WO2020097440A1

WO2020097440A1 - Methods of predicting functional recovery of tissue using circulating exosomes derived from transplanted cells

Info

Publication number: WO2020097440A1
Application number: PCT/US2019/060436
Authority: WO
Inventors: Sunjay Kaushal; Progyaparamita SAHA; Sudhish SHARMA; Prashanth VALLABHAJOSYULA
Original assignee: University of Maryland Baltimore; University of Pennsylvania Penn; University of Maryland College Park
Current assignee: University of Maryland Baltimore; University of Pennsylvania Penn; University of Maryland College Park
Priority date: 2018-11-09
Filing date: 2019-11-08
Publication date: 2020-05-14
Anticipated expiration: 2021-05-09

Abstract

The stem cell field is hindered by the inability to noninvasively monitor transplanted cells within a target organ in a repeatable, time sensitive, and condition specific manner. It was hypothesized that the quantitation and intraexosomal cargo characterization of circulating transplanted cell specific exosomes would enable a reliable, noninvasive surveillance platform to reflect the conditional activity of their cellular counterparts. To test this hypothesis, use was made of a human-into-rat-xenogeneic myocardial infarction model involving two well-studied progenitor cell types: cardiosphere-derived cells (CDCs) and c-kit+cardiac progenitor cells (CPCs), derived from the same right atrial appendage of adult humans. To noninvasively monitor the activity of transplanted CDCs or CPCs in vivo, recipient plasma exosomes were purified using antibodies to human leukocyte antigen (HLA) surface molecules only expressed on human exosome surface. After 7 days post-transplantation, a 2.5 fold increase in concentration of plasma CPC-specific exosomes was observed when compared to CDC-specific exosomes. Computational pathway analysis failed to link CPC or CDC cellular mRNA with observed myocardial recovery. However, myocardial recovery was strongly linked to the miRNA cargo of CPC exosomes purified from recipient plasma. In addition, mechanistic pathways governing myocardial recovery were identified to specific outcomes by the transplanted CPCs. Collectively, these findings demonstrate the potential of circulating progenitor cell specific- exosomes as a liquid biopsy that provides a noninvasive window into the conditional state of the transplanted cells.

Description

METHODS OF PREDICTING FUNCTIONAL RECOVERY OF TISSUE USING CIRCULATING EXOSOMES DERIVED FROM TRANSPLANTED CELLS

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with government support under Grant No. HL118491 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF INVENTION

[0002] Stem/progenitor cell-based therapies have been developed that show efficacy in treating their targeted diseases in preclinical transplantation and tissue injury models. Some of these therapies have moved beyond preclinical testing and are now showing promising results in human clinical trials [1-4] For example, it has been demonstrated that cardiac progenitor cells (CPCs, c-kit+/Lin-) and cardiosphere-derived cells (CDCs) derived from human neonatal heart tissue reduce cardiac scar size, improve myocardial function, and repress adverse myocardial remodeling secondary to myocardial infarction (MI) in preclinical models when compared to adult-derived CPCs or CDCs [5, 6] Encouraging Phase I clinical trials using either CPCs or CDCs transplantation in adults with ischemic heart disease have been reported as well.

[0003] Despite these promising innovations, a major limitation to advancing clinical efficacy is the inability to noninvasively monitor transplanted cells and their therapeutic effects during the period of tissue (e.g., myocardial) remodeling [14, 15]

[0004] Of note, the components of the stem cell secretome, including exosomes, promote myocardial recovery through donor age-dependent pathways [5, 7-13] Recently, an in-depth proteomic analysis of the CPCs secretome was performed, demonstrating that a single intramyocardial injection of the exosomes derived from neonatal CPCs promoted myocardial recovery at a level similar to that observed following neonatal CPCs injection [5] These results, among others, suggest that at least part of the functional unit of CPCs or CDCs might, in fact, be their exosomes [16, 17]

[0005] Exosomes are extracellular nanovesicles released by many tissue types into body fluids, including blood, urine, and bronchoalveolar secretions [18-21] Other studies have identified the presence of circulating tissue-specific exosomes derived from transplanted solid organs in recipient plasma [22, 23] Interestingly, exosomes contain specific proteomic and RNA signatures that reflect the conditional and functional status of their cells of origin [19, 24] Given these attributes, exosomes are being actively investigated for their functional and diagnostic potential in many medical fields. Recently, it was demonstrated that circulating transplant tissue- specific exosome characterization enables noninvasive surveillance of transplanted solid organs in a time-sensitive, condition-specific manner [21, 25, 26]

[0006] The ability to noninvasively monitor transplanted stem/progenitor cells would be an important advance in developing treatments based on such cells. The use of exosomes in these types of surveillance might form the basis of effective monitoring protocols. The present invention is directed to developing exosome-based protocols and other important goals.

BRIEF SUMMARY OF INVENTION

[0007] The present invention has been realized via the work of the inventors reported herein, demonstrating for the first time that progenitor cell-specific exosomes are present in the circulation of subjects into which such cells have been transplanted. Provided in the Examples are the results of a head-to-head comparison in a xenogeneic model of rodent myocardial infarction (MI) that studied the cardiac regenerative potential of two well-studied progenitor cells, CDCs and CPCs, derived from the same human heart biopsy. The results show the monitoring potential of a stem cell-specific exosome platform, and demonstrate that intra- exosomal microRNA cargoes reflect the functional myocardial recovery achieved by the transplanted stem cells. Data from initial studies conducted in humans is provided as well.

[0008] It will be apparent that screening plasma samples for the presence of exosomes, or for a change in the number of exosomes over time, provides a clear indication that transplanted progenitor cells have survived transfer into recipient tissue and that the cells are present in the recipient. In the broadest sense, such screens form the basis of the present invention and the following initial group of embodiments are directed to methods based on this correlation.

[0009] In a first embodiment, the present invention is directed to methods of monitoring cells transplanted into a subject. The method comprises screening a biological sample obtained from a subject into whom cells have been transplanted for the presence of transplanted cell-derived exosomes. The presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject. An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells in the subject over time. Thus, monitoring of cells transplanted into the subject can be achieved.

[0010] In a second embodiment, the present invention is directed to methods of monitoring a subject receiving cell-based therapy. The method comprises screening a biological sample obtained from a subject receiving cell therapy for the presence of transplanted cell-derived exosomes. The presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject. An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells in the subject over time. Thus, monitoring a subject receiving cell-based therapy can be achieved.

[0011] In a third embodiment, the present invention is directed to methods for predicting functional recovery of ischemic myocardium in a subject into which cells have been

transplanted. The method comprises screening a biological sample obtained from a subject that has ischemic myocardium and into which cells have been transplanted for the presence of transplanted cell-derived exosomes. The presence of exosomes in the biological sample indicates functional recovery of the ischemic myocardium in the subject is more likely than a

corresponding subject in whom such exosomes are not present. An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells, which may aid in the functional recovery of the ischemic myocardium, in the subject over time. Thus, a prediction of functional recovery of ischemic myocardium in the subject can be achieved.

[0012] In each of these embodiments, the methods may further comprise enumerating the number of exosomes present in the biological samples.

[0013] In each of these embodiments, the methods may further comprise repeating the method at one or more additional time points and enumerating the number of exosomes present in the biological samples from each time point to determine whether there is a change in the number of exosomes over time.

[0014] In each of these embodiments, the transplanted cells may be allogeneic, that is, the transplanted cells may be obtained from an individual that is different from the subject into which the cells are transplanted. When the transplanted cells are allogeneic, exosome produced by the cells can be more easily identified in the biological sample. [0015] In each of these embodiments, the transplanted cells include, but are not limited to, stem cells and progenitor cells. In particular aspects, the cells may be one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).

[0016] In each of these embodiments, the subject is one into whom cells have been transplanted. The cells will have been transplanted into a target organ or into a target organ system in the subject. Suitable target organs include, but are not limited to, heart, lungs, kidneys, liver, pancreas, spleen, brain, bladder, or lymph nodes. Suitable target organ systems include, but are not limited to, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, muscular system, nervous system, reproductive system, and respiratory system.

[0017] In each of these embodiments, the biological sample may be screened within 6, 12,

18, 24, 30, 36, 42, 48, 54, 60, 66, 72 or more hours after completion of cell therapy or transplantation of the cells into the subject. Alternatively, in each of these embodiments, the biological sample may be screened within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.

[0018] In each of these embodiments, the biological sample may be, but is not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.) urine, peritoneal fluid, and pleural fluid. In one aspect, the biological sample is blood plasma.

[0019] In each of these embodiment, the biological samples may be screened using antibodies having binding specificity for molecules displayed by the exosomes. In certain aspects of the invention, the molecules may be, for example, leukocyte antigen surface molecules, including human leukocyte antigen (ELLA) surface molecules and human mismatch ELLA surface molecules.

[0020] In each of these embodiments, the subject may be a mammal, including, but not limited to, a human.

[0021] As discussed in the Examples provided herein, a link has been established between the microRNA (miRNA) cargo of exosomes and the functional activity of the transplanted cells from which the exosomes are derived. Therefore, in the following additional embodiments of the invention, the methods of the invention include profiling the contents of exosomes collected from the biological samples. For example, polynucleotides and polypeptides within the exosomes can be isolated and characterized.

[0022] In a fourth embodiment, the invention is directed to methods of profiling exosomes derived from cells transplanted into a subject. The method comprises (i) collecting, from a biological sample obtained from a subject into whom cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing cargo of the collected exosomes. Thus, profiling of exosomes derived from cells transplanted into a subject can be achieved.

[0023] In this embodiment, the cargo may be, but is not limited to, one or more of polynucleotides, polypeptides, and lipids. In certain embodiments, the cargo is polynucleotides, such as, but not limited to, microRNA.

[0024] In a fifth embodiment, the invention is directed to methods of profiling exosomes derived from cells transplanted into a subject. The method comprises (i) collecting, from a biological sample obtained from a subject into whom cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing microRNA cargo of the collected exosomes. Thus, profiling of exosomes derived from cells transplanted into a subject can be achieved.

[0025] In each of these embodiments, characterizing microRNA cargo may comprise sequencing one or more species of the microRNA present in the collected exosomes.

Characterizing microRNA cargo may alternatively comprise otherwise identifying one or more species of microRNA present in the collected exosomes. Characterizing microRNA cargo may also comprise screening the contents of the exosomes for the presence of one or more specific species of microRNA; in some aspects of the invention, the specific species of microRNA are known to be associated with one or more activities performed by the target organ.

[0026] In certain aspects of the invention, the cargo may screened for the presence of one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27- 5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR- 623, and miR-362-5p. In other aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR- 1224, and miR-l256. In further aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224. Suitable means for identifying one or more species of microRNA present in the collected exosomes include, but are not limited to, sequencing and hybridization to labeled probes.

[0027] In both of these embodiments, the methods may further comprise repeating the method at one or more additional time points and characterizing cargo from each additional time point to determine whether there is a change in the cargo over time.

[0028] In both of these embodiments, the cells may be allogeneic, that is, the cells may be obtained from an individual that is different from the subject into whom the cells are

transplanted. When the cells are allogeneic, exosome produced by the cells can be more easily identified in the biological sample.

[0029] In both of these embodiments, the transplanted cells include, but are not limited to, stem cells and progenitor cells. In particular aspects, the cells may be one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).

