CPC EXOSOMES MIRNA373 COMBINATION THERAPIES

Abstract

Steam cell and exosome compositions via combination therapy, related gene therapy and pluripotent stem cell derived muscle regeneration as having therapeutic utility to treat a variety of diseases and disorders, e.g., cardiovascular disease, Duchenne muscular dystrophy, and fibrotic disease.

Claims

1) A method of treating cardiac disease, comprising i) administering a composition to a human patient having cardiac disease in an amount sufficient to treat said cardiac disease; ii) said composition comprising: i. allogenic or autologous cardiac progenitor cells (CPCs); ii. plus additional extracellular vesicles derived from said CPCs or another population of CPCs.

2) The method of claim 1, wherein said allogenic or autologous CPCs are made by a process comprising: i) isolating parent cells from said patient or a person allogenic to said patient, wherein said parent cells are either induced pluripotent stem cells (iPSCs) or pluripotent stem cells (PSCs); ii) treating said parent cells in vitro with ISX-9 or Danazol or other isoxazole based compound or Givinostat or the combination of Givinostat and small molecule: CHIR99021, in an amount effective to induce differentiation of said iPSCs or PSCs into CPCs and/or smooth muscle cells, myocytes, endothelial cells, or muscle progenitor cells. iii) Treating said parent cells in vitro with an isoxazole compound to induce differentiation of said iPSCs or PSCs into CPCs and/or smooth muscle cells, myocytes, endothelial cells, or muscle progenitor cells with an isoxazole formula of: ##STR00009## wherein R.sub.1 and R.sub.2 are both hydrogen or R.sub.1 is hydrogen and R.sub.2 is selected from the group consisting of substituted or unsubstituted C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, and benzyl, or where R.sub.1 and R.sub.2 may be joined together to form a ring selected from azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl; R.sub.2, R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, halogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6 cycloalkyl, substituted or unsubstituted aromatic or heteroaromatic ring, cyano, nitro and acyl; X is O, NH or S; and Y is O, NH or S.

3) The method of claim 1, wherein said allogenic or autologous CPCs are made by a process comprising: i) isolating parent cells from said patient or a person allogenic to said patient, wherein said parent cells are induced pluripotent stem cells (iPSCs) or pluripotent stem cells (PSCs) or multipotent stem cells (MSCs); ii) culturing said parent cells with 0.1-35 M ISX-9 or other isoxazole based compound for 3-10 days in a medium without insulin to induce parent cells to form CPCs; iii) culturing said CPCs in a medium without ISX-9 or other isoxazole based compound and with insulin for 3-10 days to induce differentiation of said CPC cells into a mixture comprising CPCs and one or more of cardiomyocytes, smooth muscle cells and endothelial cells.

4) A method of treating cardiac disease, comprising: i) administering a composition to a heart in a human patient having cardiac disease in an amount sufficient to treat said cardiac disease; ii) said composition comprising: i. allogenic or autologous cardiac progenitor cells (CPCs); ii. plus added extracellular vesicles derived from said CPCs; iii) said cells made by: i. isolating parent cells from said patient or a person allogenic to said patient, wherein said parent cells are induced pluripotent stem cells (iPSCs) or pluripotent stem cells (PSCs) or multipotent stem cells (MSCs); ii. culturing said parent cells with 0.1-35 M ISX-9 or isoxazole based compound for 3-10 days in a medium without insulin to induce said parent cells to form CPCs; iii. culturing said CPCs in a medium without ISX-9 or isoxazole based compound and with insulin for 3-10 days to induce differentiation of said CPC cells into a mixture comprising CPCs and one or more of cardiomyocytes, smooth muscle cells and endothelial cells.

5) The method of claim 1, wherein said CPCs are subjected to hypoxic preconditioning before use in said human patient.

6) The method of claim 1, wherein said extracellular vesicles comprise miRNA-373 or an miRNA-373 mimic or an expressible nucleotide sequence encoding miRNA-373 or an miRNA-373 mimic.

7) The method of claim 1, wherein said extracellular CPCs and/or vesicles comprise miRNA-373 or a mimic of miRNA-373 and/or a ephrinB2 protein.

8) The method of claim 1, wherein said extracellular vesicles are isolated from a culture of CPCs by ultracentrifugation, ultrafiltration, precipitation, immunoaffinity capture or combinations thereof.

9) The method of claim 1, wherein; 110.sup.8-910.sup.8 CPCs (100-900 million) and 10.sup.9-10.sup.12 (one billion-one trillion) extracellular vesicles are administered by intramyocardial injection, catheter injection or direct injection.

10) A composition for treating fibrosis or cardiac disease, said composition comprising allogenic or autologous cardiac progenitor cells (CPCs) plus added extracellular vesicles derived from said CPCs in a pharmaceutically acceptable carrier.

11) The composition of claim 10, said CPCs and said extracellular vesicles in a ratio of about 10-1000 extracellular vesicles to CPC's.

12) The composition of claim 10, said CPCs and/or said extracellular vesicles comprising an miRNA-373 or a mimic of miRNA-373 or an expressible nucleic acid encoding said miRNA-373 or said mimic of miRNA-373.

13) The composition of claim 10, further comprising an ephrinB2 protein.

14) A composition, comprising CPCs made by induction of stem cells with ISX-9, Danazol or other isoxazole based compound plus exosomes containing miRNA-373 or a mimic of miRNA-373 in a pharmaceutically acceptable carrier.

15) A method of treating fibrosis or cardiac disease, said method comprising treating a patient having fibrosis in an amount sufficient to reduce the gene expression or protein activity of growth differentiation factor 11 (GDF-11) and/or Rho-associated coiled-coil containing kinase-2 (ROCK-2).

16) A method of preparing a population of skeletal myogenic progenitors from a population of human induced pluripotent stem cells (hiPSCs) or other pluripotent stem cells, comprising contacting the hiPSCs or other pluripotent stem cells with an effective amount of Givinostat (GIV) or the combination of Givinostat and small. molecule: CHIR99021, optionally cultured in serum free media and for treating Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy, muscular dystrophy, sarcopenia, and other muscular and muscle loss diseases comprising administering to a subject in need thereof an effective amount of said population of cells and/or an effective amount of said population of cells and/or exosomes or microvesicles.

17) A method of reducing fibrosis, comprising treating a patient having fibrosis or cardiac disease with a pharmaceutically effective amount of an a MIR-373 mimic mimetic compound, oligonucleotide, recombinant AAV vector or viral vector, gene editing constructs such as CRISPR and gene therapy vectors or recombinant viral particle or other pharmaceutically acceptable carrier containing MIR-373.

18) The composition of claim 17, comprising of: 1:) A 22-26 base nucleotide strand with greater than 85% homology to the group of related miRNAs including miR-371a-5p, miR-371a-3p, miR-371b-5p, miR-371-3p, miR-372-5p, miR-372-3p, miR-373-3p, miR-373-5p, or their variants, which also contains a 6-base seed sequence identical to the conserved seed sequence found within this same group of miRNAs. 2:) A second 22-26 base nucleotide strand is significantly complementary to the first strand and has least one modified nucleotide(s), such that when the two strands bind one another the first strand has a 3 nucleotide overhang relative to the second strand

19) The method of claim 2 further comprising the steps of: iv) Treating said parent cells in vitro with an isoxazole compound with iPSCs or PSCs into CPCs and/or smooth muscle cells, myocytes, endothelial cells, or muscle progenitor cells with an isoxazole formula of: wherein the isoxazole compound has the formula: ##STR00010## wherein R1 and R2 is each selected from C1-C4 alkyl, phenyl, benzyl, trifluoromethyl or halogen, R3 is selected from hydrogen, hydroxy, C1-C4 alkyl or alkoxy, R4, in position 3 or 5, is selected from hydrogen, trifluoromethyl, C1-C4 alkoxy, C1-C4 alkyl, or C1-C4 hydroxyalkyl, Rs is selected from hydrogen or C4-C4 alkyl or R4 and Rs together form a tetramethylene group, Z at position 3 or 5 on the heterocycle is selected from: N(R6)-CO, CON(R6)-, N(R6)-CON(R6)-, CH(R6)-NHCO, or NHCOCH(R6), in which R6 is selected from hydrogen or C1-C4 alkyl. v.) Treating said parent cells in vitro with an isoxazole compound with iPSCs or PSCs into CPCs and/or smooth muscle cells, myocytes, endothelial cells, or muscle progenitor cells with an isoxazole formula of: wherein the isoxazole compound is monosubstituted at the 3, 4, or 5 position with a substitutent selected from the group: hydrogen, alkyl, aryl, alkenyl, alkynyl, heterocylic, heteroaryl, carbonyl, carboxy, halogen, amine, sulfur, oxy, hydroxyl, mercapto, sulfinyl, sulfonyl, sulfide, thioamide, nitrile, nitro, stannyl, boronic acid, carboxylic acid, carboxylic acid derivative, alkoxyphenyl, haloalkyl, haloaryl, alkylaryl, nitroaryl, and morpholinoalkyl. vi) Treating said parent cells in vitro with an isoxazole compound with iPSCs or PSCs into CPCs and/or smooth muscle cells, myocytes, endothelial cells, or muscle progenitor cells with an isoxazole formula of: wherein the isoxazole compound has the formula: wherein the isoxazole is 3,5-disubstituted, 3,4,5-tri-substituted, or 4,5-disubstituted and the substituents are selected from the group: hydrogen, alkyl, aryl, alkenyl, alkynyl, heterocylic, heteroaryl, carbonyl, carboxy, halogen, amine, sulfur, oxy, hydroxyl, mercapto, sulfinyl, sulfonyl, sulfide, thioamide, nitrile, nitro, stannyl, boronic acid, carboxylic acid, carboxylic acid derivative, alkoxyphenyl, haloalkyl, haloaryl, alkylaryl, nitroaryl, and morpholinoalkyl.

Description

BRIEF DESCRIPTION OF FIGURES

[0188] FIG. 1: Overall summary of generation of CPCs from hiPSC for cardiac repair.

[0189] FIG. 2: Schematic outline of generation of CPCs.

[0190] FIGS. 3A-3C: Characterization of CPCs: Generation and characterization of hiPSC-derived cardiac progenitor cells (CPCs). FIG. 3A, Time-dependent expression of Nkx2.5, GATA4, ISL-1, and Mef2c. *Versus Day 0, P<0.05; #versus Day 3, P<0.05, from three biological repeated experiments. FIG. 3B, Immunofluorescence staining showed that transcription factors (Nkx2.5, GATA4, and ISL-1) were highly upregulated in hiPSC treated with ISX-9. FIG. 3C, Fluorescence activated cell sorting analysis (FACS) showing 96.512.3% Nkx2.5 positive cells after ISX-9 treatment. Scale bar represents 200 mm. GATA4 indicates GATA binding protein 4; hiPSCs, human induced pluripotent stem cells; ISL-1, islet-1; ISX, isoxazole; Mef2c, myocyte enhancer factor-2; Nkx2.5, NK2 Homeobox 5.

[0191] FIG. 4: Further differentiation of CPCs into CM, EC and SMC.

[0192] FIGS. 5A-5D: Characterization of exosomes: Characterization of EV from iPSC and CPC.sup.ISX-9. FIG. 5A Secretion of EV from the CPC.sup.ISX-9 as imaged by electron microscopy. Bar=1 m. FIG. 5B EV isolated from iPSC and CPC.sup.ISX-9 visualized by transmission electron microscopy (TEM). Scale bar=200 nm. FIG. 5C Representative images of western blot for Tsg101, CD9, Hsp70, flotillin-1, and calnexin in EV lysates. C: cell lysate; E: EV. FIG. 5D Representative graph of size distribution of EV from iPSC and CPC.sup.ISX-9 as detected by TRPS.

[0193] FIGS. 6A-6B: FIG. 6A PKH 26 labeled EV from CPC.sup.ISX-9 (red) were observed inside the fibroblasts (green, Calcein AM), mostly located at the perinuclear region. The white arrows indicated the uptake of EVs. Bar=100 m. FIG. 6B Exosome effect of fibroblasts: Fibrotic gene expression in fibroblasts after TGF- stimulation. Effects of EV-CPC.sup.ISX-9 on fibrotic gene expression: role of miR-373. n=6.

[0194] FIG. 7: Expression of miRNAs in exosomes: Heatmap analysis of microarray data showing significant upregulation of miRNAs in EV-CPCISX-9 compared with EV-iPSC, EV-EB, or EV-control-CPC. Red or blue colors indicate differentially up- or downregulated miRNA, respectively (P<0:05). n=3.

[0195] FIGS. 8A-8D: CPC protective effects in ischemia: FIG. 8A Cytoprotective effects of ISX-9 on CPCs. A, Morphology of hiPSCs in RPMI/B27 medium treated with DMSO or ISX-9 under 1% 02 for 12 or 24 h. B, Representative images of TUNEL staining in Mock, DMSO, and ISX-9 treated groups after 24 h hypoxic stresses FIG. 8B Semiquantitative estimate of TUNEL positive cells. *Versus DMSO group, P<0.05. FIG. 8C, Representative images of TUNEL staining 3 days post-MI. The host cardiomyocytes were identified by -sarcomeric actinin. FIG. 8D, Quantitation of total TUNEL positive cells in the border area of infarct with different treatments. *Versus DPBS group, P<0.05; #versus hiPSC group, P<0.05.

[0196] FIGS. 9A-9D: Differentiation of CPCs when injected into the heart: FIG. 9A Engrafted CPCs were identified by PKH-26 fluorescence (red fluorescence); muscle fibers were visualized via immunostaining for human-specific cTnT or -sarcomeric actinin (Green fluorescence) at 3 M post-MI. FIG. 9B Temporal changes in FS. FIG. 9C cardiac fibrosis was evaluated by 7 levels by Masson Trichrome staining at 3 months' post MI. Sections of the representative heart were shown. FIG. 9D Quantification of scar tissue size. *P<0.05 versus DPBS treated group at the same time point, #P<0.05 versus hiPSC treated group. CPC indicates cardiac progenitor cells; hiPSCs, human induced pluripotent stem cells; MI, myocardial infarction; ISX, isoxazole.

[0197] FIGS. 10A-10D: Exosomes promote cardiac repair on injection: CPCISX-9-derived EV promoted cardiomyocyte proliferation and angiogenesis after myocardial infarction (MI) in mice. FIG. 10A Representative image of Ki67-positive cardiomyocytes (cTnT positive) in EV-CPCISX-9-treated mouse hearts 30 days after MI. Bar=50 m. FIG. 10B Quantitative estimate of proliferating cardiomyocytes as determined by Ki67 staining in the peri-infarct region 30 days after myocardial infarction. PBS group: n=940 cardiomyocytes from 3 hearts; EV-iPSC group: n=950 cardiomyocytes from 3 hearts; EV-CPCISX-9 group, n=951 cardiomyocytes from 3 hearts. * vs. the PBS group, P<0:05; # vs. the EV-iPSC group, P<0:05. FIG. 10C Representative images of arteriole density in the peri-infarct area 4 weeks after MI. Arterioles were identified by -SMA-positive staining (green) of vascular structures. Bar=100 m. FIG. 10D Quantitative analysis of arteriole density in different treatment groups.* vs. the PBS group, P<0:05; # vs. the EV-iPSC group, P<0:05, n=3.

[0198] FIGS. 11A-11D: CPC.sup.ISX-9-derived EV reversed cardiac remodeling in infarcted mice. FIG. 11A Temporal changes in FS. FIG. 11B Quantitative estimate of fibrosis. vs. the PBS group, P<0:05; # vs. the EV-iPSC group, P<0:05, n=4 FIG. 11C Representative M-mode echocardiography images from miRNA mimic negative control- (NC-) treated mice and miR-373 mimic-treated mice 30 days post-MI. FS is shown; n=8 in the NC group and n=7 in the miR-373 mimic group. FIG. 11D Representative Masson's trichrome-stained sections of hearts from NC-treated mice and miR-373 mimic mice. Quantitative analysis of fibrosis post-MI.

[0199] FIGS. 12A-12B: Images illustrating FIG. 12A EphrinB2 expression in ISX-9-CPC and FIG. 12B EphB4 expression in normal heart and infarcted heart.

[0200] FIGS. 13A-13B: Two graphs FIG. 13A and FIG. 13B illustrating combined treatment of ISX-9-CPC and EX in cardiac function 1 Month post-MI. ISOX-CPC, n=4, ISOX-CPC plus EX, n=3. The administration of CPC's together with their Exosomes improved cardiac function vs. using CPC's alone.

[0201] FIGS. 14A-14B: Two graphs FIG. 14A and FIG. 14B illustrating combined treatment of ISX-9-CPC and EX in cardiac function 1 month post-MI.

