellular vesicles contain ncRNA.

12. The method according to claim 1 1, wherein the ncRNA is selected from the group consisting of micro-RNA (miRNA), transfer RNA (tRNA), nbosomal RNA (rRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA, long noncoding RNA (lncRNA), and combinations thereof.

13. The method according to claim 1 or 2, wherein the diameter of the extracellular vesicles is about 40 nm to about 200 nm.

14. The method according to claim 1 or 2, wherein the patient is a mammal.

15. The method according to claim 14, wherein the mammal is a human.

16. The method according to claim 1 or 2, wherein the composition is administered to the patient by injection.

17. A method for decreasing cellular senescence in a senescent cell from a first host organism, comprising contacting the cell with a composition comprising extracellular vesicles obtained from stem ceils or serum from a second host organism that is younger or healthier than, and of the same species as, the first host organism.

18. The method according to claim 17, wherein the contacting occurs in vitro.

19. The method according to claim 17, wherein the contacting occurs in vivo.

20. The method according to claim 17, wherein the decreasing cellular senescence is reversing cellular senescence.

21 . A method for delaying the onset of cellular senescence in a cell from a first host organism, comprising contacting the ceil 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.

22. The method according to claim 21, wherein the cell from the first host organism expresses senescence-associated beta-galactosidase (SA-p-gal).

23. The method according to claim 21, wherein the cell from the second host organism does not express SA-p-gal.