[0030] In both of these embodiments, the subject is one into which cells have been transplanted. The cells will have been transplanted into a target organ or into a target organ system in the subject. Suitable target organs include, but are not limited to, heart, lungs, kidneys, liver, pancreas, spleen, brain, bladder, or lymph nodes. Suitable target organ systems include, but are not limited to, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, muscular system, nervous system, reproductive system, and respiratory system.

[0031] In both of these embodiments, the exosomes may be collected within 6, 12, 18, 24,

30, 36, 42, 48, 54, 60, 66, 72 or more hours after completion of cell therapy or transplantation of the cells into the subject. Alternatively, in both of these embodiments, the exosomes may be collected within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.

[0032] In both of these embodiments, the biological sample may be, but is not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.) urine, peritoneal fluid, and pleural fluid. In one aspect, the biological sample is blood plasma.

[0033] In both of these embodiments, the exosomes may be collected using antibodies having binding specificity for molecules displayed by the exosomes. In certain aspects of the invention, the molecules may be, for example, leukocyte antigen surface molecules, including human leukocyte antigen (HLA) surface molecules and human mismatch HLA surface molecules.

[0034] In both of these embodiments, the subject may be a mammal, including, but not limited to, a human.

[0035] The Examples provided herein also establish a link between the microRNA (miRNA) cargo of exosomes and myocardial recovery. Therefore, in the following additional embodiments of the invention, the methods of the invention include predicting functional recovery of ischemic myocardium in a subject based on the miRNA cargo of exosomes collected from the biological samples.

[0036] In a sixth embodiment, the invention is directed to methods of predicting functional recovery of a target organ in a subject. The method comprises (i) collecting, from a biological sample obtained from a subject having a target organ into which cells have been transplanted, exosomes derived from the transplanted cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the target organ in the subject based on characteristics of the miRNA cargo, wherein the target organ is diseased, damaged, ischemic, or in some other manner malfunctioning. Thus, predicting functional recovery of target organ in a subject can be achieved.

[0037] In a seventh embodiment, the invention is directed to methods of predicting functional recovery of ischemic myocardium in a subject. The method comprises (i) collecting, from a biological sample obtained from a subject having ischemic myocardium and into which progenitor cells have been transplanted, exosomes derived from the transplanted progenitor cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the ischemic myocardium in the subject based on characteristics of the miRNA cargo. Thus, predicting functional recovery of ischemic myocardium in a subject can be achieved.

[0038] In this embodiment, characterizing microRNA cargo may comprise sequencing one or more species of the microRNA present in the collected exosomes. Characterizing microRNA cargo may alternatively comprise otherwise identifying one or more species of microRNA present in the collected exosomes. Characterizing microRNA cargo may also comprise screening the contents of the exosomes for the presence of one or more specific species of microRNA; in some aspects of the invention, the specific species of microRNA are known to be associated with one or more activities performed by the target organ.

[0039] In certain aspects of the invention, the cargo may screened for the presence of one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27- 5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR- 623, and miR-362-5p. In other aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR- 1224, and miR-l256. In further aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224.

[0040] In certain aspects of the invention, when one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27-5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR-623, and miR-362-5p is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.

[0041] In certain aspects of the invention, when one or more of miR-378b, miR-384, miR- 515-5p, miR-525-3p, miR-623, miR-94l, miR-l224, and miR-l256 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.

[0042] In certain aspects of the invention, when one or more of miR-378b, miR-384, miR- 515-5p, miR-525-3p, and miR-l224 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.

[0043] In certain aspects of the invention, when each of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which these miRNAs are not identified as being present in the collected exosomes. [0044] In this embodiment, the methods may further comprise repeating the method at one or more additional time points and characterizing miRNA cargo from each additional time point to determine whether there is a change in the miRNA cargo over time.

[0045] In this embodiment, the cells may be allogeneic, that is, the cells may be obtained from an individual that is different from the subject into which the cells are transplanted. When the cells are allogeneic, exosome produced by the cells can be more easily identified in the biological sample.

[0046] In this embodiment, the transplanted cells include, but are not limited to, stem cells and progenitor cells. In particular aspects, the cells may be one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).

[0047] In this embodiment, the subject is one into which cells have been transplanted. The cells will have been transplanted into a target organ or into a target organ system in the subject. Suitable target organs include, but are not limited to, heart, lungs, kidneys, liver, pancreas, spleen, brain, bladder, or lymph nodes. Suitable target organ systems include, but are not limited to, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, muscular system, nervous system, reproductive system, and respiratory system.

[0048] In this embodiment, the exosomes may be collected within 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 or more hours after completion of cell therapy or transplantation of the cells into the subject. Alternatively, in this embodiment, the exosomes may be collected within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.

[0049] In this embodiment, the biological sample may be, but is not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.) urine, peritoneal fluid, and pleural fluid. In one aspect, the biological sample is blood plasma.

[0050] In this embodiment, the exosomes may be collected using antibodies having binding specificity for leukocyte antigen surface molecules displayed by the exosomes. In certain aspects of the invention, the leukocyte antigen surface molecules are human leukocyte antigen (ELLA) surface molecules and human mismatch ELLA surface molecules. [0051] In this embodiment, the subject may be a mammal, including, but not limited to, a human.

[0052] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the

accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits of the invention.

BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1: Phenotypic characterization and cardiac functional assessment in rat myocardial infarction (MI) model after adult CPCs and CDCs transplantation. (A)

Schematic diagram of the isolation of adult CPC and CDCs. (B) Flow cytometry analysis of CPCs and CDCs for stem cell-specific surface markers (CD117, CD 105, CD90, CD45 and CD31) and cardiac lineage markers (NKX2.5, Troponin I and GATA4) and mast cell marker tryptase (n=6); (See also Fig. Sl). (C) Cell proliferation of CPCs and CDCs was assessed using Alamar blue assay (n=6). (D) Senescence was assessed using b-galactosidase assay at passage 3 (n=6) of both CPCs and CDCs. b-galactosidase stained blue cells (positive) were quantified (See also Fig. S2A). (E) Transwell migration assay with CPCs (n=4) and CDCs (n=4) at passage 3 was performed and migrated cells were quantified by Image-pro software. (See also Fig. S2B). (F-I) Structural and functional parameters derived from echocardiography measurements are shown at baseline and post-MI (n=8-l0). Four weeks post-MI, heart sections were stained for (J) Masson’s trichrome, (fibrosis (blue) and viable mass (pink)) in hearts injected with CPCs, CDCs and control (IMDM) for h=10. (K) Neovessels stained by IB4 expression, and arterioles marked by a-smooth muscle actin (a-SMA) expression in myocardial sections. Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI; blue) in all images (n=8). All data represented as mean ± SEM, *P<0.05, **P<0.0l, SEPJ* * *P<0.001 and ****P<0.000l . Data analyzed using 2-way ANOVA followed by Bonferroni post-tests (B, E, G, H, I, J), l-way ANOVA followed by Kruskal -Wallis test, t-test followed by Mann- Whitney’s analysis (C, D, K).

[0054] Figure 2: Functional assessment of medium (TCM) derived from CPCs, CDCs and mixed cell population in vitro and in vivo. (A) ELISA-based quantitative analysis of 7 paracrine factors secreted by CPCs and CDCs (n=6). (B-C) Representative images (left) of wound healing assay using human mammary endothelial cells (HMEC) and its quantification (right) by ImageJ software (n=6). Treatments included basal medium (serum/growth factors free), and the secretome of CPCs and CDCs. (D). Effect of growth medium on growth properties of CPCs and CDCs as assessed by onset of senescence (E) and cellular proliferation by Alamar blue (see also Fig. S3). (F) Wound healing assay composed of groups with increasing c-kit⁺ stoichiometry (20%, 40%, 80%) in CDCs populations including unaltered CPCs and CDCs (n=4); (see also Fig. S4). (G-H). Quantification of SDF-la and VEGFA with increasing percentage of c-kit⁺ cells in CDCs (n=4) by ELISA (n=4). (I-J). Cardiac parameters of ejection fraction and fraction shortening determined by echocardiographs at 24 hours and 28 days’ post injection of mixed cells populations along with unaltered CPCs and CDCs in a rat MI model (n=5-8). * <0.05, ** <0.0l, ***_p<o.OOl, and **** ^><().0001. Grouped data are analyzed using l-way ANOVA followed by Bonferroni post-tests, and 2-way ANOVA followed by Tukey post test (C, D, E, F, G,H I and J) and t-test followed by Mann-Whitney’s analysis (A).

[0055] Figure 3: Characterization of exosomes obtained from CPCs and CDCs both in vitro and from rat plasma after injection of CPCs or CDCs respectively. (A). Transmission electron microscope (TEM) visualization of exosomes obtained in vitro from CPCs and CDCs labeled with exosome marker CD63 using immunogold. (B) FACS verification of exosomes using anti PE CD63 antibody (n=4). (C) Measurement of exosome concentration and size by Nanosight in the secretome of CDCs and CPCs (n=4). (See also Fig. S5A). (D) HMEC cell proliferation assay performed in basal medium, or CPCs and CDCs derived exosomes (n=6) as indicated. (E) Trans well cells migration assay as performed in the, basal medium or CPCs and CDCs derived exosomes (n=6) as indicated (See also Fig. S5B). Wound healing assay with basal medium or CPCs and CDCs derived exosomes (n=5) and quantification were depicted in Fig. S5C. (F) Immunoblot for the stem cell marker and cardiac associated proteins in exosomes isolated from CDCs and CPCs (n=4) (see also Fig. S6). (G) CPCs and CDCs derived exosomes were analyzed for surface expression of HLA-A on light scatter (total exosomes) and

fluorescence modes (HLA-A, goat secondary Qdot 605) by NTA (Nanosight NS300) (n=7). (H) Exosomes from rat plasma were analyzed on NanoSight nanoparticle detector on light scatter (total exosomes) and fluorescence modes (HLA Qdot 605) for transplanted CPCs and CDCs derived exosomes using anti-HLA-A. Human exosomes were isolated from rat plasma obtained after transplanted myocardial injections in rat MI model (See also schematic diagram at Fig. S7) (n=8). (I) Quantification of human exosomes retained in rat plasma 7 days post-MI (n=8). (J) Immunoblot showed the characterizations of the human exosomes isolated from rat plasma on post-operative day (POD) 0, POD 2, and POD 7 using human HLA-A1, HLA-A, c-kit, Troponin I, cytochrome C and Flotillin 1 (n=4). (See also Fig. S8). * <0.05, ** <0.0l, ***_p<o.OOl, and ****/’<0.000l Data are analyzed using t-test followed by Mann- Whitney’s analysis (C) and 2- way ANOVA followed by Tukey’s post-tests (D,E, I).