[0202] FIGS. 15A-15D: Images illustrating cytoprotection of ISX-9-CPC against ischemia. FIG. 15A In vitro effect on TUNEL staning with 24 h hypoxia (1%02) FIG. 15B Semi-quantitative estimate of TUNEL positive cells. *VS. DMSO group, P<0.05 FIG. 15C In vivo effect on TUNEL positive cells 3 days post-MI. Cardiomyocytes were identified by -dsrcomeric actinin. Bar=50 m. FIG. 15D Quantitation of TUNEL positive CM in the border area with different treatments. *vs. PBS groups, p<0.05; # vs. hiPSC groups, p<0.05

[0203] FIGS. 16A-16B: Images illustrating transdifferentiation of fibroblasts into myofibroblasts after 72 h hypoxia. FIG. 16A Transdifferentiation of fibroblasts into myofibroblasts after 72 h hypoxia as shown by immunostaining for a-smooth actin (-SMA): EX from ISOX-CPC or miR-373 mimic blocked their conversion into myofibroblasts. miR-373 inhibitor abrogated the effect of EX from ISOX-CPC. Bar-501Jm. FIG. 16B Phase-contrast images and immunofluorescence images of CD31 (red) and a-SMA (green)staining in human aortic endothelia cell (HAEC) untreated or treated with 10 ng/ml TGF-1 for 5d, or in HAEC pretreated with ISOX-CPC EX, miR-373 deficient ISOX-CPC EX or miR-373 mimic (50 nM). ISOX-CPC EX pretreatment significantly inhibited TGF-1 induced endothelial-mesenchymal transition (EndMT) with loss of CD31 expression and increase of -SMA expression, miR-373 inhibitor abrogated such effect. miR-373 mimic partly reversed TGF-1 induced EndMT.

[0204] FIGS. 17A-17C: FIG. 17A Graphs illustrating conservation of human binding sites of ROCK-2 gene with the SEQ ID NO: among species including mice and rat and FIG. 17B relative luciferase activity of 239F T cells FIG. 17C ROCK-2 expression was increased under hypoxia while MIR-373 mimic reduced effects of hypoxia.

[0205] FIGS. 18A-18B: Images illustrating FIG. 18A EphrinB2 expression in ISX-9-CPC and FIG. 18B EphB4 expression in normal heart and infarcted heart.

[0206] FIGS. 19A-19G: miR-373 mimic improved cardiac function and angiogenesis and attenuated cardiac fibrosis after MI. FIG. 19A Representative M-mode echocardiography images from miRNA mimic negative control- (NC-) treated mice and miR-373 mimic-treated mice 30 days post-MI. FS FIG. 19B and EF FIG. 19C are shown; n=8, in the NC group and n=7 in the miR-373 mimic group. FIG. 19D Representative Masson's trichrome-stained sections of hearts from NC-treated mice and miR-373 mimic mice. FIG. 19E Quantitative analysis of fibrosis post-MI. FIG. 19F Vessel density was assessed by -SMA-positive staining (green) of vascular structures. FIG. 19G Quantitative estimate of arteriole density. n=3 in each group.

[0207] FIGS. 20A-20B: Show the effect of three small molecules (ISX9, Danzol, Givinostat) on expression of cardiac and skeletal muscle genes in FIG. 20A & FIG. 20B. Real time PCR on dystrophin on small molecule (ISX9, GIV) expression in IPS cells in FIG. 20B.

[0208] FIGS. 21A-21D show that cardiac fibrobrast derived human iPS cell colonies were dissociated with accutase and plated in the presence of Y27632. FIG. 21A schematically shows cell culture conditions for the generation of cardiac fibroblasts from human iPSCs. Briefly, to generate muscle progenitor cells (MPCs) from hiPSC in vitro, human Induced Pluripotent Stem (iPS) Cells (ATCC ACS-1021) induced from human cardiac fibroblasts were cultured with mTeSR1 (STEMCELL Technologies Inc.) on Vitronectin XF (STEMCELL Technologies Inc.) coated 6-well plates. iPS Cells were passaged every 4 to 6 days with ReLeSR (STEMCELL Technologies Inc.). For differentiation of iPS Cells into MPCs, iPS Cells were dissociated into single cells with ACCUTASE (STEMCELL Technologies Inc.) into single cells and seeded at 110.sup.5 cells/cm.sup.2 with mTeSR1 supplemented with 5 M RHO/ROCK pathway inhibitor (Y-27632, STEMCELL Technologies Inc.). After 24 hr, the medium was changed to fresh mTeSR1. mTeSR1 was refreshed daily during first 3 days. After 3 days, culture medium was changed to mTeSR1 supplemented with 20 M ISX-9 (MedChemExpress). The medium was refreshed every other day. After 6 days, the medium was switched to RPMI 1640 Medium (Thermo Fisher Scientific) supplemented with N-2 Supplement (Thermo Fisher Scientific) and 20 M ISX-9 and refreshed every other day for another 3 to 6 days. Small molecules (Isx9 & GIV) were applied to initiate differentiation and analysed at day 9. FIG. 21B shows relative skeletal muscle gene expression by the treatment of Isx9 & Giv. FIG. 21C and FIG. 21D show the muscle genes (PAX3, PAX7, MYF5, MYOG, MYOD), overexpression superiority in particular of ISX-9.

[0209] FIGS. 22A-22E: Characterization of EV from iPSC and CPCISX-9. FIG. 22A Secretion of EV from the CPCISX-9 as imaged by electron microscopy. Inset shows higher magnification of secreted EV (small black arrows). Blue arrows point to EV exiting from the cells. Bar=1 m. FIG. 22B EV isolated from iPSC and CPCISX-9 visualized by transmission electron microscopy (TEM). Scale bar=200 nm. FIG. 22C Representative images of western blot for Tsg101, CD9, Hsp70, flotillin-1, and calnexin in EV lysates. FIG. 22D Average size of EV as measured by TRPS. No significant difference in average size of EV from iPSC and CPCISX-9 was observed. FIG. 22E Representative graph of size distribution of EV from iPSC and CPCISX-9 as detected by TRPS.

[0210] FIGS. 23A-23D: miRNA expression profiling and validation of microarray data. miRNA expression profiling and validation of microarray data. FIG. 23A Outline of experimental procedure. FIG. 23B Heatmap analysis of microarray data showing significant upregulation of miRNAs in EV-CPCISX-9 compared with EV-iPSC, EV-EB, or EV-control-CPC. Red or blue colors indicate differentially up- or downregulated miRNA, respectively (P<0:05). n=3. FIG. 23C Biological process of Gene Ontology (GO) enrichment analysis based on miRNA-targeted genes. GO enrichment was analyzed with mirPath v.3 software. GO biological process includes biological processes, molecular function, and cellular component of upregulated and downregulated genes. FIG. 24D Validation of microarray data using real-time PCR. Quantitative results showing significant expression of miR-373, miR-367, miR-520, miR-548ah, and miR-548q in EV-CPCISX-9. RNA samples were from three individual experiments. *P<0:001.

[0211] FIGS. 24A-24G: Fibrotic gene expression in fibroblasts after TGF- stimulation. Fibrotic gene expression in fibroblasts after TGF- stimulation. FIG. 24A Effects of EV-CPCISX-9 on fibrotic gene expression: role of miR-373. n=6. FIG. 24B Transdifferentiation of lung fibroblasts and dermal fibroblasts into myofibroblasts by hypoxia for 72 has detected by immunostaining for -smooth actin (-SMA): effects of EV-CPCISX-9 and miR-373 mimic pretreatment. Bar=50 m. FIG. 24C Schematic representation of the luciferase reporter constructs. FIG. 24D Sequence alignment of miR-373 with the human wild-type (WT) ROCK-2 3-UTR and GDF-11 3-UTR and mutated reporters. The seed sequence (red) is highlighted. FIG. 24E Relative luciferase activity (relative, firefly luciferase activity/Renilla luciferase activity) of 293FT cells cotransfected with WT 3-UTR-ROCK-2 or GDF-11 and mutant 3-UTR-ROCK-2 or GDF-11 and miR-373 mimics vs. NC. **P<0:01, n=4. UTR: untranslated region; miRNA: microRNA; NC: negative control; WT: wild type. FIG. 24F 72 h hypoxia increased GDF-11 and ROCK-2 mRNA expression in lung fibroblasts: effects of pretreatment with miR-373 mimic. ***P<0:001. n=6. FIG. 24G Pretreatment of fibroblasts during hypoxia with EV-CPCISX-9 significantly decreased the upregulation of GDF-11 and ROCK-2 similar to pretreatment with miR-373 mimic. ***P<0:001. n=6.

[0212] FIGS. 25A-25D: CPCISX-9-derived EV promoted cardiomyocyte proliferation and angiogenesis after myocardial infarction (MI) in mice. FIG. 25A Representative image of Ki67-positive cardiomyocytes (cTnT positive) in EV-CPCISX-9-treated mouse hearts 30 days after MI. Bar=50 m. FIG. 25B Quantitative estimate of proliferating cardiomyocytes as determined by Ki67 staining in the peri-infarct region 30 days after myocardial infarction. PBS group: n=940 cardiomyocytes from 3 hearts; EV-iPSC group: n=950 cardiomyocytes from 3 hearts; EV-CPCISX-9 group, n=951 cardiomyocytes from 3 hearts. * vs. the PBS group, P<0:05; # vs. the EV-iPSC group, P<0:05. FIG. 25C Representative images of arteriole density in the peri-infarct area 4 weeks after MI. Arterioles were identified by -SMA-positive staining (green) of vascular structures. Bar=100 m. FIG. 25D Quantitative analysis of arteriole density in different treatment groups. * vs. the PBS group, P<0:05; # vs. the EV-iPSC group, P<0:05, n=3.

[0213] FIGS. 26A-26G: CPCISX-9-derived EV reversed cardiac remodeling in infarcted mice. FIG. 26A Representative M-mode echocardiography images from three groups 30 days after MI. LVDs FIG. 26B, LVDd FIG. 26C, EF FIG. 26D, and FS FIG. 26E are shown. * vs. the PBS group, P<0:05; # vs. the EV-iPSC group, P<0:05. PBS group: n=10, EV-iPSC group: n=9, and EV-CPCISX-9 group: n=11. EF: ejection fraction; FS: fractional shortening; LVDd: diastolic left ventricular dimensions; LVDs: systolic left ventricular dimensions. FIG. 24F Representative Masson's trichrome-stained sections of hearts from the three groups. FIG. 24G Quantitative estimate of fibrosis. * vs. the PBS group, P<0:05; # vs. the EV-iPSC group, P<0:05,n=4.

[0214] FIGS. 27A-27G: miR-373 mimic improved cardiac function and angiogenesis and attenuated cardiac fibrosis after MI. FIG. 27A Representative M-mode echocardiography images from miRNA mimic negative control- (NC-) treated mice and miR-373 mimic-treated mice 30 days post-MI. FS FIG. 27B and EF FIG. 27C are shown; P<0:001, n=8 in the NC group and n=7 in the miR-373 mimic group. FIG. 27D Representative Masson's trichrome-stained sections of hearts from NC-treated mice and miR-373 mimic mice. FIG. 27E Quantitative analysis of fibrosis post-MI. FIG. 27F Vessel density was assessed by -SMA-positive staining (green) of vascular structures. Bar=100 m. FIG. 27G Quantitative estimate of arteriole density. P<0:05. n=3 in each group. Schematic depiction of mechanisms of protection by EV-CPCISX-9: role of miR-373 in suppressing fibrosis by targeting two genes, GDF-11 and ROCK-2, and inhibiting myofibroblast differentiation. Myocyte proliferation and angiogenesis were also promoted by EV-CPCISX-9

[0215] FIG. 28: Schematic depiction of mechanisms of protection by EV-CPCISX-9: role of miR-373 in suppressing fibrosis by targeting two genes, GDF-11 and ROCK-2, and inhibiting myofibroblast differentiation. Myocyte proliferation and angiogenesis were also promoted by EV-CPCISX-9.

[0216] FIGS. 29A-29C: Generation of muscle progenitor cell (MPC) from human iPSC using small molecules FIG. 29A Schematic outline of generation of MPC from human PSC using combination of CHIR99021 and givinostat or CHIR99021 only. FIG. 29B Morphology of differentiating cells from 4 human iPSC lines (CF-iPSC, DF-iPSC-1, DF-iPSC-2 and DMD-iPSC) at 7 days. Bar=200 m. FIG. 29C Morphology of replated MPC and differentiated myotubes from 4 human iPSC lines at day 14. Bar=200 m.

[0217] FIGS. 30A-30B: Characterization of givinostat-induced MPC. Characterization of givinostat-induced MPC. FIG. 30A The treated hiPSC at day 14 expressed Pax7 and desmin. FIG. 30B The differentiated myotubes expressed MF20 as shown by immunostaining. Bar=50 m.

[0218] FIGS. 31A-31H: Givi-MPC exhibited superior proliferation and migration capacity. FIG. 31A Representative images and quantitative estimate FIG. 31B of cell migration by adult human myoblasts, and control-MPC, Givi-MPC (arrow). Cells were stained with Calcein AM (green). Bar=1 mm. FIG. Quantitative estimate of migrated cells. Givi MPC showed highest number of cells migrated compared with human myoblasts (P<0.0001) or CHIR99021 induced MPC (P<0.0001). No significant difference was observed between human myoblasts and control-MPC. FIG. 31C Heat map of the Human RT2 motility PCR Array. FIG. 31D Differentially expressed genes related to migration in Givi-MPC vs. control-MPC using human cell motility PCR array. FIG. 31E Proliferation curves of human myoblasts vs MPC using CCK-8 assay. *P<0.05; #P<0.05 vs. control-MPC. n=6. FIG. 31F Morphology of MPC colonies. Bar=500 m. Number of colonies FIG. 31G and percentage of colonies with different cell number in color FIG. 31H control-MPC: CHIR99021 induced MPC; Givi-MPC: CHIR99021 and Givinostat induced MPC.

[0219] FIGS. 32A-32E: In vivo myogenic potential of different MPC and myoblast in Mdx/SCID mice with CTX injury. FIG. 32A Dystrophin restoration in Mdx/SCID mice by MPC transplantation at 1M after CTX injury. Bar=50 m. FIG. 32B Transplanted cells were labeled with GFP (Green) and identified with human laminin staining (Red). Quantitation of engrafted fibers at 1M: Dystrophin+fibers (n=6) FIG. 32C and human laminin and GFP double positive fibers (n=3) FIG. 32D & FIG. 32E Cross-section showing pre-synaptic staining with -bungarotoxin in dystrophin positive fibers (n=3). Bar=20 m.

[0220] FIGS. 33A-33E: Givi-MPC decrease inflammation and muscle necrosis in Mdx/SCID mice 7 days after CTX injury. FIG. 33A Representative images of HE and Trichrome Masson staining in Mdx/SCID mice with human myoblasts or control-MPC or Givi-MPC transplantation 7 days after CTX injury. Black arrows indicate infiltrated inflammatory cells. FIG. 33B Quantification of muscle fiber necrosis between PBS treated collateral TA muscle or Givi-MPC treated TA muscle 7 days after CTX injury. FIG. 33C Quantification of muscle fiber necrosis of TA muscle among human myoblast or control-MPC or Givi-MPC transplantation mice 7 days after CTX injury. FIG. 33D Quantification of CD68 positive cells in TA muscle following MPC transplantation 7 days after CTX injury. FIG. 33E Representative images of macrophages (red, CD68) and human cells in TA muscle of Mdx/SCID mice with CTX injury following MPC transplantation.

[0221] FIGS. 34A-34J: Givi-MPC decrease muscle necrosis and fibrosis in Mdx/SCID mice 1M after CTX injury. FIG. 34A Representative images of HE and Trichrome Masson staining in Mdx/SCID mice after transplantation with human myoblasts or control-MPC or Givi-MPC 1M after CTX injury. Bar=500 m (4) and Bar=100 m (20). Quantification of necrotic muscle fibers after treatment with human myoblasts FIG. 34B control-MPC FIG. 34C and Givi-MPC FIG. 34D 1M after CTX injury. FIG. 34E Comparison of muscle necrosis among human myoblasts or control-MPC or Givi-MPC transplantation mdx/SCID mice. FIG. 34F Representative images of tissue stained with Sirius red from Mdx/SCID mice. Bar=100 m. Quantification of muscle fiber fibrosis in collateral. TA muscle treated with human myoblasts FIG. 34G, control-MPC FIG. 34H and Givi-MPC FIG. 34I 1M after CTX injury. FIG. 34J Muscle fibrosis after transplantation of different MPCs in mdx/SCID mice.

[0222] FIGS. 35A-35D: Givi-MPC repopulated the muscle stem cell pool. FIG. 35A Muscle cells positive for Pax7 (green) and human nuclear antigen (red) cell under the basal lamina from Mdx/SCID mice after 1M of Givi-MPC transplantation. Bar=20 m. FIG. 35B Schematic of reinjury experiment. FIG. 35C 1M after reinjury, expression of dystrophin in Givi-MPC treated TA muscle tissue and contralateral PBS treated TA muscle tissue. Bar=50 m. FIG. 35D Representative HE stained images of Givi-MPC treated TA muscle tissue and contralateral PBS treated TA muscle tissue. Bar=50 m.