[0056] Figure 4: Computational model of covariant microRNA using the cue-signal response paradigm. Computational model and the prediction of cardiac functions of exosomes miRNA cargo. (A) Principal component analysis (PCA). In vitro derived exosomes from

CPCs/CDCs along with rat plasma purified exosomes after CPCs/CDCs transplantation, were analyzed based on their matching miRNA expression levels. (B) Predictability measurements of cardiac functions. Predictive model was created using exosome microarray data from this study and Agarwal et al, 2017 to identify the predictability of ejection fraction (EF), fibrosis, and angiogenesis functions. Top 100 miRNA VIPs from Agarwal et al study, were selected and matched with plasma (blue bars) as well as in vitro (red bars) exosome CPCs and CDCs miRNAs individually. (C-E) The predicted plasma (red bars) and in vitro (blue bars) CPCs and CDCs functional outcomes were compared with the observed functional data (green bars) for EF (C) and angiogenesis (D) in comparison with fibrosis function (E). (F). Partial least squares regression (PLSR) and miRNA target analysis. Top microRNAs with known validated targets were identified among the 60 matched miRNAs using miRTarBase and plotted in PC space. Thirty-one miRNAs with validated targets were identified by miRTarBase. Clusters of miRNAs are formed based on the functional outcome. CPCs: cardiac progenitor cells; CDCs: cardiosphere-derived cells; VIP: variable importance for projection; EF: ejection fraction.

[0057] Figure 5: Verification of functional role of miRs as identified by computational analysis. (A). Quantitative PCR depicting the enrichment of individual miRs in HMECs after transfection with miR mimics. Cells were transfected with individual miRs. (See also Fig. Sll). (B) Cell proliferation assay using Alamar blue of the transfected cells with scrambled, miR 378, miR 384, miR 515, miR 525 and miR 1224. (C-D) Trans well migration assay of the transfected cells with scrambled, miR 378, miR 384, miR 515, miR 525 and miR 1224. (E-F) Wound healing assay of the transfected cells with scrambled, miR 378, miR 384, miR 515, miR 525 and miR 1224. * <0.05, ** <0.01, ***_p<0.00l, and ****_p<0.000l. Data are analyzed using one- way ANOVA followed by Mann- Whitney’s analysis (B, D, F).

[0058] Figure 6: Schematic of donor exosome purification and identification in rat plasma.

[0059] Figure 7: Computational modeling of EXOs miRNA cargo. (A) Principal component analysis (PCA). Patients (neonate, infant, child, N=3) along with human CPCs (N=5) and CDCs (N=3) were analyzed based on their mRNA expression levels. Results were plotted in principal component (PC) space. (B) Predictability measurements of angiogenesis functional outcome. PLSR model was created with the top 300 genes of only patients (neonate, infant, child) and this model determined the predictability of CPCs and CDCs functions. (C) Predictive model was created using cardiac progenitor cells (CPCs) and cardiosphere-derived cells (CDCs) mRNA extracted from serum combined with exosomal pediatric hypoxic and normoxic CPCs [57] using hypoxic/normoxic cardiac functional data. Predictability rates were high for EF and angiogenesis and slightly less for fibrosis. (D) Canonical pathway analysis. Ingenuity pathways analysis (IP A) was used to determine top canonical pathways for these genes. Cardiac and immune response related pathways are specified by orange and brown colors respectively.

[0060] Figure 8: Transplanted MSCs release donor-specific exosomes into the recipient circulation of the HLHS patients post-operative 2 and 7, but not seen preoperatively (pre). (A) Recipient plasma total exosome pool was analyzed on NanoSight nanoparticle detector on light scatter (blue) or fluorescence mode (red) for donor MSC specific MHC signal using anti- HLA-A (n=2). (B) Total HLA-A expressing plasma exosome numbers were quantified on the NanoSight and expressed as number of nanoparticles per milliter per microgram of exosome protein at different times postoperatively (day 2 and 7) and preoperatively (pre).

DETAILED DESCRIPTION OF THE INVENTION

I Definitions

[0061] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN

0471186341); and other similar technical references.

[0062] As used herein,“a” or“an” may mean one or more. As used herein when used in conjunction with the word“comprising,” the words“a” or“an” may mean one or more than one. As used herein“another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0063] As used herein,“about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term“about” generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term“about” may include numerical values that are rounded to the nearest significant figure.

II The Present Invention

[0064] Although many cell-based therapies have been proposed for the treatment of heart failure, few clinical trials have demonstrated efficacy using the strategy of ex vivo expansion and reintroduction of cells into the injured myocardium [14, 15, 47-49] Attempts have been made to quantify exogenous cells in the recipient myocardium following transplantation using a PCR technique, but this technique cannot be applied to humans [7] The field therefore lacks a method to monitor, in real-time, the presence or remodeling abilities of therapeutic stem/progenitor cells. A noninvasive platform enabling surveillance of the cellular footprint and identification of the pathways triggered by stem/progenitor cells could facilitate the development of new therapeutic approaches for cardiac regeneration in humans. Due to the condition-specific and dynamic nature of the quantitative and cargo profiles, tissue specific exosomes are being investigated for their potential as diagnostic biomarkers and as therapeutic modulators in cardiovascular disorders [50- 52]

[0065] As reported herein, the potential of transplanted stem cell/progenitor cell-derived exosome characterization was investigated as a noninvasive tool to monitor their cellular counterpart’s presence and function in vivo. This is the first report to demonstrate that 1) transplanted human cardiac progenitor cells release HLA-specific exosomes into the recipient circulation, 2) transplant progenitor cell-derived exosomes can be isolated, quantified, and profiled noninvasively from the recipient plasma, 3) stem/progenitor cell-based remodeling of the myocardium may lead to changes in their circulating exosome signal quantity, and 4) miRNA signatures of progenitor cell-derived exosomes may reflect the mechanistic pathways in the tissue microenvironment during myocardial recovery. For these reasons, stem/progenitor cell specific exosomes can serve as a liquid biopsy of the functional stem/progenitor cell mass.

Screening Biological Samples for the Presence of Exosomes

[0066] At a base level, the present invention provides methods directed to screening biological samples from subjects into which cells have been transplanted or otherwise transferred for the presence of exosomes, or for a change in the number of exosomes over time. Such methods provide a clear indication that transplanted cells have survived transfer into recipient tissue and that the cells are present in the recipient.

[0067] Thus, and in one embodiment, the invention is directed to methods of monitoring cells transplanted into a subject. The method comprises screening a biological sample obtained from a subject into which cells have been transplanted for the presence of transplanted cell- derived exosomes. The presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject. An increase/decrease in the number of transplanted cell- derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells in the subject over time. Thus, monitoring of cells transplanted into the subject can be achieved. [0068] In another embodiment, the invention is directed to methods of monitoring a subject receiving cell-based therapy. The method comprises screening a biological sample obtained from a subject receiving cell therapy for the presence of transplanted cell-derived exosomes. The presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject. An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells in the subject over time. Thus, monitoring a subject receiving cell-based therapy can be achieved.

[0069] In a further embodiment, the invention is directed to methods for predicting functional recovery of ischemic myocardium in a subject into which cells have been

[0070] In each these embodiments, the methods may further a step of enumerating the number of exosomes present in the biological samples, whether based on the specific number of exosomes in a biological sample or based on the weight or volume of cells in the sample.

[0071] In each of these embodiments, the methods may further comprise repeating the method at one or more additional time points and enumerating the number of exosomes present in the biological samples from each time point to determine whether there is a change in the number of exosomes over time. For example, the method can be conducted on a subject within a couple of days of receiving progenitor cells, and then a week, month, etc. later to determine whether the transplanted cells continue to survive in the subject. Profiling Exosomes

[0072] At a second level, the present invention includes profiling the contents of exosomes collected from the biological samples. As discussed herein, a link has been established between the cargo of exosomes and the functional activity of the transplanted cells from which the exosomes are derived. Therefore, the methods of the invention include profiling the contents of exosomes collected from the biological samples. For example, polynucleotides and polypeptides within the exosomes can be isolated and characterized.

[0073] Thus, and in one embodiment, the invention is directed to methods of profiling exosomes derived from cells transplanted into a subject. The method comprises (i) collecting, from a biological sample obtained from a subject into which cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing cargo of the collected exosomes. Thus, profiling of exosomes derived from cells transplanted into a subject can be achieved.

[0074] In this embodiment, the cargo may be, but is not limited to, one or more of polynucleotides, polypeptides, and lipids. In certain embodiments, the cargo is polynucleotides, such as, but not limited to, microRNA.

[0075] In another embodiment, the invention is directed to methods of profiling exosomes derived from cells transplanted into a subject. The method comprises (i) collecting, from a biological sample obtained from a subject into which cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing microRNA cargo of the collected exosomes. Thus, profiling of exosomes derived from cells transplanted into a subject can be achieved.

[0076] In each of these embodiments, characterizing the cargo may be via means that include, but are not limited to, identifying specific types of cargo contained in the exosomes, e.g. the polynucleotide, polypeptide, or lipid species within the exosomes, and identifying specific species of polynucleotide, polypeptide, or lipid within the exosomes. The skilled artisan will readily understand that a variety of means are available for performing such characterizations that include, but are not limited to, Northern, Southern and Western blots, sequencing, NMR analysis, HPLC analysis, immunologic analysis, etc. [0077] When the cargo is a polynucleotide, the polynucleotide may be sequenced in order to determine its identity or subject to hybridization with an labeled probe, to name only two of the multitude of means for identifying a polynucleotide molecule.

[0078] When the cargo is one or more microRNA, characterizing the microRNAs may comprise sequencing one or more species of the microRNA present in the collected exosomes. Characterizing microRNA may alternatively comprise otherwise identifying one or more species of microRNA present in the collected exosomes. Characterizing microRNA cargo may also comprise screening the contents of the exosomes for the presence of one or more specific species of microRNA; in some aspects of the invention, the specific species of microRNA are known to be associated with one or more activities performed by the target organ.

[0079] In certain aspects of the invention, the cargo may screened for the presence of one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27- 5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR- 623, and miR-362-5p. In other aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR- 1224, and miR-l256. In further aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224.

[0080] In both of these embodiments, the methods may further comprise repeating the method at one or more additional time points and characterizing cargo from each additional time point to determine whether there is a change in the cargo over time. For example, the method can be conducted on a subject within a couple of days of receiving progenitor cells, and then a week, month, etc. later to determine whether the contents of the cargo have changed over time.

Predicting Myocardial Recovery

[0081] At a third level, the present invention is extended to include predicting functional recovery of diseased, damaged, or ischemic organs or tissues in a subject into which cells have been transplanted based on the miRNA cargo of exosomes collected from biological samples obtained from the subject. As provided in the Examples herein, a link between the microRNA (miRNA) cargo of exosomes and myocardial recovery has been established which demonstrates that the miRNA signature of transplanted cell-derived exosomes may reflect the mechanistic pathways in the tissue microenvironment during myocardial recovery. By characterizing miRNA cargo of the collected exosomes, for example determining whether specific miRNAs known to be associated with improve cardiac function are present, predictions as to whether functional recovery of ischemic myocardium in a subject will be achieved can be made. Exemplary miRNAs are those associated with enhanced angiogenesis, cellular proliferation, cellular migration, and wound healing.

[0082] Therefore, in the following additional embodiments of the invention, the invention is directed to methods of predicting functional recovery of a target organ in a subject. The method comprises (i) collecting, from a biological sample obtained from a subject having a target organ into which cells have been transplanted, exosomes derived from the transplanted cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the target organ in the subject based on characteristics of the miRNA cargo, wherein the target organ is diseased, damaged, ischemic, or in some other manner malfunctioning. Thus, predicting functional recovery of a target organ in a subject can be achieved.

[0083] The methods of the invention include predicting functional recovery of ischemic myocardium in a subject based on the miRNA cargo of exosomes collected from the biological samples. The invention is directed to methods of predicting functional recovery of ischemic myocardium in a subject. The method comprises (i) collecting, from a biological sample obtained from a subject having ischemic myocardium and into which cells have been

transplanted, exosomes derived from the transplanted cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the ischemic myocardium in the subject based on characteristics of the miRNA cargo. Thus, predicting functional recovery of ischemic myocardium in a subject can be achieved.

[0084] The skilled artisan will readily understand that a variety of means are available for performing characterization of miRNA cargo that include, but are not limited to, sequencing the miRNA species obtained from the exosome and subjecting the miRNA species to hybridization with labeled probes, to name only two of the multitude of means for identifying polynucleotide molecules.

[0085] In this embodiment, characterizing microRNA cargo may thus comprise sequencing one or more species of the microRNA present in the collected exosomes. Characterizing microRNA may alternatively comprise otherwise identifying one or more species of microRNA present in the collected exosomes. Characterizing microRNA cargo may also comprise screening the contents of the exosomes for the presence of one or more specific species of microRNA; in some aspects of the invention, the specific species of microRNA are known to be associated with one or more activities performed by the target organ.

[0086] In certain aspects of the invention, the cargo may screened for the presence of one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27- 5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR- 623, and miR-362-5p. In other aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR- 1224, and miR-l256. In further aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224.

[0087] In certain aspects of the invention, when one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27-5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR-623, and miR-362-5p is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.

[0088] In certain aspects of the invention, when one or more of miR-378b, miR-384, miR- 515-5p, miR-525-3p, miR-623, miR-94l, miR-l224, and miR-l256 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.

[0089] In certain aspects of the invention, when one or more of miR-378b, miR-384, miR- 515-5p, miR-525-3p, and miR-l224 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.

[0090] In certain aspects of the invention, when each of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which these miRNAs are not identified as being present in the collected exosomes.

[0091] In this embodiment, the methods may further comprise repeating the method at one or more additional time points and characterizing miRNA cargo from each additional time point to determine whether there is a change in the miRNA cargo over time.

[0092] In each embodiment, aspect and method of the invention, the transplanted cells may be autologous, syngeneic, allogeneic or xenogeneic cells, when consider in the context of the subject receiving the cells. However, the transplanted cells will commonly be allogeneic. When the transplanted cells are allogeneic, exosome produced by the cells can be more easily identified in the biological sample.

[0093] In each embodiment, aspect and method of the invention, the transplanted cells include, but are not limited to, stem cells and progenitor cells. The term“stem cells” refers to biological cells having the potential to differentiate into other types of cells and that retain the ability to divide to produce more of the same type of stem cell. Stem cells include embryonic stem cells and adult stem cells. As used herein“progenitor cells” refers to biological cells that are descendants of stems cells that differentiate into a specific type of cell. Progenitor cells are more limited than stem cells in their ability to divided. Suitable cells for use in the methods of the invention include, but are not limited to, one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).

[0094] In each embodiment, aspect and method of the invention, the term“biological sample” refers to a sample of biological tissue, cells, or fluid that may comprise exosomes and that can be obtained from a subject and screened for the presence of exosomes. Suitable biological samples include, but are not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.) urine, peritoneal fluid, pleural fluid, and the like. In one aspect, the biological sample is blood plasma. The sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. For example, such pretreatment may include preparing plasma from blood, diluting viscous fluids and so forth. Methods of pretreatment may also involve, but are not limited to, filtration, precipitation, dilution, distillation, mixing, centrifugation, freezing, lyophilization, concentration, inactivation of interfering components, the addition of reagents, lysing, etc.

[0095] As used herein, the term“transplanted cells” refers to one or more individual cells (e.g. a stem cell or progenitor cell, such as a cardiac progenitor cell (CPC), cardiosphere-derived cell (CDC), mesenchymal stem cell (MSC), bone marrow cell (BMC) or embryonic stem cell (ESC)) that has been isolated from its endogenous tissue or organ before being introduced into a subject in need thereof. “Transplanted cells” does not include a transplanted organ or a tissue from an organ.

[0096] As used herein, the term“transplanted cell-derived exosomes” as meaning exosomes derived from one or more cells that have transplanted into a subject in need of such cell transplantation.

[0097] In each of the relevant embodiments, aspects and methods of the invention, the biological sample may be screened within 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 or more hours after completion of cell therapy or transplantation of the cells into the subject.

Alternatively, the biological sample may be screened within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.

[0098] In each of the relevant embodiments, aspects and methods of the invention, the exosomes may be collected within 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 or more hours after completion of transplanted cell therapy or transplantation of the cells into the subject.

Alternatively, the exosomes may be collected within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.

[0099] In each embodiment, aspect and method of the invention, the biological samples may be screened for the presence of exosomes using antibodies having binding specificity for molecules displayed by the exosomes using a variety of means, including, for example, flow cytometry, enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay, magnetic immunoassay, radioimmunoassay, fluorescent immunosassay, Western immunoblot assay, dot immunoblot assay, slot immunoblot assay, and a particle analyzer (e.g., Nanosight) which detects nano-particles, etc.

[00100] In each embodiment, aspect and method of the invention, the exosomes may be isolated from a biological sample using well-known techniques such as flow cytometry, immunosorbent plates and columns, ultracentrifugation, immune precipitation, etc. [00101] In each embodiment, aspect and method of the invention, the molecules displayed by the exosomes may be, for example, leukocyte antigen surface molecules, including human leukocyte antigen (HLA) surface molecules and human mismatch HLA surface molecule.

[00102] In each embodiment, aspect and method of the invention, the subject is one into which progenitor cells have been transplanted. The progenitor cells will have been transplanted into a target organ or into a target organ system in the subject. Suitable target organs include, but are not limited to, heart, lungs, kidneys, liver, pancreas, spleen, brain, bladder, or lymph nodes. Suitable target organ systems include, but are not limited to, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, muscular system, nervous system, reproductive system, and respiratory system.

[00103] In each embodiment, aspect and method of the invention, the subject is a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.

IV. Examples

Materials and Methods

Human tissue samples and cell culture

[00104] The Institutional Review Board and the Institute of Animal Care and Use Committee at the School of Medicine, University of Maryland, approved this study. After patient consent was given, specimens (70±80 mg) from the right atrial appendage (RAA) were obtained from 14 adult patients having coronary artery disease during coronary artery bypass grafting. These tissue samples were cut into two equal halves: one half was processed for isolating c-kit⁺ cardiac progenitor cells (CPCs) and another half was used for generation of human cardiosphere-derived cells (CDCs ) via the cardiosphere development. Paired CPCs and CDCs cell lines derived from the same heart biopsies (as described above) were randomly chosen, with each biological paired sample treated as N=l .

Generation of c-kit+/CD45- cardiac progenitor cells and cardiosphere derived cells

[00105] c-kit⁺/CD45 CPCs were isolated from RAA biopsies of adult myocardium using a previously described protocol [6,28] Briefly, samples were minced and digested in Ham’s F12 (Lonza # 12-615F) basal medium containing 1-2 mg/ml of collagenase type II (Worthington # 4177) on an orbital shaker for 45 min at 37°C. Following the collagenase treatment, cells were washed twice with complete growth medium (Ham’s F12, Fetal Bovine Serum (10%), recombinant human FGF-basic (10 ng/ml), L-glutathione (0.2 mM), human erythropoietin 250 (5 U/ml)) before being plated. At sub-confluency, cells were trypsinized and sorted for c-kit⁺ cell surface antigen with Miltenyi microbeads (CD117 MicroBead Kit human #130-091-332) as per the manufacturer’s instructions. Consequently, c-kit⁺ cardiac progenitor cells (CPCs) were collected and cultured in growth medium [5]

[00106] Human CDCs were generated according to the protocol described by Smith, et al ., with modifications, as mentioned previously [6,28] Briefly, RAA biopsies are diced into small pieces (1.0 to 2.0 mm in diameter) and digested with digestion cocktail (Dispase (0.85 mg/ml), Collagenase Type II (1 mg/ml) and trypsin (0.05%) for 15 minutes at 37°C in orbital shaker for three times. Explants were collected in CDCs complete growth medium (Iscove’s Modified Dulbecco’s Media (IMDM), 20% FBC (heat inactivated), 100 mM b-mercaptoethanol) and plated in Fibronectin coated flasks. After 2 to 3 weeks, the phase-bright cells originating from explants were removed by mild trypsinization and plated on fibronectin coated flasks at low density (1.5 to 3 x lO⁴ cells/mL) in cardiospheres-growing medium (CGM: FBS (3.5%,), IMDM (35%), Pen/Strep (1%), Glutamine (1%), B27 Serum substitute (2%), Cardiotroponin I (4 ng/ml), Epidermal Growth Factor (25 ng/ml), human basic Fibroblast Growth Factor (80 ng/ml,

Thrombin (1 El/ml), b-mercaptoethanol (100 pM) and (DMEM/Ham’s F12: 1 : 1 (65%)). After a week of culture, the phase-bright cells formed cardiospheres. After 24 hours, floating

cardiospheres were collected by centrifugation and expanded on fibronectin coated flasks in CDCs complete growth medium to obtain cardiosphere derived cells (CDCs).

Flow cytometric analysis

[00107] c-kit⁺/CD45 cells derived from adult patients at passage 4 for both CPCs and CDCs were labeled with fluorochrome-conjugated primary antibodies: mesenchymal stem cell marker CD 105 or CD90, cardiac-specific transcription factors NKX2.5, GATA4, cardiac stem cell marker c-kit⁺, endothelial cell marker CD31, mast cell marker tryptase, hematopoietic cell lineage markers CD45 and CD34. Conjugated isotype antibodies were used as negative controls. The labeled cells were evaluated by flow cytometry with a Becton -Dickinson FACS Calibur (San Jose, CA), with 25,000 events/sample collected. Cell transplantation and echocardiography

[00108] Myocardial infarction was induced by permanent ligation of the left anterior descending (LAD) coronary artery in athymic nude male rats (weight, 250-300 g). The heart was exposed via a left thoracotomy, and the proximal LAD was ligated. Subsequently, 1 million aCPCs or aCDCs suspended in 100 pL of vehicle (IMDM) were injected into the myocardium at four sites adjacent to the infarct. Transthoracic echocardiograms were acquired 1 day, 7 days, and 28 days after myocardial infarct surgery. Cardiac function was evaluated based on echocardiographic parameters [5] M-mode images of the LV in the parasternal short-axis view were obtained at the level of the papillary muscles using high-resolution ultrasound (Vevo 2100: VisualSonics, Toronto, Canada) equipped with a 20-MHz scan head for calculation/estimation of LV fraction shortening and LV ejection fraction. Data were calculated from 5 cardiac cycles according to the generally accepted formula [56]

Myocardial histology

[00109] Tissues were processed as previously described [5,6,28] Briefly, rat hearts were excised under anesthesia after collection of echocardiographic data and perfused with 4% paraformaldehyde. Tissues were cryo-preserved using 30% sucrose and embedded in OCT (TissueTek). Sections were cut to 7 pm using a cryostat and immunostained for isolectin B4 (Invitrogen; Carlsbad, CA), a-SMA (Sigma; St. Louis, MO), sarcomeric a-actin (Sigma), human nuclear antigen (HNA, Millipore; Billerica, MA), human mitochondrial antigen (HMA,

Millipore; Billerica, MA) or Ki67 (Santa Cruz Biotechnology; Santa Cruz, CA). Cells and tissue sections were counterstained with DAPI (4,6-diamidino-2-phenylindole) nuclear stain (Sigma; St Louis, MO).