[0223] FIGS. 36A-36E: Extracellular vesicles derived from Givi-MPC promoted angiogenesis. FIG. 36A Representative images of CD31 (Red) and laminin (Green) staining in Mdx/SCID mice 1M post injury. Bar=50 m. FIG. 36B Quantification of capillary density (CD31 positive capillaries). FIG. 36C Representative images of tube formation by human aortic endothelia cells (HAECs) following EV treatment from human myoblasts, or control-MPC or Givi-MPC (1 g/well, 24 well plate). HAECs were labeled with Calcein AM (Green). Bar=500 m. FIG. 36D Tube formation assay. Average tube length was analyzed from 3 biological repeated experiments. FIG. 36E Heatmap showing significant upregulation of miRs in EV derived from Givi-MPC compared to EV-human myoblasts.

[0224] FIG. 37: Generation of muscle progenitor cell (MPC) from human iPSC using small molecules. Schematic outline of control-generation of MPC from human PSC using combination of CHIR99021 and givinostat or CHIR99021 only.

[0225] FIG. 38 Engrafted Givi-MPC (GFP positive) expressed dystrophin. Bar=200 m.

DETAILED DESCRIPTION OF PERFORMED EXPERIMENTS

[0226] ISX-9 is a small molecule which has been obtained from StemCell Technologies. Each reagent was aliquoted and stored per manufacturers' guidelines and under qualified supervision. Antibodies, buffers, and primers were ordered from the manufacturer catalog number and stored per manufacturer recommendations. Chemicals were authenticated by liquid chromatography.

[0227] Induced pluripotent stem (iPS) cells may hold therapeutic promise for cardiovascular diseases. The success of effective cell based therapy may lie towards generation of cardiovascular progenitors which may allow successful regeneration of infarcted tissue and replace scar tissue will fully functional myocytes integrated with host myocardium without the risk of tumor formation\[1]. The transplanted stem cells are known to differentiate into cardiac lineage cells in a cardiac ischemic environment and improve cardiac function.

[0228] Cardiac progenitor cells (CPCs) may offer a promising avenue for cardiac repair due to their multipotency and ability to proliferate. Efficient cardiac-lineage priming with small molecules prior to hiPSC or hESC differentiation decreases not only risk of tumorgenecity by reducing numbers of undifferentiated cells, but may also limit risk of immune rejection. Besides differentiation they release multiple factors involved in anti-inflammatory, fibrotic, apoptotic, and remodeling processes and rescues the injured myocardium[2], the paracrine mechanisms may involve the release of cytokines, chemokines, and exosomes[3-9, 98].

[0229] Recent discovery of exosomes for paracrine factors may prompt new approaches to accelerate the process of regeneration together with CPCs. These progenitors were assessed for therapeutic benefits in pre-clinical mouse model. These studies were intended to generate a significant quantity of multipotent progenitor cells from iPS and their exosomes for restoring damaged myocardium without the risk of tumor formation (FIG. 1). The main hypothesis of the proposal was that derived cardiovascular progenitors supplemented with their exosomes will be more effective in successful regeneration of infarcted myocardium without the risk of tumorgenicity and immune rejection.

[0230] The initial thesis was that Human iPSC reprogrammed CPCs with a specific small molecule possessing anti-oxidative, anti-inflammatory and cardiac gene promoting properties will maximize regenerative capacity of IPSC and their derivatives. To this end, efforts associated with the initial thesis we differentiated hiPS cells with a small molecule into cardiac lineage cells.

[0231] A highly efficient single small molecule, isoxazole-9 (ISX-9) capable of transforming hiPSC into cardiac lineage cells has been identified. These CPCs are multipotent and highly proliferative and meet the needs of modern regenerative medicine. These CPCs act through anti-inflammatory, immunomodulatory, pro-survival and anti-fibrotic mechanisms [10, 11].

[0232] A novel, cell free CPC based therapeutic approach utilizing paracrine signaling for the treatment of ischemic injury was then developed. Molecular and biochemical properties of exosomes are regulated by the stem cell source and environment of their tissue of origin. Exosomes can be generated in large numbers by highly proliferative and multipotent CPCs and can salvage the infarcted heart[98]. Then identified, purified, and analyzed were the exosome cargo from ISX-9-CPCs and non ISX-9-CPCs; Furthermore, associated efforts also: determined the miRNA profile in exosomes from ISX-9-CPCs and non ISX-9-CPCs. determined the efficacy of the endogenous repair of the hypoxia-injured CPCs, cardiac myocytes and endothelial cells by exosomes and miRNA mimics and also showed that anti-apoptotic and proliferative properties promoted by ISX-9 in CPCs are expressed in exosomes[98].

[0233] We also performed hypoxic preconditioning which to enhanced the release of specific cardiogenic miRs (ie MIR-373) and bioactive proteins via exosomes to stimulate regeneration which was also previously reported in our publication[98].

[0234] Also studied was the therapeutic efficacy of ISX-9 CPCs vs non ISX-9 CPCs and their exosomes in murine MI model.

[0235] Also identified was that the molecular mechanism are cardioprotection by exosomes derived from CPCs.

[0236] The small molecule N-cyclopropyl-5-(thiophen-2-yl)-isoxazole-3-carboxamide (ISX-9) has been shown to induce cardiac lineage priming in adipose-derived stem cells, which can be differentiated into CM that improve heart function when transplanted in the mouse model of myocardial infarction[23]. It is unique chemical with diverse properties of altering gene expression. A modified ISX-9 molecule forms hydrogels in vitro that bind many RNAs and RNA-binding proteins[24, 25], as ISX-9-like compounds have the potential to elicit broad-sweeping changes in mRNA stability and gene expression. This single compound was found in instances to initiate cardiac reprogramming of iPSC into CPCs expressing cardiac transcription factors within a week (FIG. 2).

[0237] We have previously shown that human iPSC derived and reprogrammed CPCs with a specific small molecule possessing anti-oxidative, anti-inflammatory and cardiac gene promoting properties will have greater efficacy in cardiac regeneration which was shown[1,98].

[0238] We also differentiated hiPS cells with a specific small molecule ISX-9 into cardiac lineage cells.; Induced pluripotent stem cells (hiPSCs) can proliferate indefinitely in an undifferentiated state and transform into many cell types in human tissues, including the heart. Therefore, hPSCs are potentially useful in cell-based therapies for heart disease. Multiple cardiac differentiation methods have been described and these procedures need animal cells, fetal bovine serum (FBS), or various cytokines.

[0239] Cardiac progenitor cells (CPCs) offer a promising avenue for cardiac repair due to their multipotency and ability to proliferate. The CPCs possess higher proliferative capacity than differentiated CMs and survive better after transplantation due to their earlier developmental stages and their relatively low demand for oxygen.

[0240] Data has shown that the overall regeneration efficiency of CPCs may be better than that of CMs because of CPC's multipotency to differentiate into CMS and vessel cells. Current strategies of human CPCs generation include isolation from atria appendage of donors and expansion in vitro[35], derivation from hiPSCs or ESCs, which have to be converted into embryoid bodies or treated with multiple small molecules and growth factors (activin, BMP4)[36, 37].

[0241] Such current strategies are labor-intensive and time-consuming, with high production costs, which limit clinical application. Despite these weaknesses, efficient cardiac-lineage priming with small molecules prior to hiPSC or hESC differentiation may decrease not only risk of tumorgenecity by reducing numbers of undifferentiated cells, but may also limit risk of immune rejection.

[0242] Small molecules can also substitute for recombinant cytokines and unknown factors in serum[38]. A number of small molecules have been examined or screened for promotion of differentiation: a BMP signaling inhibitor, a p38MAPK signaling inhibitor, a WNT signaling activator, and WNT signaling inhibitors were all reported to promote cardiac differentiation[39-43]. More recently, functional cardiomyocyte-like cells can be generated by treating human fibroblasts with a combination of nine compounds (9C) without genetic material[44]. Amongst small molecules, ISX-9 is a unique small molecule with the ability to trigger multiple signaling pathways[47] leading to conversion of iPSC into different lineage cells.

[0243] Here, based on data it was proposed that ISX-9 strongly induces expression of mesodermal and ectodermal fates leading to formation of cardiac progenitors capable of cardiac regeneration. These CPCs exhibit spontaneous contraction within 10 days after induction.

[0244] We had two experimental designs whereby in Group 1: Commercial CPCs obtained from iCell or generated using published techniques[1,98]; Group 2: hiPSC line+ 10-20 uM ISX-9; Group 3: validation in additional hiPSC lines using ISX-9.

[0245] Data showed: hiPSCs cell line (ACS-1021) was used to generate CPCs with the treatment of ISX-9 as outlined in FIG. 1-2. hiPSCs cultured in mTeSR1 were dissociated into single cells using accutase (Invitrogen) at 37 C. for 10 min and then were seeded on to a vitronectin-coated six-well plate at 110.sup.6 cell/well in mTeSR1 supplemented with 5 uM ROCK inhibitor (Y-27632, Stem Cell Technology) for 24 h. The medium was changed to RPMI/B27 minus insulin supplemented with ISX-9 (20 uM, dissolved in DMSO) for 7 days. Within 3-day ISX-9 treatment, Nkx2.5, GATA4, ISL-1 and Mef2c are upregulated (FIG. 3). By day 7 of ISX-9 treatment cells were 96.5 2.3% Nkx2.5 positive (FACS) and were multipotent and directly differentiated into all three cardiovascular lineages, including CMs, ECs and SMCs in basal differentiation conditions without any specific induction signaling molecules (FIG. 4). For endothelial cells (ECs) differentiation, culture medium was switched to EGM-2V medium (Lonza) for another 10 days. For smooth muscle cells (SMCs) differentiation, culture medium was replaced by DMEM-F12 medium supplemented with TGF (2 ng/ml, R&D) and PDGFBB (10 ng/ml, R&D) for 10 days.

[0246] Efforts described herein differentiated hiPSC cell lines with ISX-9 into cardiac progenitor cells and their derivatives (FIG. 2): hiPSCs cultured in mTeSR1 were dissociated into single cells using accutase (Invitrogen) at 37 C. for 10 min and were seeded on to a vitronectin-coated six-well plate at 110.sup.6 cell/well in mTeSR1 supplemented with 5 M ROCK inhibitor (Y-27632, Stem Cell Technology) for 24 h. Afterwards cells were cultured in mTesR1, and changed daily. At day 0, the medium is changed to RPMI/B27 minus insulin supplemented with ISX-9 (20 M, dissolved in DMSO) for 7 days. Next, for CMs differentiation, after 7-10 days of ISX-9 treatment, culture medium was switched to RPMI/B27 with insulin for another 10 days. Long term cultured CM were maintained in STEMdiff Cardiomyocyte Maintenance medium. For endothelial cells (ECs) differentiation, culture medium was switched to EGM-2V medium (Lonza) for another 10 days. For smooth muscle cells (SMCs) differentiation, culture medium was replaced by DMEM-F12 medium supplemented with TGF (2 ng/ml, R&D) and PDGFBB (10 ng/ml, R&D) for 10 days.

[0247] For ISX-9-CPCs characterization, mRNA-Sequencing transcriptome analysis and global miRNA expression profiles analysis was performed. Expression of cardiac transcription factors, such as GATA4, Mef2C, ISL-1, Nkx2.5 were analyzed by RT-PCR, immunostaining and FACS[FIG. 3].

[0248] Further characterized were ISX-9-CPCs derived CMs with cardiomyocyte markers by immunofluorescence, sarcomeric structure by transmission electron microscopy and intracellular electrical recording of action potentials from single beating CM by patch clamp.

[0249] We were able to obtain fully reprogrammed iPSC over 90% expressing CPCs markers. These were consistent and reproducible results over several repeats.

[0250] A novel, cell free CPC based therapeutic approach utilizing paracrine signaling for the treatment of ischemic injury was further developed.

[0251] Cell free exosomes (Ex) were generated in large numbers by highly proliferative and multipotent CPCs and these can salvage the infarcted heart. Two kinds of exosomes from CPCs were compared; exosomes from isoxazole initiated CPCs (ISX-9 CPC) vs non ISX-9 CPCs as we observed superior results due their anti-inflammatory, anti-apoptotic and proliferative properties promoted by isoxazole in CPCs compared to non ISX-9 CPCs. Exosomes are small microvesicles, 30-200 nm in diameter, and are stored within multivesicular bodies and released into the environment by fusion with the cell membrane (FIG. 5)[49, 50].

[0252] Exosomes are produced by all cells and possess adhesion molecules on their surface which may guide to target delivery of their cargo into specific cell types. They contain a distinct cargo that not only represents the cell of origin but may also be differentially-enriched in specific nucleic acid or lipid species[51]. Integrin activation, sonic hedgehog signaling, and microRNA transfer are among the possible mediators for exosome-induced biological effects[9]. exosomes are internalized as intact vesicles in target cells[52] or EVs fuse with target cells and dump their bioactive cargo (specifically microRNAs), which in turn alters the transcriptome potential of the target cells[53]. EV-target cell interaction is restricted to merely surface interaction[54].

[0253] Exosomes internalization by EC[55] have been shown. Since miRs are the major components of exosomes that regulate the function of target cells[56], plans include investigation of the differential expression of specific miRNAs residing within CPC (ISX-9, non ISX-9) exosomes vs. hiPSC by microarray and deep sequencing screening.

[0254] Also determined were the miRNA profile in exosomes from ISX-9-CPCs and non ISX-9-CPCs both under normoxic and hypoxic conditions.

[0255] We also showed that certain anti-apoptotic and proliferative properties promoted by isoxazole in CPCs are also expressed in exosomes.

[0256] Data support the notion that the endogenous exosomes generated from multipotent, proliferative, regenerative CPCs exert potentially anti-fibrotic effects by transferring their exosomes to cardiac fibroblasts (FIG. 6,[58]).

[0257] CPC-derived exosomes represent a mechanism of action of progenitors to enable endogenous self-repair of the damaged hearts by cell to cell transfer of proteins, mRNAs, and miRNAs. Data demonstrates that the primary mechanism of myocardial restoration by cardiac progenitors is both regeneration and paracrine and that the exosomes effectively salvage the injured myocardium. There is direct link/correlation between ISX-9 derived CPCs and their exosomes in their action to promote endogenous repair.

[0258] We also determined the miRNA profile of exosome cargo from ISX-9-CPCs and non ISX-9-CPCs

[0259] ISX-9-CPCs and non-ISX-9-CPCs were generated as described[1, 48, 98]. Exosomes were purified from the media by qVE size exclusion column following standard protocols, visualized by transmission electron microscopy, and western blot (FIG. 5) and conjugated to 3.92-um latex beads for flow cytometric confirmation of exosomal surface marker CD63. Exosome particle number were then quantified using qNano (IZON). There were differences in protein and/or RNA content of the different exosome populations which we compared.

[0260] Then quantified were the miRNA expression with a two-step polymerase chain reaction (PCR) process hybridized to microarrays with probes to hundreds of miRNA targets and protein contents by proteomic analysis.

[0261] Then used was a ultracentrifugation approach to copurify many other extracellular species such as protein aggregates and other vesicle types[21] which can cause inflammatory response. then were purified exosomes from other extracellular vesicles (EVs) and large protein aggregates through ultracentrifugation.

[0262] CPCs are multipotent cells capable of forming cardiac cells including myocytes, endothelial cells. These are highly proliferative and have the ability to regenerate the ischemic myocardium.

[0263] Treatment consisted of plated or 110.sup.8 of iCPCs: 1) 1*10.sup.8 non ISX-9-CPCs exosomes/well, 2) 1*10.sup.8 ISX-9-CPCs exosomes/well, 3) non ISX-9-CPCs miRNA mimic (selected from highly expressed miR) (50 nM), 4) ISX-9-CPCs miRNA mimic (selected from highly expressed miR) (50 nM), and 5) CPCs culture medium (control).

[0264] The following data was obtained: To determine which miRNA is involved in proliferation, CMs were treated with specific miRNA-373 mimic particle/compound. The number of proliferating CMs were compared amongst groups.

[0265] A protocol was developed to purify these vesicles, free from extracellular protein components and other vesicles secreted by the same cells or present in the media. Therefore, it was expected that characterization of exosome size and charge by TEM, cryo-EM, and TRPS accurately reflects the properties of the exosome populations being studied.

[0266] Further showed was that anti-apoptotic and proliferative properties promoted by isoxazole treatment in CPCs are expressed in exosomes compared to non ISX-9 CPCs.

[0267] Hypoxic preconditioning enhances release of cardiogenic miRs and bioactive proteins via exosomes to stimulate regeneration.

[0268] Studies strongly suggested specific exosomes released from the treatment of iPSC with ISX-9 included anti-fibrosis miRs important in attenuating fibrosis (FIG. 11). In this study it was hypothesized that HPC enhances survival and regeneration by CPCs by secreting bioactive molecules and cardiogenic miRs via exosomes. Isoxazole, a small molecule with biologically potent properties[1,98] was used in this research.