Myocardial viability

[00110] To calculate infarct size, Masson trichrome-stained sections at various levels along the long axis were analyzed for collagen deposition. The midline technique for infarct size determination was used as previously described [27] The stained sections were analyzed by Image-Pro software [27] To calculate the amounts of viable and non-viable tissue, the number of pink pixels (viable tissue) and blue pixels (non-viable tissue) were measured and the ratio of non-viable tissue/overall number of the pixels was presented. 6 sections per animal and at least 15 animals per group were analyzed.

Preparation of cell mixtures with increasing stoichiometry of CPCs in CDCs populations

[00111] CPCs and CDCs are mixed together in the proportion of 20%, 40% and 80% of CPCs in the CDCs population for in vivo and in vitro studies. CDCs populations inherently contain 10% c-kit⁺ cells, which was factored into the proportions presented below, and which is why artificial mixtures started from 20% CPCs in CDCs. One million adult CPCs and CDCs suspended in 100 pL of vehicle (IMDM) were injected into the rat myocardium as a control. In case of 20% CPCs group (2 x 10⁵ CPCs + 8 x 10⁵ CDCs), 40% CPCs group (4 x 10⁵ cells from CPCs + 6 x 10⁵ cells from CDCs), 80% (8 x 10⁵ cells from CPCs + 2 x 10⁵ cells from CDCs) totaling 1 million cells (in 100 pL) were injected in the rat myocardium.

Paracrine factor quantification

[00112] Adult CPCs and CDCs at passage three (P3) were grown in complete media until they reached 85% confluence (~l x 10⁶ cells) in a T-25 flask. The conditioned medium (CM) was collected and filtered through 0.22 pm filters and concentrated using 3 kDa filters (Millipore Inc, Billerica, MA). Protein content was quantified using the Pierce bicinchoninic assay (BCA) method (Thermo Fisher; Waltham, MA). To normalize the protein content, the following formula was used: (concentration factor) x (total volume of medium)/(total protein content) of conditioned medium [5] The conditioned medium was quantified using BCA method and normalized to a total of 1 mg protein. ELISA was performed for human VEGFA, SDF-la, PDGFB, IGF-l, ANG-l, bFGF, and HGF in the core facility at the University of Maryland School of Medicine using human-specific ELISA kits (Millipore and R&D systems), according to the manufacturers’ protocols.

Trans-well migration assay

[00113] CPCs and CDCs were placed on the upper layer of a cell culture insert with permeable fluorescence block (8.0 um pore size, Cat # 351152) membrane and the media with serum and without serum are placed below the cell permeable membrane in a 24 well cell culture plate (Cat # 353504). Following an incubation period (6-7hours) at 37°C, the cells that migrated through the membrane were stained with Calcein (Calcein AM C3100MP, Thermo Fisher;

Waltham, MA), imaged as whole wells by an automated EVOS microscope, and quantified by Image-Pro software. In case of endothelial migration assay by HMECs, exosomes (50 ng) isolated from CPCs and CDCs were incubated along with the HMECs on the cell culture insert (upper chamber). After 3 hours of the incubation, migrated cells were imaged by EVOS Systems (Thermofisher, Inc) and quantified using Image Pro software.

Alamar blue cell proliferation assay

[00114] Cell proliferation was assessed using Alamar blue as per manufacturer’s instructions (Alamar Blue 10% of the total volume of the medium). Briefly, 5000 cells/well were seeded in 96 well plates in their respective medium. After overnight incubation at 37°C, 10 ul of Alamar- blue cell viability reagent (Invitrogen cat # 1933424) was added per well and absorbance was taken immediately (basal absorbance) and after 3 hours (proliferation absorbance) of incubation at 37°C. To obtain the actual absorbance, basal absorbance was subtracted from proliferation absorbance.

Senescence-associated b-galactosidase staining

[00115] Cellular senescence was identified as described previously [5] Briefly, adult CPCs and CDCs (5.0 x 10⁴) were plated in a 24 well plate at P6. A b-galactosidase staining kit (Cat #9860, Cell Signaling Technology; Danvers, MA) was used to stain the enzyme using the manufacturer’s protocol. Cell growth medium was removed from the cells; cells were rinsed with PBS and fixed with fixative solution (4% paraformaldehyde) for 15 minutes at room

temperature. Cells were incubated with 1 ml of b-galactosidase staining solution. The b- galactosidase positive (blue) cells were imaged in EVOS microscope (20x). The number of positive cells was quantified by Image Pro software.

In vitro wound healing assay

[00116] An in vitro scratch assay was performed to assess the relative migratory potential of CPCs, CDCs, and several mixtures of CPCs in CDCs. Cells were seeded in a l2-well plate to create a confluent monolayer. After 12 hours of serum starvation with basal medium, a 1 mL pipette tip was used to create linear scratches along the cell monolayer to simulate a wound. Cell debris was removed by washing the cells once with basal medium. Images of each wound were taken at specific reference points along the scratch at times 0 h and 6-7 h after treatment. Image- Pro software was used to measure the total wound area before and after treatment to calculate percentage change in wound closure. For endothelial wound-healing assay, HMEC was seeded in 24 well to create confluent monolayer. HMEC scratches were treated with basal medium

(MCDB 131) or basal medium containing exosomes derived from CPCs or CDCs. Cells were fixed in their wells after 16 hours; migrated distances were calculated using image Pro software.

Immunoblotting

[00117] Exosome proteins were separated using NuPAGE 4-12% Bis-Tris Gels and transferred onto nitrocellulose membranes (Life Technologies, CA, EISA). The blots were blocked with 5% non-fat dry milk at room temperature for 1 hour and incubated overnight at 40°C with desired primary antibodies at concentration per manufacturer’s protocol, followed by incubation with HRP-conjugated secondary antibodies (Santa Cruz Biotechnologies Inc.) at room temperature for 1 hour. The membrane blots were developed with ECL detection reagent (Luminata Forte, Millipore Corporation, Billerica, MA) per manufacturer’s protocol and detected through Chemiluminescence using Image quant LAS 400 Phospho-Imager (GE Health, USA).

As a standard protein marker, Thermo Scientific PageRuler Plus Prestained protein Ladder (# 26619) was used. Antibodies specific to c-kit (Cat# 18696-1-AP, Protein Tech Labs), Troponin-I (ab56357, Abeam), HLA-A (ab52922, Abeam), Flotilin-l (3253, Cell Signaling Technology), HLA-A1 (BIH0331, One Lambda Inc), NKX2.5 (SC-376565) (according to company’s data sheet this antibody recognizes two bands in some cell lines), CD-63 (Sc-7080), and Cytochrome- C (Sc-l3 l56) were purchased from Santa Cruz Biotechnology, Inc.

Affinity antibody coupled purification of human specific exosomes

[00118] HLA-A specific antibodies were covalently conjugated to N-Hydroxy Succinamide magnetic beads (NHS beads, Pierce Inc.) as per manufacturer’s suggestions. 50 pg protein equivalent of exosomes were incubated with antibody -magnetic-beads complex for overnight at 4°C on a rocker platform. The bead bound exosomes were washed using PBS and eluted using manufacturer’s protocol and utilized for downstream analysis. Isolation procedure of exosomes and exosomal micro RNA

[00119] Exosomes were isolated from CPC and CDC conditioned media (48 hours) by size exclusion chromatography and micro RNA immediately isolated from the exosomes using Exo RNeasy kit (Cat# 77023, Qiagen Inc.) as per manufacturer instruction. Total RNA was quantified on a NanoDrop ND-1000 spectrophotometer followed by RNA quality assessment on an Agilent TapeStation. Micro RNA labeling was performed by FlashTag Biotin HSR RNA Labeling Kit (Applied Biosystems). GeneChip miRNA Arrays 4.0 Arrays were hybridized with Flash Tag Biotin Labeled total RNA (100 ng) from experimental and control samples in 100 pl

hybridization cocktail. Target denaturation was performed at 99°C for 5 min. and then 45°C for 5 min. followed by hybridization for 18 hrs at 48°C. Arrays were washed and stained using Genechip Fluidic Station 450 according to protocol. Chips were scanned on an Affymetrix Gene Chip Scanner 3000, using Command Console Software. These studies were performed at Cancer Genomics Laboratory of the Thomas Jefferson University.

Nanosight particle analysis for exosomes

[00120] Exosomes from CDCs and CPCs were isolated and analyzed as previously described [22] Briefly, exosomes were isolated using by size exclusion chromatography using a Sepharose 2B column (Sigma-Aldrich) and eluted fractions were analyzed using nanosight NS300 (405 nm laser diode) for the presence of 40-120 nm diameter vesicles. For cell based in vitro assays, exosomes from CDCs and CPCs were used at the constant number 0.5 x 10⁹/100 ul, equivalent to 10 ng/lOO ul proteins. The surface expression of HLA class I was analyzed using exosomes (2 x 10⁸) incubated with anti-HLA class I (0.5 ug, Cat #311402, Bio Legend) for 2 hrs. Thereafter, goat anti-mouse Qdot 605 (1 :50 dilution, Q-11001MP, Thermo Fisher) was added as fluorescent secondary antibody and incubated for 2 hours. The unbound primary and secondary antibodies were removed using Exosquick plus (EQPL10A-1, System Biosciences) exosome isolation kit according to the manufacturer protocol. Total exosomes were counted in bright field emission and the HLA class I labeled exosomes were counted using fluorescent emission in Nanosight. HLA-specific exosome signal was quantified using following formula: (HLA Flourescence/HLA light scatter) - (POD 0 Flourescence/POD 0 light scatter) - (IgG isotype Flourescence/IgG isotype light scatter). miRNAs mimic transfections

[00121] To identify the functional role of shortlisted miRNAs, human miRIDIAN mimics (miRNA 378, miRNA 384, miRNA 515-5p, miRNA 525-3p and miRNA 1224) along with the transfection control -Dy547 (cat # CP-004500-01-05), positive control (cat # CP-001000-02-05) and scrambled (non-targeting) miR, were procured from Dharmacon. Cells were transfected with 50 nM of each miR mimic using reverse transfection protocol of lipofectamine RNAiMAX® (cat # P/N 100014472).

Partial least-squares regression modeling

[00122] Principal component (PC) analysis and partial least squares regression (PLSR) were performed using SIMCA-P software (UMetrics, now part of Sartorius Stedim Biotech;

https://umetrics.com) that solves the PLSR problem with the nonlinear partial least squares algorithm [57] Gene and miRNA data were analyzed by ingenuity pathway analysis (IP A). miRTarBase was used to identify miRNAs with known targets (validated by at least 3 assays, http://mirtarbase.mbc.nctu.edu.tw).