[0269] Stem cell therapy may offers hope for cardiac tissue repair and regeneration following heart attack. Exosomes are regarded as the critical agents of cardiac regeneration triggered by stem cells. Exosomes are nano-sized biological membrane-enclosed vesicles (30-200 nm) that contain a cell-specific cargo of proteins, lipids, and nucleic acids and act as mediators of cell-cell communication.

[0270] Efforts have successfully generated induced cardiac progenitor cells (iCPCs) & induced muscle progenitor cells (iMPCs) from human induced pluripotent stem cells (iPSCs) using a cardiogenic small molecule ISX-9 and other isoxazole based compounds such as Danazol & other non isoxazole based compounds such as Givinostat(FIGS. 8, 20, 21). Efforts have demonstrated that these iCPCs were strongly resistant to oxidative stress in ischemic environment (FIG. 8) and showed strong engraftment, growth and long-term improvement on cardiac function after transplantation in the infarcted mouse heart after three months (FIG. 9). The transfer of the antioxidative, proliferation and regenerative properties of Isoxazole into the exosomes of ISX-9-CPCs may be important to successful propagation of ISX-9-CPCs in the infarcted area. Therefore, exosomes secreted by these CPCs (FIG. 5) also exhibit strong cardioprotective effects in damaged heart. Work included testing whether ISX-9-CPCs had any effects on myocyte proliferation, fibrosis and function post MI. The data was compelling and supportive of our hypotheses (FIG. 10-11). miRNA-373, one of the enriched miRNAs, showed strong anti-fibrosis effects in vitro and in vivo and ultimately improved cardiac function. It was proposed that the exosomes and the corresponding miRNAs are expected to provide high levels of pleiotropic effects to reduce fibrosis and promote regeneration in the scar area.

[0271] Efforts included studying the in vivo fate of transplanted CPCs in murine heart model and; evaluating the therapeutic efficacy of exosomes together with their parent CPCs on ischemic injury and fibrosis in the infarcted heart.

[0272] MI was created in NOD/SCID mice by ligating left anterior descending coronary artery. Post-engraftment tracking of the transplanted cells and determination of their fate, the cells were labeled with PKH26 (Sigma, Product #PKH26-GL) according to manufacturer's instructions as described earlier[83-85]. For cell transplantation and their fate determination, mice were randomized in groups (n=8 each): 1) control DMEM medium; 2) hiPSCs; 3) non ISX-9-CPCs or commercial CPCs; 4) ISX-9-CPCs; 5) non ISX-9-CPCs plus their exosomes 6) ISX-9-CPCs plus their Ex; 7) 8) ISX-9-CPCs (pretreated with GW4869 which block exosomes release from CPC); 9) Group-6=CPC-derived endothelial cells.

[0273] Cardiac function after MI by serial high-resolution two-dimensional echocardiography were also recorded.

[0274] Data showed that ISX-9-CPCs expressed ephrin B2 (FIG. 12A, while inflammatory endothelial cells in infarcted hearts strongly expressed EphB4[87] and our data also showed acute infarcted heart expressed EphB4 (FIG. 12B).). We have found that EphB4/ephrinB2 signaling is involved in guiding the delivery of Exosomes to the infarcted heart leading to angiogenesis.

[0275] Exosomes were delivered by intromyocardium injection (2*10.sup.9 particles) and/or IV injection (2*10.sup.10 particles) after ligation; 1*10.sup.6 iCPCs were injected into the border zones immediately after induction of MI. Exosomes were be labeled using RNASlect Green Fluorescent cell stain to analyze exosome retention/distribution in the heart. Surviving transplanted iCPCs will be analyzed by labeling iCPCs with LuminiCell Tracker before injection. Cardiac function after MI by serial high-resolution two-dimensional echocardiography was performed.

[0276] It was expected that the overall regeneration efficiency of CPCs would be better than that of CMs because of CPC's multipotency to differentiate into CMS and vessel cells which supports our previously published strong ejection fraction improvement from our CPC's in a murine model[1].

[0277] Specific Methods used. Methods are referred to by previous publications c[1] cited herein[1,98,99]. Total RNA were isolated from cells using RNeasy Mini Kit. RT-PCR and PCR[83, 85, 95]; miRNA isolation and detection and transfection[75]; Flow cytometry[75];PCR for sry-gene[83]. Heart function[96], angiogenesis assays[95] miRNAmicroarray[83].

[0278] For post-engraftment tracking of the transplanted cells and determination of their fate, the cells were labeled with PKH26 (Sigma, Product #PKH26-GL) according to manufacturer's instructions and also previously reported[1,98].

[0279] Procedure for echocardiography in mice is described as follows. Echocardiography was performed with mice first anesthetized in an anesthesia chamber at room temperature. Induction of anesthesia is with isoflurane (4%). After the animals have been anesthetized (approximately 1 minute), they are weighed, and placed supine onto a heated (98 F.) imaging platform.

[0280] The imaging system used is HDI-5000 SONOS-CT (HP) ultrasound machine with a 7-MHz transducer. The heart was imaged in the two-dimensional mode in the parasternal long-axis and/or parasternal short-axis views which were subsequently used to position the M-mode cursor perpendicular to the ventricular septum and left ventricle posterior wall, after which M-mode images were obtained.

[0281] For each animal, measurements were obtained from 4-5 consecutive heart cycles. Measurements of ventricular septal thickness (VST), left ventricle internal dimension (LVID), and left ventricle posterior wall thickness (LVPW) were made from two-dimensionally directed M-mode images of the left ventricle in both systole and diastole. The average value from all measurements in an animal were used to determine the indices of left ventricle contractile function, i.e., left ventricle fractional shortening (LVFS) and left ventricle ejection fraction (LVEF) using the following relations LVFS=(LVEDdLVESd)/LVEDd100 and LVEF=[(LVEDd.sup.3LVESd.sup.3)/LVEDd.sup.3]100 and expressed as percentages. The scoring system we utilize is patterned after the American Society of Echocardiography's scoring system used conventionally in interpreting clinical echocardiographic studies.

Detailed Description of Actual Data

[0282] Previously identified were highly efficient small molecules, isoxazole (ISX) and isozazole-9 (ISX-9), that are capable of transforming hiPSC into multipotent cardiac lineage cells that are highly proliferative and generate large numbers of exosomes (EX) containing miRNAs to elicit anti-oxidant, anti-inflammatory, immunomodulatory, pro-survival and anti-fibrotic effects. Now used is ISX-9 coupled with hypoxic preconditioning to generate large numbers of multipotent CPC and their EX from hiPSC for therapeutic testing in a pre-clinical animal model of myocardial infarction. Shown herein is that hiPSC pharmacologically reprogrammed into CPC and supplemented with their EX are optimally effective to regenerate infarcted myocardium.

[0283] Also shown is:

[0284] 1) We have maximized the vascular and myocyte lineage differentiation by reprogramming hiPSC with ISX-9, thereby enhancing anti-oxidative, anti-inflammatory and cardiac gene promoting properties. HiPSC was differentiated into multipotent CPC with ISX-9.

[0285] 2) A novel, cell free therapeutic approach utilizing CPC exosomes for promoting angiomyogenesis and cytoprotection of ischemic heart was developed. These CPC derived EXosomes actually protect CPC, cardiac myocytes and endothelial cells against ischemia.

[0286] Cell Culture:

[0287] Human iPSC cell line (ACS-1021, ATCC, USA) was maintained in mTeSR1 media (Stem Cell Technology) on vitronectin coated six-well plates with daily medium changes. CPCs were differentiated in RPMI/B27 minus insulin supplemented with ISX-9 (20 M, dissolved in DMSO, Stem Cell Technology) for 7 days. Embryoid bodies (EB) were generated using the hanging drop method in RPMI/B27 minus insulin medium. Commercial human CPCs derived from human iPS cells (Catalog: R1093, Cellular Dynamics International) were maintained in serum-free William's E Medium supplemented with Cocktail B (CM400, Life Technologies).

[0288] Isolation of Exosomes:

[0289] Human iPSC cell line ACS-1021 (ATCC, USA), and CPCs induced by ISX-9 were cultured as described(15). In some cases, EB and commercial human CPCs were also cultured. Conditioned media was collected and exosomes were isolated by centrifugation at 3000 rpm for 30 min to remove cells and debris, followed by filtration through a 0.22 m filter to remove the remaining debris. Then the medium was further concentrated to 500 l using Amicon Ultra-15 100 kDa centrifugal filter units (Millipore). Isolation of exosomes in the concentrated medium was carried out through qEV size exclusion columns (Izon Science). Exosome fractions were collected and concentrated by Amicon Ultra-4 10 KDa centrifugal filter units to a final volume of <100 l. The purified exosomes were stored at 80 C. and subsequently characterized by particle size, exosome markers and electron microscopy.

[0290] Particle Size and Concentration:

[0291] Particle size and concentration distribution were performed using tunable resistive pulse sensing (TRPS) technique with a qNano instrument (Izon Science). Briefly, the number of particles were counted (at least 600 to 1000 events) using 20 mbar pressure and NP200 nanopore membranes stretched between 46.5-47.5 mm. Calibration was performed using known concentration of beads CPC200 (diameter: 210 nm). Data were processed using Izon Control Suite software.

[0292] Transmission Electron Microscopy:

[0293] Exosome pellets were fixed with 4% paraformaldehyde (PFA). Following a total of 8 washes using PBS, grids were contrasted with a uranyl-oxalate solution for 5 minutes, and transferred to methyl-cellulose-uranyl acetate for 10 minutes on ice as previously described(16). Samples were examined on a JEOL JEM-1220 transmission electron microscope (TEM) (JEOL USA, Inc.)

[0294] Exosome Uptake by Fibroblasts:

[0295] To track exosome uptake by cultured fibroblasts, purified exosomes were labeled with PKH26, a red membrane dye (Sigma-Aldrich), according to the manufacturer's protocol. Briefly, 300 l of exosomes was suspended into 100 l of Diluent C, which was mixed with 1.4 l of PKH26 dye. The labeling reaction was stopped by adding an equal volume of exosome-free FBS. Exosomes were pelleted using an exosomes column. The cultured fibroblasts in the slide chamber were incubated with labeled exosomes at 37 C. for 24 h. After incubation, cells were stained with Calcein AM (5 M). Cells were fixed with 2% formaldehyde for 5 min and mounted with DAPI containing prolong Gold Antifade medium (Thermo Fisher Scientific). Images were taken with FV1000 confocal microscope (Olympus, Japan).

[0296] Cell Transfection and In Vitro Fibrosis Assay:

[0297] Experiments were performed using CPC.sup.ISX-9 grown in RPMI/B27 minus insulin, 25 nM miRNA-373 mimic, anti-miRNA-373, negative controls and RNAiMAX (Invitrogen) according to the manufacturer's instructions. miRNA-373 mimic and anti-miRNA-373 (inhibitor) were synthesized by Ambion (Life Technologies). The sequence depicted as SEQ ID NO:13 of miRNA-373 inhibitor was as follows: Anti-miRNA-373, 5-ACACCCCAAAAUCGAAGCACUUC-3, miRNA mimic negative control (#4464066, Ambion) and miRNA inhibitor negative control (#4464076, Ambion) were obtained from Life Technologies company. After 24 hour transfection, cells remained in culture for 24 hours and exosomes from different cell groups were collected for experimentation. The transfection efficiency was analyzed using real-time PCR. In order to test anti-fibrotic potential of exosomal miRNA-373 from CPC.sup.ISX-9, fibroblasts were co-cultured with exosomes (1*10.sup.8/ml) from anti-miR373 inhibitor treated CPC.sup.ISX-9 or negative control treated CPC.sup.ISX-9 or miRNA-373 mimic for 48 h, and then fibroblasts were grown in serum free DMEM medium with or without TGF- (10 ng/ml, R&D) for 48 h. Expression of pro-fibrotic genes was analyzed by real-time PCR. For the hypoxia assay, lung fibroblasts and dermal fibroblasts in culture were randomly divided into five groups and treated with: miRNA-NC, anti-miR, miRNA-373 mimic, Exo-CPC.sup.ISX-9 and Exo-CPC.sup.ISX-9+anti-miRNA-373. After 24 hours of different pretreatments, cells were subjected to 1% O.sub.2 in hypoxic chamber (INVIVO.sub.2500) for 72 hours. Then, cells were fixed with 4% formaldehyde for 10 mins, and stained with -SMA (ab5694, abcam, 1:200). Signals were visualized with Alexa Fluor 488 secondary antibodies (Life Technologies).

[0298] miRNA Array Analysis:

[0299] The NanoString nCounter Human v3 miRNA Expression Assay was used to perform the microRNA profiling analysis.

[0300] miRNA Target Gene Prediction, Gene Ontology(GO) Analysis and Luciferase Activity Assay:

[0301] miRNA target genes prediction and gene ontology analysis were carried out using DIANA mR-microT and mirPath software.

[0302] Myocardial Infarction Model:

[0303] Animal experiments were carried out both at University of Illinois at Chicago and Augusta University according to experimental protocols approved by the University of Illinois at Chicago and Augusta University Animal Care and Use Committee, and the methods were performed in accordance with the guide for the Care and Use of Laboratory Animals by the Institute of Animal Resources. MI model was generated as previously described(15). Briefly, MI was induced in 8-9-week-old NOD/SCID mice (The Jackson Laboratory) or C57/B6 mice which were anaesthetized with 2% isoflurane (isoflurane USP, HENRY SCHEIN), intubated and ventilated. The left anterior descending coronary artery (LAD) was permanently ligated with a prolene #8-0 suture. 10 mins after LAD ligation, exosomes (1*10.sup.12/ml) from hiPSC or CPC.sup.ISX-9 were injected into the myocardium along the border zone with a total of 20 l. The same volume of PBS was injected in the control group. miRNA-373 mimic in vivo transfection was performed in C57/B6 mice. In vivo-jetPEI system (POLYPLUS TRANSFECTION SA) was used for intracardiac miRNA delivery. 200 pMoles miRNA-373 mimic complexed with in vivo-jetPEI at a N/P ratio of 7 in a volume of 20 l were injected into the myocardium along the border zone immediately after LAD ligation.

[0304] Echocardiography:

[0305] Echocardiography was performed in mice anesthetized mildly with inhaled isoflurane (0.5%) using Philips iE33 ultrasound machine, equipped with L15-7io probe as described previously(15).

[0306] Histology:

[0307] Histological analysis was performed in randomly selected hearts from mice subjected to MI and treatment groups (PBS, Exo-hiPSC or Exo-CPC.sup.ISX-9 (n=3 per group). Mice were sacrificed after 1 month of treatment with exosomes.

[0308] Western Blot:

[0309] Exosomes and cell extracts were lysed with radio immunoprecipitation assay (RIPA) buffer supplemented with Complete Protease Inhibitor Mixture tablets (Roche Diagnostics). Protein concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Scientific).

[0310] RNA Extraction and Real Time PCR:

[0311] Total RNA from exosomes was isolated using miRNeasy Micro Kit (Qiagen). Reverse transcription was performed using miScript II RT Kit (Qiagen).

[0312] Statistical Analysis:

[0313] Data are expressed as mean SD. Test for normality of data was performed. Statistical analysis of differences was compared by ANOVA with Bonferroni's correction for multiple comparisons. Comparisons between two groups were evaluated with Students t-test. A probability value of P<0.05 was considered statistically significant. Statistical analyses were performed using Graphpad Prism 6.0 (Chicago, USA).

[0314] Based on the functional improvement observed with exosome treatment alone, we tested the capacity of CPC-EX to enhance cell transplantation. Indeed, the combination of ISX-9-CPCs plus EX further increased LVEF and LVFS, with continued improvement observed at 30 days post MI (FIG. 13).

[0315] The combined administration of CPC with their EX adjunctively enhanced CPC survival and engraftment in the ischemic myocardium (FIG. 13).

[0316] Moreover, ISX-9-CPC exhibited strong protection against apoptosis both in in vivo and in vitro conditions (FIG. 1). ISX-9-CPC are highly resistant to ischemia and able to efficiently engraft and grow in the oxidative environment. ISX-9-CPC have displayed resistance to oxidative and inflammatory stress, with lower percentage of apoptotic cells.

[0317] Treatment with miRNA-373 mimic, one of the miRNAs enriched in EX, prevented fibroblast stimulation by TGF-, thereby reducing expression of fibrotic genes and their transdifferentiation into myofibroblasts(FIG. 16), while its in vivo administration promoted new vessel formation and prevented fibrosis and ultimately improved cardiac function. Under ischemic conditions it was shown that fibroblasts assume a myofibroblast-like phenotype which was surprisingly blocked by ISX-9-CPC EX and its major exosomal miRNA(miRNA-373) mimic (Fig.). Moreover, similar transformation in human aortic endothelial cells (HAEC) with TGF- stimulation exhibiting -SMA expression occurred (Fig.) and was also blocked by ISX-9-CPC EX and miRNA-373 mimic. FIG. 12: Treatment with miR-373 mimic, one of the miRNAs enriched in EX, prevented fibroblast stimulation by TGF-, thereby reducing expression of fibrotic genes and their transdifferentiation into myofibroblasts, while its in vivo administration promoted new vessel formation and prevented fibrosis and ultimately improved cardiac function. It is shown here that under ischemic conditions, fibroblasts assume a myofibroblast-like phenotype which was surprisingly blocked by ISX-9-CPC EX and its major exosomal miR (miR-373) mimic (FIG. 12). Moreover, similar transformation in human aortic endothelial cells (HAEC) with TGF- stimulation exhibiting -SMA expression occurred (FIG. 12) and was also blocked by ISX-9-CPC EX and miR-373 mimic. We have identified ROCK-2 as a target gene of miRNA-373 using luciferase reporter assay (FIG. 17B). Thus, exosomal miRNA-373 inhibits myofibroblast transdifferentiation and EndMT elicited by TGF- signaling via targeting the Rho/ROCK pathway.

[0318] Novel data visualizing surface marker localization by immunohistology suggest that ISX-9-CPC are strongly positive for EphrinB2 (FIG. 18). EphrinB2 and EphB4 belong to a large family of cell surface receptor tyrosine kinases (RTKs) signaling molecules. EphB4/EphrinB2 signaling was reported to be involved in early stage of cardiac lineage development [78], cell migration [79] and angiogenesis [80]. Data show that ISX-9-CPC express EphrinB2 (FIG. 18A), while activated endothelial cells in infarcted hearts strongly express EphB4 (FIG. 18B).].

[0319] FIG. 19 miRNA-373 mimic improved cardiac function and angiogenesis and attenuated cardiac fibrosis after MI. (A) Representative M mode echocardiography images from miRNA mimic negative control (NC) treated mice and miRNA-373 mimic treated mice 30 days post-MI. FS (B) and EF (C) are shown; (P<0.001), n=8 in NC group and n=7 in miRNA-373 mimic group. (D) Representative Masson's trichrome-stained sections of hearts from NC treated mice and miRNA-373 mimic mice. (E) Quantitative analysis of fibrosis post MI. (F) Vessel density was assessed by -SMA positive staining (green) of vascular structures. Bar=100 m. (G) Quantitative estimate of arteriole density. P<0.05. n=3 in each group

[0320] FIG. 20 demonstrates an effect of three small molecules (ISX-9, Danzol, Givinostat) on expression of cardiac and skeletal muscle genes. Real time PCR on dystrophin on small molecule (ISX9, GIV) expression in IPS cells.

[0321] FIG. 21 demonstrates that cardiac fibrobrast derived human iPS cell colonies were dissociated with accutase and plated in the presence of Y27632. FIG. 21A schematically shows cell culture conditions for the generation of cardiac fibroblasts from human iPSCs. Briefly, to generate muscle progenitor cells (MPCs) from hiPSC in vitro, human Induced Pluripotent Stem (iPS) Cells (ATCC ACS-1021) induced from human cardiac fibroblasts were cultured with mTeSR1 (STEMCELL Technologies Inc.) on Vitronectin XF (STEMCELL Technologies Inc.) coated 6-well plates. iPS Cells were passaged every 4 to 6 days with ReLeSR (STEMCELL Technologies Inc.). For differentiation of iPS Cells into MPCs, iPS Cells were dissociated into single cells with ACCUTASE (STEMCELL Technologies Inc.) into single cells and seeded at 110.sup.5 cells/cm.sup.2 with mTeSR1 supplemented with 5 M RHO/ROCK pathway inhibitor (Y-27632, STEMCELL Technologies Inc.). After 24 hr, the medium was changed to fresh mTeSR1. mTeSR1 was refreshed daily during first 3 days. After 3 days, culture medium was changed to mTeSR1 supplemented with 20 M ISX-9 (MedChemExpress). The medium was refreshed every other day. After 6 days, the medium was switched to RPMI 1640 Medium (Thermo Fisher Scientific) supplemented with N-2 Supplement (Thermo Fisher Scientific) and 20 M ISX-9 and refreshed every other day for another 3 to 6 days. Small molecules (Isx9 & GIV) were applied to initiate differentiation and analysed at day 9. FIG. 21B shows relative skeletal muscle gene expression by the treatment of Isx9 & Giv. FIGS. 21C and 21D show the muscle genes (PAX3, PAX7, MYF5, MYOG, MYOD), overexpression superiority in particular of ISX-9.

[0322] The following publication is fully incorporated into the present disclosure. [0323] miRNAs in Extracellular Vesicles from iPS Derived Cardiac Progenitor Cells Effectively Reduce Fibrosis and Promote Angiogenesis in Infarcted Heart [0324] Wanling Xuan.sup.1, Lei Wang.sup.2, Meifeng Xu.sup.3, Neal L. Weintraub.sup.1, Muhammad Ashraf.sup.1*Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, Ga., USA *Correspond to Prof Muhammad Ashraf, Email: mashraf@augusta.edu [0325] Department of Pharmacology, University of Illinois at Chicago College of Medicine, Chicago, Ill., USA [0326] Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, USA. [0327] Short title: Extracellular vesicles microRNAs in prevention of cardiac fibrosis

[0328] Abstract

[0329] Cardiac stem cell therapy offers the potential to ameliorate post-infarction remodeling and development of heart failure but requires optimization of cell-based approaches. Cardiac progenitor cell (CPC) induction by ISX-9, a small molecule possessing antioxidant, prosurvival and regenerative properties, represents an attractive potential approach for cell-based cardiac regenerative therapy. Here, we report that extracellular vesicles (EV) secreted by ISX-9-induced CPCs (EV-CPC.sup.ISX-9) faithfully recapitulate the beneficial effects of their parent CPCs with regard to post-infarction remodeling. These EV contain a distinct repertoire of biologically-active miRNAs that promoted angiogenesis and proliferation of cardiomyocytes while ameliorating fibrosis in the infarcted heart. Amongst the highly enriched miRNAs, miR-373 was strongly antifibrotic, targeting 2 key fibrogenic genes, GDF-11 and ROCK-2. miR-373 mimic itself was highly efficacious in preventing scar formation in the infarcted myocardium. Together, these novel findings have important implications with regard to prevention of post-infarction remodeling.

[0330] Keywords: cardiac progenitor cells; extracellular vesicles; microRNAs, miR-373, fibrosis, functional recovery

[0331] Introduction

[0332] Myocardial infarction (MI) and subsequent heart failure is a leading cause of death worldwide (1). Despite advances in medical and device therapies, heart failure continues to be associated with a 5-year mortality of 50%. Stem cell therapy thus offers a great potential for cardiac tissue repair and regeneration, which might ultimately improve symptoms and longevity (2).

[0333] Notably, the beneficial effects of cardiac stem cell therapy are largely attributed to a paracrine mechanism of action that involves the release of cellular factors from the transplanted stem cells (3-5). More recent studies show that these factors are packed into small membrane bound vesicles known as extracellular vesicles (EV, 30-200 nm), which can invoke a multitude of signals (6,7). The EV contents vary amongst stem cells. Cardiac progenitor cells (CPCs) are of particular interest due to their inherent properties of cell protection, cell development, differentiation, and desirable effects imparted into the host tissue (8-10). EV from newborns improved ventricular remodeling post-MI significantly more than those derived from aging patients (11). Similarly, EV secreted from young cardiosphere-derived cells exerted stronger anti-senescent effects than those derived from aged animals (12).

[0334] Recent studies demonstrated that effects of CPCs on cardiac repair and regeneration can be faithfully recapitulated by their EV (6,13). Multiple miRNAs in EV act as mediators of cell-cell communication within the cardiovascular system (2) and can be transferred into recipient cells to regulate gene expression, thus leading to cardioprotection (11,13,14). We reported that a small molecule, ISX-9, could render CPCs (CPC.sup.ISX-9) highly resistant to oxidative stress, thus permitting better survival and engraftment in the infarcted myocardium (15). Development of CPC.sup.ISX-9 may represent a significant advance in the cardiac stem cell field, as ISX-9 treatment circumvents the need to genetically reprogram the cells in order to enhance their function. Since CPC.sup.ISX-9 are well positioned for therapeutic application in humans, characterizing EV secreted from these cells is not only important to provide insight into their mechanisms of action, but also may help to identify novel miRNAs involved in cardioprotection.

[0335] Since the EV cargo contents are unique to each cell type and consequently their effectiveness is variable. Considering this limitation, we have generated multipotent CPCs from human induced pluripotent stem cells (hiPSCs) using a unique small molecule with anti-oxidant and regenerative properties capable of successfully propagating in the infarcted myocardium. Since CPC are the cells of choice for regeneration, their EV would be considered to be more effective in cardiac repair than EV from non CPC. Therefore, the purpose of the study was to exploit EV from hiPSC-CPC induced with ISX-9 and not the role of ISX-9 per se on EV release from CPC. Here, we tested the hypothesis that EV secreted by ISX-9-induced CPCs (EV-CPC.sup.ISX-9) will be highly efficacious in cardiac repair owing to the unique properties of their parent cells. EV-CPC.sup.ISX-9 exerted strong effects on fibrosis and angiogenesis in the infarcted myocardium of mice. Mechanistically, we identified miR-373 enriched in EV-CPC.sup.ISX-9, which elicited strong anti-fibrotic effects by targeting two genes, growth differentiation factor 11 (GDF-11) and Rho-associated coiled-coil containing kinase-2 (ROCK-2), and showed that miR-373 mimic effectively inhibits post-infarct cardiac remodeling.

[0336] Materials and Method

[0337] Cell Culture

[0338] Human iPSC cell line (ACS-1021, ATCC, USA) was maintained in mTeSR1 media (Stem Cell Technology) on vitronectin coated six-well plates with daily medium changes. Cells were passaged with ReLeSR reagent every 4-7 days according to the manufacturer's protocol (Stem Cell Technology). For CPC generation, briefly, hiPSCs maintained on vitronectin coated six-well plates in mTeSR1 media (Stem Cell Technology) were dissociated into single cells using accutase (Invitrogen) at 37 C. for 10 min and then were seeded on to a vitronectin-coated six-well plates at 110.sup.6 cells/well in mTeSR1 supplemented with 5 M ROCK inhibitor (Y-27632, Stem Cell Technology) for 24 h. The following day, cells were cultured in mTesR1 with daily medium change for 3 days. Afterwards, the medium was switched to RPMI/B27 minus insulin supplemented with ISX-9 (20 M, dissolved in DMSO, Stem Cell Technology) for 7 days. Embryoid bodies (EB) were generated using the hanging drop method in RPMI/B27 minus insulin medium. Human dermal fibroblast cell line (CC-2511) and lung fibroblast cell line (CC-2512) were obtained from Lonza Company. Briefly, fibroblasts were maintained in FibroGRO Complete Media (Millipore Sigma). Cells were passaged with accutase; passages 2-4 were used for experiments. Commercial human CPCs (control-CPC) derived from human iPS cells (Catalog: R1093, Cellular Dynamics International) were maintained in serum-free William's E Medium supplemented with Cocktail B (CM400, Life Technologies). Passage 2 was used for experiments.

[0339] Isolation of EV

[0340] Human iPSC cell line ACS-1021 (ATCC, USA), and CPCs induced by ISX-9 were cultured as described(15). In some cases, EB and commercial human CPCs were also cultured. Conditioned media was collected and EV were isolated by centrifugation at 3000 rpm for 30 min to remove cells and debris, followed by filtration through a 0.22 m filter to remove the remaining debris. Then the medium was further concentrated to 500 l using Amicon Ultra-15 100 kDa centrifugal filter units (Millipore). Isolation of EV in the concentrated medium was carried out through qEV size exclusion columns (Izon Science). EV fractions were collected and concentrated by Amicon Ultra-4 10 KDa centrifugal filter units to a final volume of <100 l. The purified EV were stored at 80 C. and subsequently characterized by particle size, EV markers and electron microscopy.

[0341] Particle Size and Concentration Distribution Measurement with Tunable Resistive Pulse Sensing

[0342] Particle size and concentration distribution were performed using tunable resistive pulse sensing (TRPS) technique with a qNano instrument (Izon Science). Briefly, the number of particles were counted (at least 600 to 1000 events) using 20 mbar pressure and NP200 nanopore membranes stretched between 46.5-47.5 mm. Calibration was performed using known concentration of beads CPC200 (diameter: 210 nm). Data were processed using Izon Control Suite software.

[0343] Transmission Electron Microscopy

[0344] EV pellets were fixed with 4% paraformaldehyde (PFA). Following a total of 8 washes using PBS, grids were contrasted with a uranyl-oxalate solution for 5 minutes, and transferred to methyl-cellulose-uranyl acetate for 10 minutes on ice as previously described (16). Samples were examined on a JEOL JEM-1220 transmission electron microscope (TEM) (JEOL USA, Inc.)

[0345] EV Uptake by Fibroblasts

[0346] To track EV uptake by cultured fibroblasts, purified EV were labeled with PKH26, a red membrane dye (Sigma-Aldrich), according to the manufacturer's protocol. Briefly, 300 l of EV was suspended into 100 l of Diluent C, which was mixed with 1.4 l of PKH26 dye. The labeling reaction was stopped by adding an equal volume of EV-free FBS. Exosome Spin Columns (Cat. 4484449, Thermo Fisher Scientific) was used to remove unincorporated PKH26. The cultured fibroblasts in the slide chamber were incubated with labeled EV at 37 C. for 24 h. After incubation, cells were stained with Calcein AM (5 M). Cells were fixed with 2% formaldehyde for 5 min and mounted with DAPI containing prolong Gold Antifade medium (Thermo Fisher Scientific). Images were taken with FV1000 confocal microscope (Olympus, Japan).

[0347] Cell Transfection and In Vitro Fibrosis Assay

[0348] Experiments were performed using CPC.sup.ISX-9 grown in RPMI/B27 minus insulin, 25 nM miR-373 mimic, anti-miR-373, negative controls and RNAiMAX (Invitrogen) according to the manufacturer's instructions. miR-373 mimic and anti-miR-373 (inhibitor) were synthesized by Ambion (Life Technologies). The sequence of miR-373 inhibitor identified as SEQ ID NO:13 was as follows: Anti-miR-373, 5-ACACCCCAAAAUCGAAGCACUUC-3. miRNA mimic negative control (#4464066, Ambion) and miRNA inhibitor negative control (#4464076, Ambion) were obtained from Life Technologies company. After 24 h transfection, cells remained in culture for 24 h and EV from different cell groups were collected for experimentation. The transfection efficiency was analyzed using real-time PCR. In order to test anti-fibrotic potential of miR-373 from EV-CPC.sup.ISX-9, fibroblasts were co-cultured with EV (1*10.sup.8/ml) from anti-miR373 inhibitor treated CPC.sup.ISX-9 or negative control treated CPC.sup.ISX-9 or miR-373 mimic for 48 h, and then fibroblasts were grown in serum free DMEM medium with or without TGF- (10 ng/ml, R&D) for 48 h. Expression of pro-fibrotic genes was analyzed by real-time PCR. For the hypoxia assay, lung fibroblasts and dermal fibroblasts in culture were randomly divided into five groups and treated with: miR-NC, anti-miR, miR-373 mimic, EV-CPC.sup.ISX-9 and EV-CPC.sup.ISX-9-9+anti-miR-373. After 24 h of different pretreatments, cells were subjected to 1% O.sub.2 in hypoxic chamber (INVIVO.sub.2 500) for 72 h. Then, cells were fixed with 4% formaldehyde for 10 mins, and stained with -SMA (ab5694, abeam, 1:200). Signals were visualized with Alexa Fluor 488 secondary antibodies (Life Technologies).

[0349] miRNA Array Analysis

[0350] The NanoString nCounter Human v3 miRNA Expression Assay was used to perform the microRNA profiling analysis. The assay allows measurement of 800 different microRNAs at the same time for each sample. 3.5 l of suspension RNA was annealed with multiplexed DNA tags (miR-tag) and bridges target specifics. Mature microRNAs were bonded to specific miR-tags using a Ligase enzyme, and excess tags were removed by enzyme clean-up step. The tagged microRNA product was diluted 1 to 5, and 5 l was combined with 20 l of reporter probes in hybridization buffer and 5 l of Capture probes overnight (17 hours) at 65 C. to permit hybridization of probes with specific target sequences. Excess probes were removed using two-step magnetic bead-based purification on an automated fluidic handling system (nCounter Prep Station) and target/probe complexes were immobilized on the cartridge for data collection. The nCounter Digital Analyzer took images of immobilized fluorescent reporters in the sample cartridge with a CCD camera through a microscope objective lens. For each cartridge, a high-density scan encompassing 325 fields of view was performed. NanoString raw data was analyzed with nSolver software, provided by NanoString Technologies. The mean plus 2 times the standard deviation of Negative Control Probes was used to perform background subtraction; positives were used to perform technical normalization to adjust lane by lane variability due to differences in hybridization, purification or binding. Data was then normalized by calculating the geometric mean of the spikes present in each sample, as recommended by NanoString. One-way ANOVA was used to calculate the P value; targets with P<0.05 were selected.