Transmission electron microscopy

[00123] Exosomes were negatively stained after absorption onto carbon-coated copper grids for 2 minutes. Grids were washed twice for 1 minute each in dELO and stained for 1 minute with 1% aqueous uranyl acetate (Ted Pella; Redding, CA). Samples were viewed on a JEOL 1200EX transmission electron microscopy (JEOL USA; Peabody, MA) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques; Woburn, MA). For immunogold labeling of exosomes, grids were incubated with mouse anti-CD63 antibody (AB193349, Abeam), for 30 minutes, followed by secondary goat anti-mouse IgG Ab conjugated to colloidal gold (Jackson Immuno Research Laboratories; West Grove, PA) for 30 minutes. Grids were washed and stained with uranyl acetate and viewed by transmission EM as described above.

Statistical analyses

[00124] Data were analyzed using GraphPad Prism 7 software. When comparing two conditions, Student’s t-tests with Mann-Whitney’s test were used. More than two comparisons were made using one-way analysis of variance (ANOVA) followed by Dunn’s or Tukey’s post hoc test. Two-way ANOVA with Bonferroni correction was used for grouped analysis of echocardiographic data. P-values of less than 0.05 were considered significant and two-sided tests were performed. Data are represented as mean ± standard error of the mean (SEM).

Other Methods

[00125] Immunoblotting, induction of MI, cell transplantation, heart function, and histological analysis were performed as previously described [27,28]

Results

Functional characteristic of CPCs and CDCs in vitro and in vivo

[00126] Human myocardial biopsies were obtained from the right atrial appendage (RAA) at the time of cardiac surgery from adult patients (68+10 years) undergoing coronary artery bypass grafting for severe atherosclerotic disease. Adult CPCs and CDCs were isolated from the same RAA biopsy sample using a previously modified protocol involving explant plating, selection processing, and expansion [5,6] (Fig. 1A). At passage 3 (P3), CPCs and CDCs were

characterized for cell surface markers and had similar cellular morphologies as previously described for each cell type (Fig. IB) [5,6] Both cell types expressed mesenchymal stem cell markers (CD 105 and CD90) and the cardiomyocyte lineage-specific markers, transcription factor NKX2.5, and Troponin I. They did not express hematopoietic markers (CD34 and CD45), mast cell marker (tryptase), or cardiomyocyte lineage-specific transcription factor GATA4, however, CPCs were 85% c-kit⁺ positive (CD117; Fig. IB).

[00127] Growth properties and functional characteristics of these cells during expansion provide relevant metrics that may reflect their functionality after transplantation in a rodent MI model. At P3 (passage 3), CPCs were approximately more than four times more proliferative than CDCs derived from the same patient (Fig. 1C). When the two progenitor cell types were allowed to proliferate until P6 (passage 6), the majority of the adult CDCs developed senescence as evident by increased b-gal activity when compared to CPCs (Fig. ID).

[00128] Another key functional property of the progenitor cells is their intrinsic migration ability after transplantation into the infarcted myocardium. To recapitulate this functional activity in vitro , a trans-well migration assay was performed to measure the migrator}' response of both progenitor cell types. Derived from same RAA of four biological replicates, CPCs migration was significantly more as compared to CDCs (Fig. I E) after 6 hours of incubation in presence of serum.

[00129] To estimate whether the growth and migratory properties of CPCs are associated with an improved ability to recover myocardial function after infarction, both CPCs (P3) and CDCs (P3) from the same heart biopsy were transplanted in the rodent MI model and groups were evaluated for cardiac function and left ventricular remodeling by echocardiogram and post- mortem histology. After ligation of the left anterior descending artery, 1 million CPCs or 1 million CDCs or cell-free Iscove’s Modified Dulbecco’s Medium (IMDM) was injected into the injured myocardium of the left ventricle (LV) as described previously [5,6] The consistency of the MI model was verified for each treatment group by performing echocardiography 24 hours after MI, which demonstrated similar ejection fraction (EF) among the 3 groups at this time point (Fig. IF). Compared to IMDM control, transplanted CPCs and CDCs significantly improved cardiac function and structure as indicated by increased EF and fractional shortening (FS) and decreased end-diastolic volume (EDV) and end-systolic volume (ESV). But the LV functional improvement was significantly greater with CPCs as compared to CDCs (Fig. 1F-1I). The functional improvement was apparent 1 week after MI and sustained for the entire 4 weeks of follow-up. Structural changes in the LV were further evaluated by histologic analysis at 28 days post-MI, focusing on fibrosis (Masson trichrome), arteriolar density (smooth muscle actin), and total vascular density (Isolectin IB4). Representative images of myocardial fibrosis and quantification of the three different treatment groups are shown in Fig. 1 J. At 4 weeks post-MI, infarct size was analyzed by measuring the area of fibrosis relative to total stained myocardial area. Hearts treated with either CPCs or CDCs had significantly smaller infarcted areas relative to IMDM controls, and the fibrosis was significantly reduced in CPC-treated hearts when compared with CDCs (Fig. 1 J). Although both CPCs and CDCs treatments significantly increased arteriolar (SMA) and total neovascular density (IB4) compared to IMDM control (Fig. IK), CPCs outperformed CDCs in regard to neovascular density.

Increased c-kit⁺ stoichiometry in CPC population improved therapeutic efficacy

[00130] Recent reports have demonstrated that the most significant functional unit of the transplanted progenitor cells is their secretome, containing independently secreted proteins and exosomes [5,29] Initially, the secretion levels of 7 well-studied paracrine factors (HGF, IGF1, SDF-la, ANG-l, VEGFA, PDGFB and bFGF) released by CPCs and CDCs into the total conditioned medium (TCM) were compared. These factors have previously been suggested to play a role in stem/progenitor cell-mediated repair [29-34] ELISA-based quantitative analysis of these 7 paracrine factors in the TCM of CPCs and CDCs revealed that HGF, IGF-l, SDF-la, ANG-l, VEGFA, PDGFB, and bFGF are secreted at significantly higher levels by CPCs compared to CDCs (Fig. 2A). To assess the angiogenic potential of the TCM derived from CDCs and CPCs, wound healing assays were performed on Human Mammary Epithelial Cells

(HMECs) keeping TCM proteins concentration constant (50 ng/ul proteins concentration).

Wound area was significantly reduced in the presence of TCM derived from CPCs compared to CDCs (Fig. 2B, C).

[00131] These data suggested that the presence of the higher concentration of independently secreted molecules is a critical property for the c-kit⁺ cells within the CPCs. To test this hypothesis, the c-kit⁺ cell stoichiometry was increased by varying the c-kit⁺ cell concentration from 20% to 80% within the CDCs population while maintaining the total cell number at a constant level in these combination groups. Since growth medium composition can affect the biochemical cellular and functional properties of CPCs and CDCs-including the c-kit⁺ cell concentration, growth of CDCs in CPCs’ culture medium and vice versa at passage 3 of the cells was attempted. Change to the non-designated growth medium formulation of CDCs and CPCs resulted in early onset of senescence, reduced cellular proliferation, loss of mesenchymal cell markers (CD90 and CD 105) (Fig. 2D, E). It was also demonstrated that CPCs and CDCs could not be derived from RAA explant in the non-designated growth medium as the outgrowth of the cells was limited (data not shown). Consequently, CPCs and CDCs were cultured in their prescribed medium and the c-kit⁺ cell population was mixed within the CDCs population just before the in vitro assays or intramyocardial injection into the rat MI model. ETsing a scratch migration assay, each CDCs combination group with increasing c-kit⁺ cell concentration demonstrated a progressively enhanced wound healing process (Fig. 2F).

[00132] Next, TCM was harvested after 48 hours of incubation at 37°C from the mixed populations of CDCs. Two well-established paracrine factors, SDF-la and VEGFA, were shown to increase with increasing c-kit⁺ cell concentration by ELISA (Fig. 2G-H) [35-37] Lastly, the myocardial recovery potential of these combination groups were tested in an established rodent MI model while maintaining the same total number of transplanted cells (1 million) [5,6] Ejection fraction and fractional shortening were significantly higher with increasing concentrations of c-kit⁺ cells (Fig. 21, J). Taken together these data suggested that the higher levels of cardioprotective factors in the secretome of c-kit⁺ cells may contribute to their superior functional abilities when compared to CDCs.

CPCs and CDCs release progenitor cell-specific human major histocompatibility complex

-containing exosomes into the recipient plasma

[00133] Although, the roles of CDCs- or CPCs-derived exosomes in cardiac repair are being actively investigated [5,8,9], in vivo validation of their exosomes production and functional potential remains to be investigated. It was hypothesized that transplanted human CDCs or CPCs would release exosomes with their parent cell-specific constituents into the recipient circulation during myocardial recovery in the xenogeneic rodent MI model. To test this hypothesis in vitro , >85% confluent CPCs or CDCs derived from the same heart biopsy were conditioned in serum- free media for 48 hours and their exosome output was analyzed. Subsequent to conditioning in serum free basal medium, both cell types retained viability. Exosomes were purified from the conditioned medium using size exclusion chromatography [5,38] Transmission electron microscopy (TEM) confirmed that the isolated extracellular vesicles were in the size range of exosomes and expressed canonical exosome marker CD63, as identified by immunogold staining (Fig. 3A). Flow cytometry using CD63 -conjugated magnetic beads demonstrated higher CD63 expression on CDCs derived exosomes as compared to CPCs derived exosomes (Fig. 3B).

Further characterization of these exosomes by nanoparticle tracking analysis (NT A, Nanosight NS300) revealed that CDCs-derived exosomes were not only larger in size (average size 165 nm) than CPCs-derived exosomes (average size 124 nm), but also existed at a higher concentration as compared to CPCs-derived exosomes (Fig. 3C). However, CPCs-derived exosomes showed more proliferation of HMEC and exhibited more angiogenic potential when compared to CDCs- derived exosomes in transwell migration assay and wound healing assay, when keeping exosome numbers constant and equal (Fig. 3D-E. It was concluded that despite being fewer in number, CPCs-derived exosomes maybe more potent for myocardial repair as compared to CDCs derived exosomes.

[00134] Next, immunoblot analyses were performed to determine whether progenitor cells would release exosomes containing progenitor-specific cellular markers as shown in Fig. IB. The exosomes derived from the CPCs and CDCs contained NKX2.5, HLA, Troponin I, c-kit⁺, and exosomes markers flotillin-l and CD63 (Fig. 3F). Importantly, cytochrome C, a marker for apoptotic bodies, could not be detected showing the homogenous population of exosomes.

Similar to its cellular counterpart, HLA-A is selectively present on the surface of human stem/progenitor cell exosomes. Therefore, the MHC class I specificity was utilized to quantify and purify the human exosome subpopulation (Fig. 6) from the rat plasma after cellular transplantations. This platform was tested in vitro on exosomes derived from CDCs or CPCs in culture. Purified exosomes were analyzed on the NanoSight in fluorescence mode (Qdot 605) for HLA-detection [39,40] HLA-A specific signal using goat secondary Qdot 605, was detected on the exosomes derived from CPCs or CDCs (Fig. 3G). To validate this concept in vivo , progenitor cells specific exosome signal in the recipient rat plasma from days 2 and 7 post- progenitor cell transplantation was quantified in the total exosomes using anti-HLA-A

antibodies and Qdot 605 (secondary antibodies) on the nanoparticle detector (Nanosight NS300). These time points were chosen because previous analysis had demonstrated that despite the loss of majority of transplanted progenitor cells, improved cardiac function of the injured

myocardium was noticed by day 7 after transplantation [5,41] At day 7, total plasma exosomes were similar between the IMDM (negative control), CDCs, and CPCs groups, but progenitor cell specific exosome signal was detected only in the CDCs and CPCs groups as shown in the representative pictures (Fig. 3H). Further, significantly higher progenitor cells specific HLA-A exosome signal was seen in CPCs group compared to CDCs group by approximately 2.0 fold (Fig. 31), Importantly, HLA-A signal was not detected in the control group with IMDM injection.