[0351] miRNA Target Gene Prediction, Gene Ontology(GO) Analysis and Luciferase Activity Assay

[0352] miRNA target genes prediction and gene ontology analysis were carried out using DIANA mR-microT and mirPath software. Differentially miRNA target genes in significant GO and pathway categories, obtained from GO and pathway analyses, were analyzed with mirPath v.3 software. GO biological process includes biological processes, molecular function and cellular component of upregulated and downregulated genes.

[0353] For luciferase activity assay, using standard procedures, wild-type (WT) or mutant 3untranslated regions (UTRs) of GDF-11 or ROCK-2 were subcloned into the pLenti-UTR-Dual-Luc vector (abm, Canada) obtaining the sequence as shown in SEQ ID NO: 14 (FIG. 24C) downstream of the luciferase gene. The predicted binding sites and mutant sequences from SEQ ID NO: 15 to SEQ ID NO:20 are shown in FIG. 24D. GDF-11-3UTR-WT (SEQ ID NO: 18), GDF-11-3UTR-Mut (SEQ ID NO: 20), ROCK2-3UTR-WT (SEQ ID NO: 15), or ROCK2-3UTR-Mut (SEQ ID NO:17) vectors were co-transfected with miR-373 mimic or negative control into 293FT cells using Lipofectamine 3000 for 48 h. Transfected cells were analyzed using the dual-luciferase reporter assay system (Promega). The luciferase activity was normalized using Renilla activity. Myocardial infarction model

[0354] Animal experiments were carried out both at University of Illinois at Chicago and Augusta University according to experimental protocols approved by the University of Illinois at Chicago and Augusta University Animal Care and Use Committee, and the methods were performed in accordance with the guide for the Care and Use of Laboratory Animals by the Institute of Animal Resources. MI model was generated as previously described(15). Briefly, MI was induced in 8-9-week-old NOD/SCID mice (The Jackson Laboratory) or C57/B6 mice which were anaesthetized with 2% isoflurane (isoflurane USP, HENRY SCHEIN), intubated and ventilated. The left anterior descending coronary artery (LAD) was permanently ligated with a prolene #8-0 suture. 10 mins after LAD ligation, EV (1*10.sup.12/ml) from hiPSC or CPC.sup.ISX-9 were injected into the myocardium along the border zone with a total of 20 l. The same volume of PBS was injected in the control group. miR-373 mimic in vivo transfection was performed in C57/B6 mice. In vivo-jetPEI system (POLYPLUS TRANSFECTION SA) was used for intracardiac miRNA delivery. 200 pMoles miR-373 mimic complexed with in vivo-jetPEI at a N/P ratio of 7 in a volume of 20 l were injected into the myocardium along the border zone approximately 10 mins after LAD ligation.

[0355] Echocardiography

[0356] Echocardiography was performed in mice anesthetized mildly with inhaled isoflurane (0.5%) using Philips iE33 ultrasound machine, equipped with L15-7io probe as described previously(15). Hearts were imaged in 2D in the parasternal long-axis and/or parasternal short-axis views at the level of the highest LV diameter. Measurements of left ventricular end diastolic diameter (LVDd), and left ventricular end systolic diameter (LVDs) were made from 2D M-mode images of the left ventricle in both systole and diastole. Left ventricle fractional shortening (LVFS) was calculated using the following formula: LVFS=(LVDd-LVDs)/LVDd100. Ejection fraction (EF), Left ventricular end diastolic volume (LVEDv) and left ventricular end systolic volume (LVESv) were calculated using the following formula: 7.0LVEDd/(2.4+LVDd) and 7.0LVESd/(2.4+LVDs) respectively; left ventricular ejection fraction (LVEF) was calculated as (LVEDvLVESv)/LVEDv100%. LVFS and EF were expressed as percentages.

[0357] Histology

[0358] Histological analysis was performed in randomly selected hearts from mice subjected to MI and treatment groups (PBS, EV-hiPSC or EV-CPC.sup.ISX-9 (n=3 per group). Mice were sacrificed after 1 month of treatment with EV. For immunostaining, hearts were fixed with 4% PFA for 1 hour at room temperature and replaced by 30% sucrose overnight at 4 C. Afterwards, hearts were cryopreserved in an optical cutting temperature (OCT) compound (Tissue Tek) at 80 C. Hearts were sliced into 5-m-thick frozen sections and incubated with primary antibodies including -sarcomeric actinin (A7811, Sigma, 1:200), ki67 (ab16667, abeam, 1:500), cTnT (13-11, Thermo fisher Scientific, 1:300) and SMA (ab5694, abeam, 1:300). Signals were visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies (Life Technologies). Images were recorded on a confocal microscope (FV1000, Olympus, Japan). For fibrosis analysis, hearts were embedded in paraffin and cut at 5-m-thick sections. Masson trichrome staining was performed according to the manufacturer's protocol (HT-15, Sigma). The size of LV area and scar area were measured using the ImageJ software. 4 sections (EV treated mice) and 6 sections (miR-373 mimic treated mice) were analysed per heart. The fibrosis area was determined as the ratio of scar area to LV area and expressed as percentage. Vessel density was assessed in 9 animals (3 in each group) in NOD/SCID mice, and 6 animals in C57/B6 mice (3 in each group) which were sacrificed at 1M after MI. The number of vessels was blindly counted on 27 sections (3 sections per heart) in NOD/SCID mice or 18 sections (3 sections per heart) in C57/B6 mice in the infarct and border areas of all mice after staining with an antibody -SMA using a fluorescence microscope at a 400 magnification. Vascular density was determined by counting -SMA positive vascular structures. The number of vessels in each section was averaged and expressed as the number of vessels per field (0.2 mm.sup.2)

[0359] Western Blot

[0360] EV and cell extracts were lysed with radio immunoprecipitation assay (RIPA) buffer supplemented with Complete Protease Inhibitor Mixture tablets (Roche Diagnostics). Protein concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Scientific). 10 g proteins were separated by SDS/PAGE and transferred to PVDF membrane (BioRad). Membranes were incubated with primary antibodies against the following proteins overnight at 4 C.: mouse anti-tsg101 (sc-365062, Santa Cruz), mouse anti-Calnexin (sc-23954, Santa Cruz), goat-anti-Hsp70 (EXOAB-Hsp70A-1, SBI), rabbit anti-CD9 (#13174, CST), rabbit anti-Flotillin-1(#18634, CST), mouse anti-GADPH (sc-365062, Santa Cruz). The membrane was then washed, incubated with an anti-mouse/rabbit/goat peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized by the enhanced chemiluminescence method (Pierce, Thermo Scientific) with a western blotting detection system (Fluorchem E, ProteinSimple USA) and were quantified by densitometry with ImageJ software.

[0361] RNA Extraction and Real Time PCR

[0362] Total RNA from EV was isolated using miRNeasy Micro Kit (Qiagen). Reverse transcription was performed using miScript II RT Kit (Qiagen). Quantification of mRNA and selected miRNAs were performed by real-time system quantstudio3 (ABI) using miScript SYBR Green PCR Kit (Qiagen). miRNA primer sequences are shown in Table S1, and mRNA primer sequences are shown in Table S2. Expression levels of selected miRNAs were quantified, validated with RT-PCR and values are expressed as 2.sup.CT with respect to the expression of the reference U6. The primer of U6 was provided in the PCR kit.

[0363] Statistical Analysis

[0364] Data are expressed as mean SD. Test for normality of data was performed. Statistical analysis of differences was compared by ANOVA with Bonferroni's correction for multiple comparisons. Comparisons between two groups were evaluated with Students t-test. A probability value of P<0.05 was considered statistically significant. Statistical analyses were performed using Graphpad Prism 6.0 (Chicago, USA).

[0365] Results

[0366] Characterization of EV Secreted by ISX-9 Induced Cardiac Progenitors

[0367] Electron microscopy analysis showed that secreted EV measured 160-170 nm in diameter (FIGS. 22A, 22B and 22D). No significant difference in size was observed amongst the groups (FIG. 1D, IE). Additionally, EV were enriched in EV-specific markers Tsg101, CD9, Hsp70 and Flotillin-1. Calnexin was absent in isolated EV (FIG. 22C), confirming their purity.

[0368] EV-CPC.sup.ISX-9 Exhibit a Unique miRNA Profile

[0369] Next, we performed miRNA array to determine whether miRNA cargo content of EV-CPC.sup.ISX-9 differs from that of hiPSCs, EBs and commercial CPCs (FIG. 23A). Global miRNA profiling showed that EV-CPC.sup.ISX-9 had a unique miRNA expression signature very different from that of the other derived EV. miR-520/-373 family members, including miR-371, miR-302, miR-372, miR-373 and miR-520, as well as miR-512, miR-548 and miR-367, were significantly upregulated in EV-CPC.sup.ISX-9 compared to EV from other parent cells (FIG. 23B) (GEO number is pending). Furthermore, the expression of these enriched miRNAs (miR-373/miR-548/miR-367/miR-520) was validated with real-time PCR (FIG. 23D). The target genes of the differentially expressed miRNAs control a broad range of biological functions. Biological process of gene ontology (GO) enrichment analysis based on enriched miRNA-targeted genes demonstrated that some target genes were significantly enriched in responses to stress and cell cycle (FIG. 23C).

[0370] Anti-Fibrotic Effects Mediated by miR-373 Derived from EV-CPC.sup.ISX-9

[0371] We hypothesized that the enriched miR-373 EV from CPC.sup.ISX-9 exert anti-fibrotic effects. First, using PKH26 labeling, we confirmed that EV were internalized by fibroblasts and localized in the perinuclear region. miR-373 expression level was markedly higher in EV than in their donor cells, CPC.sup.ISX-9. Inhibition of miR-373 in CPC.sup.ISX-9 resulted in decreased miR-373 expression in EV-CPC.sup.ISX-9 and reduced miR-373 expression in fibroblasts incubated with these EV compared to those from control cells. Stimulation of fibroblasts with TGF- led to significant upregulation of fibrotic genes (MMP-2, TIMP-2, TIMP-1, FN1, CTGF and MMP-9). Upon pretreatment of fibroblasts with EV from control CPC.sup.ISX-9, upregulation of these fibrotic genes by TGF- was inhibited. However, inhibition of miR-373 in CPC.sup.ISX-9 abrogated the capacity of the EV to inhibit fibrotic gene expression. Conversely, pretreatment of fibroblasts with miR-373 mimic inhibited TGF- induced expression of fibrotic genes (FIG. 24A). We also performed experiments in a second cellular model of fibrosis by exposing the fibroblasts to hypoxia. FIG. 24B shows that with 72 h exposure in a hypoxic environment, both lung and dermal fibroblasts differentiated into myofibroblasts expressing -SMA. As expected, miR-373 mimic significantly inhibited fibroblast transdifferentiation into myofibroblasts under hypoxia. Similarly, fibroblasts failed to transdifferentiate into myofibroblasts when they were pretreated with EV from CPC.sup.ISX-9. Taken together, these results suggest that miR-373 contained in EV-CPC.sup.ISX-9 suppresses fibrosis both in in vitro and in vivo levels.

[0372] Although a previous study reported miR-373 might target TGF-(17), here we identified two new potential target genes of miR-373, GDF-11 and ROCK-2, using DIANA mR-microT software and dual-luciferase reporter assay. The 3-UTR binding sites are shown in FIG. 3D. Next the predicted binding sites of GDF-11 and ROCK-2 were cloned into 3-UTR of the dual luciferase vector respectively (FIG. 24C) and transiently transfected into 293FT cells. miR-373 mimic transfection significantly decreased the relative luciferase activity when co-transfected with GDF-11 and ROCK-2 3-UTR vectors. When 3-UTR binding sites were mutated, the repression of GDF-11 and ROCK-2 3-UTR by miR-373 mimic was attenuated (FIG. 24E). Notably, the human miR-373 3 UTR binding sites for GDF-11 and ROCK-2 are conserved among several species, including mice and rats.

[0373] Moreover, under hypoxic conditions, the expression of GDF-11 and ROCK-2 was increased in lung fibroblasts (FIG. 24F), while pretreatment with miR-373 mimic or EV-CPC.sup.ISX-9 significantly inhibited expression of these two genes, supporting the contention that miR-373 targets GDF-11 and ROCK-2 (FIG. 24).

[0374] EV-CPC.sup.ISX-9 Promoted CM Proliferation and Angiogenesis, and Reversed Ventricular Remodeling, in Mice Post MI

[0375] Next, we determined the effects of treatment with EV-CPC.sup.ISX-9 in a mouse model of MI. compared to PBS and EV-hiPSCs, EV-CPC.sup.ISX-9 treatment boosted cardiomyocyte proliferation in the infarcted hearts. FIG. 25A & 25B show representative images and quantitative data of proliferating Ki67 and -actinin positive cardiomyocytes in the peri-infarct region. We further determined the impact of EV-CPC.sup.ISX-9 on angiogenesis using tube formation assay. We found that EV-CPC.sup.ISX-9 indeed increased average tube length of human aortic endothelia cells (HAECs) in vitro. Remarkably, EV-CPC.sup.ISX-9 also reduced oxidant induced changes in HAEC. Similarly, the vessel density as identified by -SMA staining and tube like structures (FIG. 25C, 25D) in the infarcted region was also increased by treatment with EV-CPC.sup.ISX-9. The deterioration in cardiac function, as noted by rise in LVEDD and LVESD as well as a progressive decline in LVFS 1-month post-MI, was attenuated by EV-CPC.sup.ISX-9 treatment. EV-CPC.sup.ISX-9 slowed the progression of left ventricle enlargement (LVDs, 2.350.31 mm vs. 2.790.30 mm and 3.0470.35 mm; LVDd, 3.540.40 mm vs. 3.790.33 mm and 3.910.38 mm in EV-hiPSC, PBS and EV-CPC.sup.ISX-9 treated groups, respectively) and improved cardiac function (LVFS: 33.772.42% vs. 26.442.79% and 22.162.78% in EV-hiPSC, PBS and EV-CPC.sup.ISX-9 treated groups, respectively; LVEF: 70.823.12% vs. 52.674.78% and 60.054.58% in EV-hiPSC and PBS treated groups) (FIG. 26 A-E). Moreover, smaller scar size was observed in mice treated with EV-CPC.sup.ISX-9 compared with PBS and EV-hiPSC (P<0.01; FIG. 26F, 26G).

[0376] miR-373 Mimic Attenuated Cardiac Fibrosis and Improved Cardiac Function and Angiogenesis after MI

[0377] Having demonstrated the anti-fibrotic effects of miR-373 in vitro, we further explored and validated direct effects of miR-373 on post-infarct remodeling and fibrosis. We delivered miR-373 mimic by intramyocardial injection after LAD ligation. After 1 month, miR-373 mimic treatment significantly improved cardiac function compared to control mice (LVFS: 33.381.72% vs. 19.984.45% in NC treated mice; LVEF: 62.252.16% vs. 40.878.17% in NC treated mice.) FIG. 27A-C). In addition, miR-373 mimic dramatically attenuated cardiac fibrosis in comparison to NC treatment (FIG. 27D-E and). Moreover, the vessel density as identified by -SMA staining and tube like structures (FIGS. 27F and G) in the infarcted region was increased by miR-373 treatment. Despite the effectiveness of EV-CPC.sup.ISX-9 or miR-373, no difference in survival rate between the two groups was observed due to death because of cardiac rupture.

[0378] Discussion

[0379] Stem cell based therapy has been well recognized to improve cardiac function following MI. While this therapy has merits, it also suffers from several limitations, particularly lack of suitable stem cell type and their insufficient engraftment and growth, ranging from no new cell formation to sparse newly formed cells in the infarcted tissue (18-20). Cellular therapy has been propelled by the invention of iPS cells, which have the ability to transform into different progenitor cells types. The cardiac progenitors derived from iPS cells and their counter parts have been used both in animal models of MI (21,22) and in humans (23) with promising results. While the underlying mechanisms of beneficial effects of stem cell therapy remain a point of debate, increasing evidence suggests that paracrine factors play a key role by reducing cell death and stimulating cell migration and proliferation (24,25). This paracrine signaling involves the secretion of small vesicles or EV harboring multiple miRNAs, proteins and other factors that mediate protection in the heart. Secreted extracellular vehicles (EVs), such as EV, are packed with potent pro-repair proteins and RNA cargo that are both cell type-specific as well as differentially produced and secreted according to the cellular environment. Additionally, miRNA profiles of EV might be distinct from cellular miRNA patterns (26).

[0380] In this study, EV derived from CPC.sup.ISX-9 were found to be highly cardioprotective, and the effect can in part be attributed to their specific miRNA content. CPC.sup.ISX-9 derived EV were highly enriched with miR-520/-373 family members including miR-371, miR-372, miR-373 and miR-520, as well as miR-512, miR-548 and miR-367, compared to EV derived from other parent cells. miR-373, which was particularly highly enriched in EV-CPC.sup.ISX-9, was first identified as a human embryonic stem cell (ESC)-specific miRNA, implicated in the regulation of cell proliferation, apoptosis, senescence, migration and invasion, as well as DNA damage repair following hypoxia stress (27).