[00135] Given these promising findings, an assessment was undertaken as to whether circulating progenitor cell exosomes co-expressed surface markers and proteins that reflect the cellular constituents of CDCs and CPCs. If so, this would validate the in vitro findings and that the MHC mismatch enables purification and characterization of progenitor cell exosomes and their cargoes in vivo. Human progenitor cell specific exosomes were purified from rat plasma using anti-HLA A antibody conjugated beads from post-operative day (POD) 2 and POD 7 time points post-transplantation. Immunoblot analysis demonstrated that both enriched progenitor cell specific exosome subpopulations expressed HLA molecules (HLA-A and HLA-A1), flotillin 1 (exosome marker), c-kit (progenitor cell marker), and cardiomyocyte marker troponin I (Fig. 3J). But, they were negative for cytochrome C (cellular/ apoptotic body contamination). Importantly, pre-transplant samples (POD 0) failed to show expression of these markers specifically seen in the CPC and CDC exosome populations.

[00136] Taken together these data demonstrated that human progenitor cells transplanted into ischemic myocardium release distinct, quantifiable exosome signal into the peripheral circulation, with CPCs having a significantly higher exosome contribution than CDCs in this model. Progenitor cell exosomes carry protein markers specific to the cytoplasmic and membrane constituents of their cellular counterparts. Collectively, these findings support the concept that circulating progenitor cell specific exosome subpopulation may reflect the conditional state of the transplanted progenitor cells, and its quantitative and intra-exosomal cargo characterization may enable noninvasive monitoring of the functional transplanted progenitor cell mass.

Computational modeling of miRNA and system biology of the genes involved in myocardial recovery

[00137] To understand the mechanisms by which CPCs or CDCs might potentiate myocardial recovery, computational analyses were performed on: 1) cellular mRNA from in vitro CDCs and CPCs cultures, 2) miRNA cargoes of in vitro CDCs and CPCs culture supernatant exosomes, and, 3) miRNA cargoes of circulating progenitor cell specific exosomes purified from recipient total plasma exosome pool from day 7 samples of CPCs and CDCs transplants. Analyses of cellular mRNA profiles of in vitro cultured CPCs and CDCs could not predict the outcomes involved with improved cardiac function purely based on the differential expression patterns of mRNA specified genes (Table 1).

Table 1: Top 50 mRNA changes (CPC compared to CDC)

[00138] Previously, computational tools such as principal component analysis (PCA) and partial least squares regression (PLSR) modeling of pediatric CPCs cellular mRNA content and CPCs exosome miRNA cargo harvested from in vitro cultures was used to understand potential mechanisms and predictors of myocardial remodeling [42,43] The top 300 mRNA signals were identified and used to generate a predictive model to quantitatively link signals to functional outcomes triggered by the transplanted CPCs and CDCs [43] Therefore, in this study the same top 300 genes were selected and matched with the CDCs and CPCs mRNA genes using PC A (Fig. 7A). Individuals in each age/cell type group are clustered together and are localized in close proximity. Despite clustering uniquely from prior studies and matching signals, because of low correlation between the observed and expected ratio the model was unable to predict most endpoints for this study, including angiogenesis (Fig. 7B). Therefore, a new PLSR prediction model was re-trained by combining the adult CDCs and CPCs mRNA signals with data from Agarwal, et al. , to create a new model to understand potential shared mechanisms [42] Then, PLSR analysis was used to establish a relationship between the covarying mRNA signals and responses of angiogenesis, fibrosis reduction, migration, and proliferation that were shared among the studies (Fig. 7C). Ingenuity Pathway Analysis (IP A, Qiagen; Redwood City, CA) of the top genes revealed a prominent role for inflammatory and cardiac development genes being involved in the reparative process. Finally, loading plot from PLSR analysis demonstrated specific shared cardiac and immune system related genes from the past [42] and present studies that clustered with cardiac functions (Fig. 7D).

[00139] In addition, a model was developed to predict functional effects based on exosome miRNA content, this time using both CPCs and CDCs exosomes isolated in vitro from TCM, as well as circulating CPC and CDC specific exosome subpopulations purified from the rat plasma [42] PC A showed that in vz/roderived exosomes, whether from CPCs or CDCs, clustered very closely together, while progenitor cell exosomes purified from recipient plasma had divergent expression patterns (Fig. 4A). Interestingly, both datasets fit the previously published

computational model quite well with high predictability ranging from 82-97% (Fig. 4B). When the computational model was used to predict outcomes of the current study (improvement in ejection fraction and angiogenesis, reduction in fibrosis), the model using plasma CPCs and CDCs exosome subsets more closely matched to the observed results than the model using in vitro CDCs and CPCs exosomes (Figs. 4C-E and Table 2). Table 2: Exosome microRNA variable importance of projection (VIP)

[00140] Due to the high predictability, the model was retrained on all data sets to determine miRNAs likely involved in the response using PLSR and plotted them in PC space along with the cellular mRNA signals (Fig. 4F and Table 3). Table 3: miRs with their role in cardioprotective processes

[00141] miRNAs were identified associated with improvement in ejection fraction (miRs 378b, 623, and 941), reduction in fibrosis (1256 and 384), and induction of angiogenesis (525- 3p, 515-5p, and 1224). Functional analyses of these miRNAs using IPA identified a total number of 45 cardioprotective pathways that were upregulated (Table 4) favoring cell growth and proliferation (PI3/AKT, mTOR, HIF-la, JAK/STAT, HMGB1, PDGF, IGF1, FGF, TGF-b), resistance to stress (oxidative stress response, nitric oxide signaling, PXR/RXR), antifibrosis and anti-inflammation (inhibition of MMPs and IL-6), cardiomyocyte regeneration (NOTCH and HIPPO), angiogenesis (FAK/PAK, HIF-la, VEGF), and cardiomyocyte proliferation (NOTCH and HIPPO). Collectively, these data demonstrated that the miRNA cargo of CPCs exosomes isolated from recipient plasma was different than the cargo isolated from in vitro collected exosomes, and that the in vivo exosomal cargo was more predictive of mechanistic

cardioprotective potency than the in vitro exosome profiles.

Table 4: List of canonical signaling pathways affected by VIP miRs

Experimental Validation of miRNAs function predicted by computational analyses

[00142] Bioinformatics tools facilitate the study of miRNAs by providing a list of potential functions, however, due to multi-targeted approach of miRNAs, it is important to validate the predicted functions of miRNA. Based on computational analyses, five miRNAs (miRNAs 378, 384, 515, 525, and 1224 [10,9,15,11,14, respectively]) were identified and predicted to improve cardiac function after MI by enhancing angiogenesis. These miRNAs were not detected in the in vitro cultured CPCs exosomes but were enriched in the circulating CPCs exosome subset purified from rat plasma in the MI model. Literature based software analyses (TargetScan and Diana Tools) and previous reports also suggested angiogenic potential of these miRNAs [44] To validate the predictions, HMECs were transfected with the mimics of these miRNAs and their angiogenic potential was assessed by three well -accepted angiogenic assays: a) endothelial cell proliferation, b) transwell migration, and c) wound healing assay [45,46] Transfection with specific miRNA mimics resulted in multifold enrichment of that specific miRNA in the transfected cells (Fig. 5A). All the shortlisted miRNAs, as predicted, significantly induced cellular proliferation (Fig. 5B) as compared to non-specific miR transfection control. Next, transwell migration assay (Fig. 5C, D) demonstrated that miRNAs 384, 525 and 1224 significantly enhanced the migration of HMECs, while no significant migration was observed with miRNA 378 and 515 transfected HMECs. For wound healing assay, HMECs transfected with mimics of miRNAs 384, 525, and 1224 significantly reduced the wound area, while miRNAs 378 and 515 transfected HMECs had no significant effect on the healing of wound area (Fig. 5E, F). Taken together, these results showed that the computational model can predict the functional potential of miRNAs with high validity in a variety of well-established angiogenesis in vitro assays.

Donor MSC exosomes isolated from recipient serum

[00143] The ELPIS Phase I study is an open-label study to primarily determine the safety and feasibility of injecting allogeneic human mesenchymal stem cells (MSCs) into the right ventricle (RV) of human patients having hypoplastic left heart syndrome (HLHS) and undergoing the Stage II operation. The secondary objective is to determine the efficacy of MSC treatment from baseline to 12 month follow-up in all MSC-treated subjects by serial cardiac magnetic resonance (CMR). Efficacy endpoints included RV regional and global cardiac function, ventricular volume, heart failure status, size of fibrosis, and somatic growth.

[00144] In the ELPIS trial, blood samples are being collected from the enrolled HLHS patients after their stage II operation where transplanted MSCs were injected into the RV. The HLA mismatch between the MSC donor and HLHS recipient was utilized. First, the total exosome population was isolated using a PEG precipitation from the serum. This population was verified using the bright field on a NanoSight machine (bright blue peaks, Fig. 8A). Second, donor exosomes secreted from the donor transplanted MSC were selected out using HLA specific-antibody conjugated beads and detected by Quantum Q dot (red peaks) on the

NanoSight (Fig. 8A). This process has been quantified for the first two patients enrolled in the ELPIS trial. It has been determined that there were donor exosomes present after MSC treatment from the transplanted MSC. The number of exosomes was the highest after two days post transplantation (Fig. 8B). This is the first documentation that transplantation of human MSCs secretes exosomes not only in the heart but also in the recipient’s serum. TGF 1 b identified as a master regulator of donor-MSC EXO-cargo

[00145] The TGF i p superfamily plays a key role in attenuation of cardiac hypertrophy, cardioprotection, and remodeling after myocardial infarction (MI) as an autocrine/paracrine factor [67-69] Similar to the technique described for isolating tissue-specific exosomes for monitoring immunologic rejection for solid organ transplantation, mismatched anti-HLA antibody were used to isolate and quantify MSC-specific donor exosomes in the serum of ELPIS HLHS patients. After miRNA-microarray analysis on these exosomes (EXO), enrichment of miRs up-regulating the proteins of TGF 1 b family, including GDF15, was found in donor MSCs- EXOs after MSCs transplanted in HLHS patients at day7 (Table 5, N=3). This is the first demonstration that transplanted stem cell donor exosomes can be detected in the recipient serum and the cargo can be interrogated. Further, this data supports the concept that serum exosome detection may be a platform for allogeneic MSC treatment.