[0381] Little has been published regarding the putative role of miR-373 in regulating cardiac pathology or function. In a mouse model of type 1 diabetic cardiomyopathy, miR-373 was found to be significantly downregulated, and application of a miR-373 mimic to neonatal cardiomyocytes exposed to elevated glucose in vitro suppressed cell hypertrophy (28). Fibrosis is also an important pathological feature of diabetic cardiomyopathy, but effects of miR-373 on fibrosis were not investigated in that study. Fibrosis plays a prominent role in ventricular remodeling and ultimately in the pathogenesis of heart failure after MI. A previous study reported that miR-373 targeted the members of TGF- signaling including TGF- receptor2 and Smad2, and promoted mesoderm differentiation in human embryonic stem cells (17). miR-373-3p expression was low in hypertrophic myocardium with diffuse myocardial fibrosis (29), suggesting that miR-373 may function as an anti-fibrotic miRNA. Thus, we hypothesized that because of their enrichment in miR-373, EV-CPC.sup.ISX-9 might produce strong anti-fibrotic effects to modulate cardiac remodeling.

[0382] Our results indicate that both EV-CPC.sup.ISX-9 and miR-373 mimic inhibited TGF-- and hypoxia-induced fibrotic gene expression in vitro. With inhibition of miR-373 in EV-CPC.sup.ISX-9, or treatment with miR-373 inhibitor, the effects on fibrotic gene expression were abrogated. The luciferase activity assay confirmed that miR-373 targeted GDF-11 and ROCK-2, both known to be involved in fibrosis. An isoform of Rho-associated coiled-coil forming protein kinase 2, ROCK-2 is reportedly a critical mediator of organ fibrosis. Inhibition of ROCK-2 protected ROCK-2-haploinsufficient mice from bleomycin-induced myofibroblast differentiation and pulmonary fibrosis (30), while its activation was implicated in development of idiopathic pulmonary fibrosis (31). Additionally, fibroblast-specific ROCK2 was reported to promote cardiac hypertrophy, fibrosis, and diastolic dysfunction due to upregulation of profibrotic gene (CTGF) and promyofibroblast differentiation (-SMA) genes (32). Mutant mice with elevated fibroblast ROCK activity exhibited enhanced Ang II-stimulated cardiac hypertrophy and fibrosis (32). The role of the second identified target gene, GDF-11 is more controversial. It was reported to beneficially reverse age-related cardiac hypertrophy and skeletal muscle dysfunction (33,34), while other reports suggest that it promotes cardiac and skeletal muscle dysfunction and wasting (35), inhibits skeletal muscle regeneration (36), exerts pro-fibrotic effects (37), and renal failure and interstitial fibrosis(38). Therefore, our data suggest that miR-373 inhibited profibrotic gene upregulation and myofibroblast differentiation in fibroblasts by targeting GDF-11 and ROCK-2.

[0383] In vivo data showed that compared with EV-hiPS and PBS, EV-CPC.sup.ISX-9 treatment reduced fibrosis and improved cardiac function, thus supporting a therapeutic role for EV-CPC.sup.ISX-9 in cardiac remodeling. Given that EV-CPC.sup.ISX-9 were found to be highly enriched in miR-373, we tested the effects of EV-CPC.sup.ISX-9 injection in the heart and miR-373 mimic treatment in vivo on miR-373 expression level and found that miR-373 expression level in the heart was increased and it significantly decreased fibrosis and improved cardiac function post MI. Moreover, the miR-373 mimic also promoted angiogenesis, which was likely mediated by its ability to activate HIF downstream signaling (39). These findings suggest that the anti-fibrotic effects of EV-CPC.sup.ISX-9 are, at least in part, mediated by miR-373, and they also support the notion that miR-373 mimic might represent a novel therapeutic strategy for controlling fibrosis and cardiac remodeling post infarction and perhaps in other disorders, such as diabetic cardiomyopathy.

[0384] The second major effect of EV-CPC.sup.ISX-9 was on cardiomyocyte proliferation in the infarcted myocardium. A previous study reported miR-294 (miR-290 cluster), the mouse homolog of human miR-371/372/373 cluster, had a strong effect on cardiac progenitor cell proliferation (40), and that its overexpression led to differentiation towards the mesendoderm lineage (17). It should be borne in mind, however, that these effects could also be attributed to other miRNAs present in the EV, including miR-302, miR-548, miR-512 and miR-367. Further studies are required to dissect the role of individual EV-CPC.sup.ISX-9 miRNAs in regulating cardiac fibrosis, cardiomyocyte proliferation, and other pathological events in the context of post-infarction remodeling.

Conclusion

[0385] Several clinical and investigational reports have demonstrated the therapeutic application of cardiac progenitor cells for the treatment of ischemic heart. Consequently, these studies led to advance new cell free (EV) strategies to overcome the limitations of cell-based approaches with the same effectiveness and outcomes. The intracoronary administration of EV eliminates the need for open heart surgery for intramyocardial administration of stem cells. However, the promise of EV does not establish the fact whether their effect is continuous and permanent or future efforts should continue on strategies directed towards successful engraftment and survival of iPSC derived cardiac progenitors as a source for new myofiber growth and EV for paracrine effects as well.

[0386] In summary (FIG. 28), we report that EV-CPC.sup.ISX-9 exhibit a unique miRNA profile, and that miR-373 is particularly highly enriched in these EV. EV-CPC.sup.ISX-9 elicit strong anti-fibrotic effects, which is attributed at least in part to their enrichment in miR-373. Treatment with EV-CPC.sup.ISX-9 in vivo reduced post-infarction fibrosis and remodeling, promoted cardiomyocyte proliferation and angiogenesis, and improved cardiac function, findings which were at least in part recapitulated by direct application of miR-373 mimic. These findings have important implications for understanding the paracrine mechanisms of stem cell function and advancing the field of cardiac stem cell therapeutics.

[0387] Data Availability:

[0388] The raw data of miRNA array is deposited in GEO database (GSE126347). Other data are available from the authors upon reasonable request.

[0389] Funding Statement:

[0390] This study was supported by National Institutes of Health grants, RO1 HL126516.sup.1, RO1 HL134354.sup.2 and RO1 AR070029.sup.2 (to M Ashraf.sup.1,2, Y Tang.sup.2, N Weintraub.sup.2), and HL124097 and HL126949 (to N Weintraub.sup.2).

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[0432] Figure Legends for Material Discussed Beginning in Paragraph 0298

[0433] FIG. 22. Characterization of EV from iPSC and CPC.sup.ISX-9. (A) Secretion of EV from the CPC.sup.ISX-9 as imaged by electron microscopy. Inset shows higher magnification of secreted EV (small black arrows). Blue arrows point to EV exiting from the cells. Bar=1 m. (B) EV isolated from iPSC and CPC.sup.ISX-9 visualized by transmission electron microscopy (TEM). Scale bar=200 nm. (C) Representative images of western blot for Tsg101, CD9, Hsp70, Flotillin-1, and Calnexin in EV lysates. C: cell lysate; E: EV. (D) Average size of EV as measured by TRPS. No significant difference in average size of EV from iPSC and CPC.sup.ISX-9 was observed. (E) Representative graph of size distribution of EV from iPSC and CPC.sup.ISX-9 as detected by TRPS.

[0434] FIG. 23 miRNA expression profiling and validation of microarray data. (A) Outline of experimental procedure. (B) Heatmap analysis of microarray data showing significant upregulation of miRNAs in EV-CPC.sup.ISX-9 compared with those in EV-iPSC, EV-EB or EV-control-CPC. Red or blue colors indicate differentially up- or downregulated miRNA, respectively (P<0.05). n=3. (C) Biological process of Gene Ontology (GO) enrichment analysis based on miRNA-targeted genes. GO enrichment was analyzed with mirPath v.3 software. GO biological process includes biological processes, molecular function and cellular component of upregulated and downregulated genes. (D) Validation of microarray data using real-time PCR. Quantitative results showing significant expression of miR-373, miR-367, miR-520, miR-548ah, and miR-548q in EV-CPC.sup.ISX-9 RNA samples were from three individual experiments. *P<0.001.

[0435] FIG. 24 Fibrotic gene expression in fibroblasts after TGF- stimulation. (A) Effects of EV-CPC.sup.ISX-9 on fibrotic gene expression: role of miR-373. n=6 (B) Transdifferentiation of lung fibroblasts and dermal fibroblasts into myofibroblast by hypoxia for 72 h as detected by immunostaining for -smooth actin (-SMA): effects of EV-CPC.sup.ISX-9 and miR-373 mimic pretreatment. Bar=50 m. (C) Schematic representation of the luciferase reporter constructs. (B) Sequence alignment of miR-373 with the human wild type (WT) ROCK-2 3-UTR and GDF-113-UTR and mutated reporters. The seed sequence (Red) is highlighted. (D) Relative luciferase activity (relative, firefly luciferase activity/Renilla luciferase activity) of 293FT cells co-transfected with WT 3-UTR-ROCK-2 or GDF-11 and mutant 3-UTR-ROCK-2 or GDF-11 and miR-373 mimics vs. NC. ** P<0.01, n=4. UTR, untranslated region; miRNA, microRNA; NC, negative control; WT, wild type. (E) 72 h hypoxia increased GDF-11 and ROCK-2 mRNA expression in lung fibroblasts: effects of pretreatment with miR-373 mimic. *** P<0.001. n=6.

[0436] FIG. 25 CPC.sup.ISX-9-derived EV promoted cardiomyocyte proliferation and angiogenesis after myocardial infarction (MI) in mice. (A) Representative image of ki67 positive cardiomyocytes (cTnT positive) in EV-CPC.sup.ISX-9 treated mouse hearts 30 days after MI. Bar=50 m. (B) Quantitative estimate of proliferating cardiomyocytes as determined by Ki67 staining in peri-infarct region 30 days after myocardial infarction. PBS group: n=940 cardiomyocytes from 3 hearts; EV-iPSC group: n=950 cardiomyocytes from 3 hearts; EV-CPC.sup.ISX-9 group, n=951 cardiomyocytes from 3 hearts. * vs. PBS group, P<0.05; # vs. EV-iPSC group, P<0.05. (C) Representative images of arteriole density in peri-infarct area 4 weeks after MI. Arterioles were identified by -SMA positive staining (green) of vascular structures. Bar=100 m. (D) Quantitative analysis of arteriole density in different treatment groups. * vs. PBS group, P<0.05; # vs. EV-iPSC group, P<0.05, n=3.

[0437] FIG. 26 CPC.sup.ISX-9 derived EV reversed cardiac remodeling in infarcted mice. (A) Representative M mode echocardiography images from three groups 30 days after MI. LVDs (B), LVDd (C), EF (D) and FS (E) are shown. * vs. PBS group, P<0.05; # vs. EV-iPSC group, P<0.05, PBS group: n=10, EV-iPSC group, n=9, EV-CPC.sup.ISX-9, n=11. EF, ejection fraction; FS, fractional shortening; LVDd, diastolic left ventricular dimensions; LVDs systolic left ventricular dimensions. (F) Representative Masson's trichrome-stained sections of hearts from three groups. (G) Quantitative estimate of fibrosis. * vs. PBS group, P<0.05; # vs. EV-iPSC group, P<0.05, n=4.

[0438] FIG. 27 miR-373 mimic improved cardiac function and angiogenesis and attenuated cardiac fibrosis after MI. (A) Representative M mode echocardiography images from miRNA mimic negative control (NC) treated mice and miR-373 mimic treated mice 30 days post-MI. FS (B) and EF (C) are shown; (P<0.001), n=8 in NC group and n=7 in miR-373 mimic group. (D) Representative Masson's trichrome-stained sections of hearts from NC treated mice and miR-373 mimic mice. (E) Quantitative analysis of fibrosis post MI. (F) Vessel density was assessed by -SMA positive staining (green) of vascular structures. Bar=100 m. (G) Quantitative estimate of arteriole density. P<0.05. n=3 in each group. (H) Schematic depiction of mechanisms of protection by EV-CPC.sup.ISX-9: role of miR-373 in suppressing fibrosis by targeting two genes, GDF-11 and ROCK-2 and inhibiting myofibroblast differentiation. Myocyte proliferation and angiogenesis were also promoted by EV-CPC.sup.ISX-9.

[0439] FIG. 28 Schematic depiction of mechanisms of protection by EV-CPC.sup.ISX-9: role of miR-373 in suppressing fibrosis by targeting two genes, GDF-11 and ROCK-2 and inhibiting myofibroblast differentiation. Myocyte proliferation and angiogenesis were also promoted by EV-CPC.sup.ISX-9.

[0440] The following publication is incorporated herein in its entirety

Human iPS Cells Derived Skeletal Muscle Progenitor Cells Promote Myoangiogenesis and Restore Dystrophin in Duchenne Muscular Dystrophic Mice

[0441] Abstract

[0442] Background and Objective:

[0443] Duchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. Loss of dystrophin leads to severe and progressive muscle-wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) and their derivatives offer important opportunities to treat a number of diseases. Here, we investigated whether givinostat, a histone deacetylase inhibitor (HDACi), could reprogram hiPSCs into muscle progenitor cells (MPC) for DMD treatment.

[0444] Methods and Results:

[0445] MPC generated by CHIR99021 and givinostat (Givi) small molecules from multiple hiPSCs expressed myogenic makers (Pax7, desmin) and were differentiated into myotubes expressing MF20 upon culture in specific differentiation medium. These MPC exhibited superior proliferation and migration capacity determined by CCK-8, colony and migration assays compared to control-MPC generated by CHIR99021 and fibroblast growth factor (FGF). Upon transplantation in hind limb of Mdx/SCID mice with cardiotoxin (CTX) induced injury, these MPC showed higher engraftment and restoration of dystrophin than treatment with control-MPC and human myoblasts. In addition, treated muscle with these MPC showed significantly limited infiltration of inflammatory cells and reduced muscle necrosis and fibrosis. A number of these cells were engrafted under basal lamina expressing Pax7, which were capable of generating new muscle fibers after additional injury. Extracellular vesicles released from these cells promoted angiogenesis after reinjury.

Conclusion

[0446] We successfully generated highly expandable and integration free MPC from multiple hiPS cell lines using CHIR99021 and Givi. Givinostat induced MPC showed marked and impressive regenerative capabilities and restored dystrophin in injured tibialis muscle compared to control MPC. Additionally, MPC generated by Givi also seeded the stem cell pool in the treated muscle. It is concluded that hiPSCs pharmacologically reprogrammed into MPC with a small molecule, Givi with anti-oxidative, anti-inflammatory and muscle gene promoting properties might be an effective cellular source for treatment of muscle injury and restoration of dystrophin in DMD.

[0447] Keywords: Duchenne Muscular Dystrophy; Human induced pluripotent stem cells; muscle progenitor cells; histone deacetylase inhibitor, angiogenesis

[0448] Introduction

[0449] Duchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. Loss of dystrophin leads to severe and progressive muscle-wasting in both skeletal and heart muscles. Cell replacement gives a promising hope for DMD therapy. Satellite cells (SCs) are endogenous skeletal muscle stem cells, which are responsible for muscle maintenance and muscle regeneration after injury (1,2). A previous study reported that xenotransplantation of human SCs into mice achieved efficient engraftment and populated the satellite niche (3). However, a biopsy is needed for procurement of SCs. In addition, freshly isolated SCs progeny though can be propagated in vitro but their transplantation potential becomes limited during in vitro expansion (4-6). Therefore, procurement of larger number of SCs for transplantation becomes an obstacle for clinical application. Human induced pluripotent stem cells (hiPSCs) derived derivatives offer important sources to treat a number of diseases. Efforts have been made in the past few years for generation of muscle progenitor cells (MPC) from hiPSCs either by genetic modification or small molecules. Nevertheless, generation of MPC from hiPSCs by viral vectors remains a safety concern. High percentage of Pax7 positive MPC can be generated from hiPSC by small molecules (CHIR99021, LDN19389 and FGF) (7,8), but their limited engraftment was observed in vivo upon transplantation (9). Interestingly, it has been recently reported that MPC can be generated from teratoma which showed high engraftment efficiency in muscle dystrophy model (10). However, human teratoma derived MPC poses safety concerns for clinical application. Therefore, it seems more appropriate to look for alternate approaches for inducing MPC from hiPSCs with high engraftment and differentiation properties.

[0450] Givinostat is a histone deacetylase inhibitor (HDACi) that has been shown to increase muscle regeneration in a mouse model of DMD (11). Interestingly, most of the beneficial effects of HDACi arise from its ability to redirect fibroadipogenic lineage commitment toward a myogenic fate (12). Using genome-wide Chip-seq analysis in myoblasts, it was demonstrated that HDACi induced myogenic differentiation program in myoblasts (i.e., Myosin 7, Enolase 3 and Myomesin1) (13). Therefore, here we propose that Givi could reprogram hiPSCs into MPC for DMD treatment.