Table 5

[00146] The data presented herein demonstrates that plasma-derived progenitor cell exosome profiling more accurately predicts the clinical outcomes seen with stem cell therapy than the RNA profiles of cultured progenitor cells in vitro. In support of this idea, in the xenogeneic rat MI model, the total plasma exosome numbers and exosome particle size were similar between the CDCs and CPCs groups, but the transplanted CPCs exosome signal was significantly increased during the early postoperative period when compared to the CDCs exosome signal. This higher CPCs-specific exosome signal correlated with better functional and biochemical properties of the transplanted CPCs on myocardial recovery. The diagnostic potential of this platform is further validated by the ability to noninvasively enrich for the progenitor cell specific exosomes containing the cellular constituents of the transplanted CPCs and CDCs. [00147] As mentioned above, initial data recently obtained from humans again demonstrates that exosome produced by donor stem cells can be isolated and collected from the plasma of cell recipients. The number of exosomes isolated from subjects receiving allogenic mesenchymal cells was the highest after two days post-transplantation. Thus, transplanted stem cells, such as human MSCs, secrete exosomes not only in the heart but also in the recipient’s blood stream.

[00148] Further, in the model the miRNA cargo of progenitor cell exosomes in recipient plasma most accurately represented how transplanted cells may be remodeling the myocardium.

[00149] When the model was trained on either data set (exosomes derived from cultured cells or progenitor cell specific exosomes from rat plasma), predictability values were high regardless of exosome source, indicating the validity of the model. However, only progenitor cell exosomes derived from the plasma predicted the outcomes seen in the current study. Interestingly, when just looking at the in vitro exosome data, the results would predict that exosomes derived from CDCs would be much more reparative than CPCs, suggesting that to better understand the clinical potential of CPCs versus CDCs more head-to-head comparison of purified exosomes are warranted. Importantly, these findings suggest that progenitor cells release different exosomes in vivo versus in vitro , and that the microenvironmental cues in vivo may play a role in triggering release of different exosome quantities with different cargos.

[00150] The miRNAs enriched in the circulating progenitor cell exosomes may be

contributing to the responses seen. Pathways identification by KEGG analysis using top 10 VIP miRNAs (Table 2) identified modulation of various pathways promoting cellular metabolism, cell survival and proliferation, especially Hippo and FoxO signaling pathways [54] miRNAs within the CPC exosomes that may have roles in cardiac repair include miR-384, miR-378, miR- l224-5p, miR-525-3p, and miR-5l5.

[00151] Computational modeling provides an insightful avenue for determining the mechanistic pathways driving progenitor cell mediated remodeling of the MI myocardium. Similar to a previous validation study for the computational modeling of transplanted pediatric progenitor cells [42,55], the initial validation of the model was performed by examining miRNAs involved in angiogenesis. The angiogenesis endpoint was selected from the model because its high significance for cardiac function recovery and well-established in vitro angiogenesis assays. Although miRNAs 378 and 525 failed to promote cell migration (Fig. 5), all the shortlisted miRNAs induced cellular proliferation, suggesting that the methodology adopted by the computational analyses presented herein is accurate and the exosomes secreted after cellular transplantation are more effective in predicting miRNAs involved in angiogenesis.

[00152] By direct head-to-head comparison of miRNA in vitro functionality, functional superiority of CPCs as compared to CDCs was demonstrated. Since both cell types were derived from the same heart biopsy sample, the approach presented herein eliminated patient variability, strengthening the observed comparative data. CPCs were superior in terms of paracrine factor secretion, angiogenesis, myocardial tissue preservation, and functional improvement. As expected, increasing the c-kit+ cell concentration in the CDCs increased myocardial recovery, supporting the critical function of the c-kit+ cell population. Despite increased exosome secretion by CDCs in vitro , CPCs demonstrated increased exosome secretion in vivo in this model. Further, the cardioprotective pathways identified by the miRNA-driven pathways potentially mediated by exosome transfer gives an unparalleled insight into the mechanisms of cellular recovery following CPCs and CDCs administration.

* * * *

[00153] While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.

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Claims

WHAT IS CLAIMED IS:

1. A method of monitoring cells transplanted into a subject comprising screening a biological sample obtained from a subject into whom cells have been transplanted for the presence of transplanted cell-derived exosomes.

2. A method of monitoring a subject receiving cell-based therapy comprising screening a biological sample obtained from a subject receiving cell-based therapy for the presence of transplanted cell-derived exosomes.

3. A method for predicting functional recovery of ischemic myocardium in a subject into which cells have been transplanted comprising screening a biological sample obtained from a subject that has ischemic myocardium and into which cells have been transplanted for the presence of transplanted cell-derived exosomes.

4. The method of any one of claims 1-3, wherein the presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject.

5. The method of any one of claims 1-3, further comprising enumerating the number of exosomes present in the biological sample.

6. The method of any one of claims 1-3, further comprising screening one or more additional biological samples obtained from the subject at different time points for the presence of transplanted cell-derived exosomes and enumerating the number of exosomes present in the additional biological samples.

7. The method of claim 6, wherein an increase/decrease in the number of transplanted cell-derived exosomes over time indicates a corresponding increase/decrease in the number of transplanted cells in the subject over time.

8. The method of claim 3, wherein the presence of exosomes in the biological sample indicates functional recovery of the ischemic myocardium in the subject is more likely than a corresponding subject in whom such exosomes are not present.

9. The method of claim 6, wherein an increase in the number of transplanted cell- derived exosomes over time indicates an increased likelihood of functional recovery of the ischemic myocardium in the subject.

10. The method of claim 6, wherein a decrease in the number of transplanted cell- derived exosomes over time indicates a decreased likelihood of functional recovery of the ischemic myocardium in the subject.

11. The method of any one of claims 1-10, wherein the transplanted cells are allogeneic to the subject.

12. The method of any one of claims 1-11, wherein the transplanted cells are stem cells or progenitor cells.

13. The method of any one of claims 1-12, wherein the transplanted cells are one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).

14. The method of any one of claims 1-13, wherein the transplanted cells have been transplanted into an organ.

15. The method of claim 14, wherein the organ is a heart.

16. The method of any one of claims 1-15, wherein the biological sample is screened within 7 days after completion of cell therapy or transplantation of the cells into the subject.

17. The method of any one of claims 1-16, wherein the biological sample is plasma.

18. The method of any one of claims 1-17, wherein the biological sample is screened using antibodies having binding specificity for molecules displayed by the exosomes.

19. The method of claim 18, wherein the antibodies having binding specificity for human leukocyte antigen (ELLA) surface molecules or human mismatch ELLA surface molecules.

20. The method of any one of claims 1-19, wherein the subject is a human.

21. A method of profiling exosomes derived from cells transplanted into a subject comprising (i) collecting, from a biological sample obtained from a subject into which cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing cargo of the collected exosomes.

22. A method of profiling exosomes derived from cells transplanted into a subject comprising (i) collecting, from a biological sample obtained from a subject into which cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing microRNA cargo of the collected exosomes.

23. The method of claim 21, wherein the cargo is one or more of polynucleotides, polypeptides, and lipids.

24. The method of any one or claims 21-23, wherein characterizing cargo comprises identifying one or more species of polynucleotide, polypeptide or lipids present in the collected exosomes.

25. The method of claim 22, wherein characterizing microRNA cargo comprises sequencing one or more species of microRNA present in the collected exosomes.

26. The method of claim 22, wherein characterizing microRNA cargo comprises identifying one or more species of microRNA present in the collected exosomes.

27. The method of claim 24 or 25, wherein the species of microRNA identified as being present in the exosomes is one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR- 1270, miR-384, miR-2355-3p, miR-3 l27-5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR- 337-5p, miR-5l5-3p, miR-767-5p, miR-623, and miR-362-5p.

28. The method of claim 24 or 25, wherein the species of microRNA identified as being present in the exosomes is one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR-l224, and miR-l256.

29. The method of claim 24 or 25, wherein the species of microRNA identified as being present in the exosomes is one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224.

30. The method of any one of claims 21-29, wherein the characterizing is via the use of hybridization to labeled probes.

31. The method of any one of claims 21-30, further comprising repeating the method at one or more additional time points and characterizing cargo from each additional time point, thereby determining whether there is a change in the cargo over time.

32. The method of any one of claims 21-31, wherein the transplanted cells are allogeneic to the subject.

33. The method of any one of claims 21-32, wherein the transplanted cells are stem cells or progenitor cells.

34. The method of any one of claims 21-33, wherein the transplanted cells are one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).

35. The method of any one of claims 21-34, wherein the cells have been transplanted into an organ.

36. The method of claim 35, wherein the organ is a heart.

37. The method of any one of claims 21-36, wherein the biological sample is screened within 7 days after completion of cell therapy or transplantation of the cells into the subject.

38. The method of any one of claims 21-37, wherein the biological sample is plasma.

39. The method of any one of claims 21-38, wherein the exosomes are collected using antibodies having binding specificity for molecules displayed by the exosomes.

40. The method of claim 39, wherein the antibodies having binding specificity for human leukocyte antigen (HLA) surface molecules or human mismatch HLA surface molecules.

41. The method of any one of claims 21-40, wherein the subject is a human.

42. A method of predicting functional recovery of a target organ in a subject comprising (i) collecting, from a biological sample obtained from a subject having a target organ into which cells have been transplanted, exosomes derived from the transplanted cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the target organ in the subject based on characteristics of the miRNA cargo, wherein the target organ is diseased, damaged, or in some other manner malfunctioning.

43. A method of predicting functional recovery of ischemic myocardium in a subject comprising (i) collecting, from a biological sample obtained from a subject having ischemic myocardium and into which cells have been transplanted, exosomes derived from the

transplanted cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the ischemic myocardium in the subject based on

characteristics of the miRNA cargo.

44. The method of claim 42 or 43, wherein characterizing microRNA cargo comprises sequencing one or more species of microRNA present in the collected exosomes.

45. The method of claim 42 or 43, wherein characterizing microRNA cargo comprises identifying one or more species of microRNA present in the collected exosomes.

46. The method of any one of claims 42, 43 and 45, wherein the species of microRNA identified as being present in the exosomes is one or more of miR-4649-3p, miR- 548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27-5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR-623, and miR-362-5p.

47. The method of any one of claims 42, 43 and 45, wherein the species of microRNA identified as being present in the exosomes is one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR-l224, and miR-l256.

48. The method of any one of claims 42, 43 and 45, wherein the species of microRNA identified as being present in the exosomes is one or more of miR-378b, miR-384, miR-5 l5-5p, miR-525-3p, and miR-l224.

49. The method of any one of claims 42-48, wherein the characterizing is via the use of hybridization to labeled probes.

50. The method of any one of claims 42-49, further comprising repeating the method at one or more additional time points and characterizing cargo from each additional time point, thereby determining whether there is a change in the cargo over time.

51. The method of any one of claims 42-50, wherein the transplanted cells are allogeneic to the subject.

52. The method of any one of claims 42-51, wherein the transplanted cells are stem cells or progenitor cells.

53. The method of any one of claims 42-52, wherein the transplanted cellsare one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).

54. The method of any one of claims 42-53, wherein the cells have been transplanted into an organ.

55. The method of claim 54, wherein the organ is a heart.

56. The method of any one of claims 42-55, wherein the biological sample is screened within 7 days after completion of cell therapy or transplantation of the cells into the subject.

57. The method of any one of claims 42-56, wherein the biological sample is plasma.

58. The method of any one of claims 42-57, wherein the exosomes are collected using antibodies having binding specificity for molecules displayed by the exosomes.

59. The method of claim 58, wherein the antibodies having binding specificity for human leukocyte antigen (ELLA) surface molecules or human mismatch ELLA surface molecules.

60. The method of any one of claims 42-59, wherein the subject is a human.