[0451] Methods

[0452] Human iPSC Culture

[0453] The Human iPSC cell lines from ATCC Company CYS0105 and DYS0100 were used. CYS0105 was reprogrammed from human cardiac fibroblasts of a 72 years old healthy donor, while DYS0100 was reprogrammed from human foreskin fibroblasts of a normal newborn. DMD-iPS cell line (SC604A MD) was purchased form SBI Company, which was generated from a DMD patient with Exon 3-7 deletion of dystrophin. The forth iPS cell line was reprogrammed from human dermal fibroblasts (CC-2511, Lonza) of a 45 years old healthy donor in our lab using Cyto Tune iPS 2.0 sendai reprogramming kit (A16517, Thermo fisher Scientific) as previously described (14). iPSCs were grown and maintained on vitronectin coated six-well plate in mTeSR1 medium (Stem Cell Technologies) with daily change.

[0454] Differentiation Protocols to Generate Muscle Progenitor Cells (MPC) and their Characterization

[0455] Human iPSCs at passage 20-30 were used for conversion to MPC. Human iPSCs were dissociated into single cells using Accutase (Stem Cell Technologies) at 37 for 10 min and then were seeded onto a vitronectin-coated six-well plate at 310.sup.5 cell/well in mTeSR1 supplemented with 5 M ROCK inhibitor (Y-27632, Stem Cell Technology) for 24 h. Afterwards cells were switched into E6 medium (Thermo Fisher Scientific) supplemented with CHIR99021 (10 M) for two days followed by Givi (100 nM) for 5 days. The differentiating cells were cultured in E6 medium for 7 days. The schematic outline is shown in FIG. 1A. At Day 14, cells were replated on 0.1% galectin coated coverslips and expression of Pax7 and desmin were analyzed by immunostaining. MPC were expanded in SKGM-2 medium plus FGF-2 (2.5 ng/ml) and cells at passage 2-4 were used for experiments. Here, we referred the givinostat induced MPC as Givi-MPC. To further enhance muscle differentiation, at Day 14, cultured cells were replated and switched into high glucose DMEM medium supplemented with 2% horse serum (Thermo fisher Scientific) and 1% ITS (Thermo fisher Scientific) for 7 days. Immunostaining of cultured cells for MF20 was performed. To generate control-MPC (7), a previously reported method using only CHIR99021 was used. The schematic outline is shown in supplemental FIG. S1. Control-MPC were expanded using the same condition as Givi-MPC and passage 2-4 of cells were used for experiments.

[0456] CCK-8 Assay for Proliferation

[0457] CCK-8 assay was used for evaluation of cell proliferation. Briefly, 2000 cells were seeded into 96 well plate per well and cell proliferation was analyzed at 0 h, 24 h, 48 h and 72 h respectively by using CCK-8 kit (ab228554, abcam) according to the manufacturer protocol.

[0458] Colony Formation

[0459] Thirty cells (single cell) were seeded in one well of six-well plate. After 7 days, cells were stained with crystal violet dye. Number of colonies and size of cell growth were analyzed and compared between control-MPC and Givi-MPC groups.

[0460] Cell Migration

[0461] For cell migration experiment, human myoblasts, control-MPC and Givi-MPC were seeded in 35 mm dish with culture-insert 2 well (ibidi company) at 110.sup.5/ml concentration in SKGM-2 medium with 2% Fetal Bovine Serum (FBS). The next day, a confluent layer was observed and culture-inserts were removed, and after 24 h the number of migrated cells were analyzed.

[0462] Human Endothelial Cell and Human Myoblast Culture

[0463] Human aortic endothelial cells (HAEC, CC-2535) and human skeletal myoblasts (HSMM-Muscle Myoblasts, CC-2580) were obtained from Lonza Company. HAEC were maintained in endothelial cell growth medium V-2 (213-500, CELL APPLICATIONS, Inc.) and cells at passage 2-6 were used for experiments. Human myoblasts were maintained in SKGM-2 medium (Lonza) and cells at passage 2-4 were used for experiments.

[0464] Cardiotoxin Injury and Cell Transplantation

[0465] Animal experiments were carried out according to experimental protocol approved by the Augusta University Animal Care and Use Committee. 6-8 weeks old Mdx/SCID mice (Stock No: 018018, The Jackson Laboratory) were used in the present study. One-day prior to cell transplantation, mice were anaesthetized using 2% isoflurane and tibialis anterior (TA) muscle was injured with 50 l of 10 M cardiotoxin (Naja mossambica-mossambica, Sigma). For cell transplantation experiments, control-MPC and Givi-MPC were differentiated from the same human iPS cell line, DYS0100. For transplantation, myoblast, control-MPC and Givi-MPC were dissociated using Accutase (Stem Cell Technologies) and resuspended in Dulbecco's phosphate-buffered saline (DPBS) at 110.sup.5 per 20 l. Cells were injected into the left TA muscle while the same volume of DPBS was injected into the right TA as control. In some cases, cells were transfected with Green Fluorescent Protein (GFP) Lentivirus (abm company, Canada) for cell tracking. Some Mdx/SCID mice transplanted with Givi-MPC were subjected to CTX reinjury at 2M after first injury and cell transplantation.

[0466] Immunostaining for Cells

[0467] Cells were fixed with 4% PFA, and blocked with 10% FBS, followed by incubation with anti-Pax7 antibody (ab187339, abcam, 1:300), anti-desmin antibody (ab32362, abcam, 1:500) and anti-Myosin Heavy Chain Antibody (MF20) antibody (Novus, MAB4470, 1:200) respectively at 4 C. overnight and secondary antibody conjugated to Alexa Fluor 594 or Alexa Fluor 488 (Life Technologies) at room temperature for 1 h. Images were taken by a florescent microscope (Olympus, Japan).

[0468] Immunostaining for Muscle Sections

[0469] After 7 days or 30 days of cell transplantation, Mdx/SCID mice were euthanized and TA muscles were harvested and fixed with 4% paraformaldehyde (PFA) for 1 h at room temperature and then immersed in 30% sucrose overnight at 4 C. At day two, hearts were cryopreserved in an optical cutting temperature (OCT) compound (Tissue Tek) at 80 C. TA muscle samples were sliced into 5-m-thick frozen cross-sections using a Leica CM3050 cryostat. Muscle sections were incubated with primary antibodies including Laminin (L9393, Sigma, 1:500), dystrophin (D8168, Sigma, 1:200), human specific laminin (LAM-89, Novus, 1:200), GFP (#2956, Cell Signal Technologies, 1:500), dystrophin (ab15277, abcam, 1:200), human nuclear antigen (NBP2-34342, Novus, 1:100), CD68 (NB600-985, Novus, 1:200) and CD31 (NB600-562, Novus, 1:200) at 4 C. overnight respectively and anti-rabbit/mouse secondary antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 647 or Alexa Fluor 488 (Life Technologies) at room temperature for 1 h. Images were taken using a confocal microscope (FV1000, Olympus, Japan). For cell engraftment quantification, 4 sections at 150 m interval in each TA muscle were analyzed. Dystrophin or laminin staining was used to define the physical boundaries of muscle fibers. The number of muscle fibers and cross-section area were measured using Image J with the colocalization plugin (NIH). Capillary density was assessed in 4 sections cut at 150 m interval by counting CD31 positive vascular structures using a fluorescence microscope at a magnification of 400. The number of capillaries in each TA muscle was expressed as the number of capillaries per field (0.2 mm.sup.2). For quantification of inflammatory cells, number of CD68 positive cells were counted in 3 sections cut at 150 m interval after 7 days' post cell transplantation and was expressed as the number of CD68 positive cells per field (0.2 mm.sup.2). Staining of presynaptic marker -bungarotoxin (-BTX) was carried out using -bungarotoxin, Alexa Fluor 594 conjugate (Invitrogen) according to the manufacturer's instruction.

[0470] Histology

[0471] Histological staining was performed at Electron Microscopy and Histology Core of Augusta University. After 7 days or 30 days of cell transplantation, TA muscle were harvested and embedded in paraffin. 5-m-thick sections of TA muscle were cut and stained with hematoxylin and eosin (H and E), Masson trichrome and Sirius red according to the manufacturer protocol (abcam). Images were taken by a vertical microscope (Olympus, Japan). Fibrosis and necrosis were determined using the ImageJ software (NIH) and expressed as the ratio of total area of the cross-section and normalized with the ratio of control lateral TA muscle section. Myofiber necrosis was identified with fragmented sarcoplasm (15) and/or increased inflammatory cell infiltration, and was measured using non-overlapping tile images of transverse muscle sections that provided a view of the entire muscle cross section.

[0472] Isolation of Extracellular Vesicles (EV)

[0473] EV were isolated using size exclusion column method as we described previously (16). Briefly, conditioned media was collected and EV were isolated by centrifugation at 3000 rpm for 30 min to remove cells and debris, followed by filtration through a 0.22 m filter to remove the remaining debris. Then the medium was further concentrated using Amicon Ultra-15 100 KDa centrifugal filter units (Millipore). Isolation of EV in the concentrated medium was carried out through qEV size exclusion columns (Izon Science). EV fractions were collected and concentrated by Amicon Ultra-4 10 KDa centrifugal filter (Millipore). The purified EV were stored at 80 C. and subsequently characterized by particle size and electron microscopy.

[0474] Concentration and Particle Size Measurement with Tunable Resistive Pulse Sensing

[0475] Particle size and concentration were analyzed using tunable resistive pulse sensing (TRPS) technique with a qNano instrument (Izon Science) as described in previous studies (16,17). Briefly, the number of particles were counted (at least 600 to 1000 events) at 20 mbar pressure. Beads CPC200 (200 nm) were used for calibration. Data were analyzed using Izon Control Suite software.

[0476] Transmission Electron Microscopy (TEM)

[0477] Tissue samples were processed for TEM by the Electron Microscopy and Histology Core Laboratory at Augusta University as described previously (16). Briefly, EV suspension was fixed with an equal volume of 8% paraformaldehyde to preserve ultrastructure. Ten l of suspended/fixed exosomes was applied to a carbon-formvar coated 200 mesh copper grid and allowed to stand for 30-60 seconds. The excess was absorbed by Whatman filter paper. 10 l of 2% aqueous uranyl acetate was added and treated for 30 seconds. Grids were allowed to air dry before being examined in a JEM 1230 transmission electron microscope (JEOL USA Inc., Peabody, Mass.) at 110 kV and imaged with an UltraScan 4000 CCD camera & First Light Digital Camera Controller (Gatan Inc., Pleasanton, Calif.).

[0478] RNA Extraction and PCR Array

[0479] Total RNA from cells was isolated using miRNeasy Kit (Qiagen). Reverse transcription was performed using QuantiTect Reverse Transcription kit (Qiagen). Human cell motility RT2 profiler PCR Array (Qiagen) for control-MPC and Givi-MPC was performed. Data was analysed using RT2 Profiler PCR Array Data Analysis Webportal (Qiagen). Genes with a fold change >2.0 were considered overexpressed.

[0480] RNA Extraction from EV and miRNA Array Analysis

[0481] Total RNA from EV was isolated using miRNeasy Micro Kit (Qiagen). The miRNA Array analysis was performed in the Integrated Genomics and High Performance Computer Server center at Augusta University. RNA purity and concentration were evaluated by spectrophotometry using Nanodrop ND-1000 (Thermo Fisher Scientific). Quality and the related size of small RNA was assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif.). 130 ng of total RNA was labeled with biotin using the FlashTag Biotin HSR RNA Labeling Kit (Applied Biosystems) according to manufacturer's procedure. The labeled samples were then hybridized to the GeneChip miRNA 4.0 array (Thermofisher) that contains 2,578 and 2,025 human mature and premature miRNA, respectively. Array hybridization, washing, and scanning of the arrays were carried out according to Affymetrix's recommendations. Data was obtained in the form of CEL file. The CEL files were imported into Partek Genomic Suites version 6.6 (Partek, St. Louis, Mo.) using standard import tool with RMA normalization. The differential expressions were calculated using ANOVA of Partek Package.

[0482] Tube Formation Assay

[0483] Human aortic endothelia cells (HAEC, 110.sup.5 cells/well) were seeded on Matrigel (Corning) in a 24-well plate and treated with or without 1 g EV from different groups of Givi-MPC, control-MPC and human myoblast in EGM-2V basal medium (Lonza). After 16 h, cells in Matrigel were stained with Calcein AM, and images were taken with fluorescent microscope. Tube formation was analysed by Image J software with the angiogenesis analyzer plugin (NIH).

[0484] Statistical Analysis

[0485] Data were expressed as mean SD. After test for normality, statistical analysis of differences among different groups was compared by ANOVA with Bonferroni's correction for multiple comparisons. Percentage of different size of colony was compared using Chi-squared test. The Differences were considered statistically significant at P<0.05. Statistical analyses were performed using Graphpad Prism 6.0 (Chicago, US).

[0486] Results

[0487] Generation of muscle progenitor cells from human iPSC using small molecules

[0488] As outlined in FIG. 1A, we used 3 iPSC lines from healthy donors with different ages, and one iPSC line from DMD patient with frameshift deletions of exons 3-7 in the dystrophin gene for MPC generation. After 2 days treatment with CHIR99021, the morphology of the differentiating cells from 4 cell lines was dramatically altered indicative of epithelial to mesenchymal transition (EMT) (FIG. 29B). Following treatment with givinostat for 5 days, cells became confluent and clustered (FIG. 29B). FIG. 1C showed the morphology of differentiated MPC after replating and terminal muscle differentiation for 7 days in 2% horse serum differentiation medium. Using immunostaining, the MPC derived from 4 iPSC lines expressed the myogenic markers Pax7 and desmin. The MPC during terminal differentiation exhibited elongated shape (FIG. 29C) and expressed MF20 (FIG. 30B), indicating their myogenic differentiation potential.

[0489] Givinostat Induced MPC (Givi-MPC) Expressed High Proliferation and Motility Properties In Vitro

[0490] Next, we explored whether MPCs were proliferative and possessed self-renewal and motility properties. Using migration assay, compared to normal adult human myoblasts or control-MPC, Givi-MPC exhibited superior migration capability in low serum medium culture (FIG. 31A) with highest number migrated compared to other MPCs (FIG. 31B). Genes related to migration as determined by cell motility PCR array increased multifold in Givi-MPC as compared with MPCs (ITGA4 (25.61 fold), RAC2 (7.48 fold), FGF2 (6.75 fold) AND ENAH (5.53 fold)). FIG. 31C and FIG. 31D show heatmap and the list of upregulated genes related to migration (>2 fold). In addition, CCK-8 assay at 48 h and 72 h time points Givi-MPC showed higher OD value compared with human myoblasts and control-MPC (FIG. 31E). These data suggest that Givi-MPC possess better self-renewal potential. Colonies formed by Givi-MPC were bigger and had a higher cell density compared to control-MPC (FIG. 31F). Quantitative data (FIGS. 31G and 31H) showed that Givi-MPC formed more colonies with higher density of cells (>200 cells) compared to control-MPC. These data support the notion that Givi-MPC possessed highly proliferation and self-renewal capabilities.

[0491] In Vivo Engraftment of Givi-MPC Restores Dystrophin and Integrated into the Recipient Environment

[0492] We transplanted human myoblast, control-MPC and Givi-MPC into Mdx/SCID mice with CTX injury, respectively. One-month post-transplant, Givi-MPC showed increased engraftment capacity and restoration of dystrophin than treatment with control-MPC and human myoblasts (FIGS. 32A and 32B). FIG. S2 showed the engrafted Givi-MPC (GFP positive) expressed dystrophin. Quantitative data showed Givi-MPC treated TA muscle had significantly higher number of dystrophin positive muscle fibers (FIG. 32C) and GFP and human laminin double positive muscle fibers (FIG. 32D). To determine the functionality of the newly formed muscle fibers from Givi-MPC, we tested whether they were integrated into the recipient environment with innervation. Positive staining of presynaptic marker -BTX was observed in close proximity to dystrophin positive muscle fibers in Givi-MPC treated TA muscle, suggesting the presence of neuromuscular junction in these muscle fibers (FIG. 32E).

[0493] Givi-MPC Limited Inflammation, Muscle Necrosis and Reduced Fibrosis in Mdx/SCID Mice Post CTX Injury

[0494] Hematoxylin and eosin and trichrome Masson staining revealed infiltration of inflammatory cells, and necrotic muscle fibers in Mdx/SCID mice 7 days' post CTX injury (FIG. 33A). A significant decrease in muscle necrosis was observed in Givi-MPC treated TA muscle compared to collateral PBS treated TA muscle (FIG. 33B). Amongst different MPCs which were transplanted, Givi-MPC reduced muscle necrosis the most (FIG. 33C) with reduced number of CD68 positive macrophages as compared with human myoblast and control-MPC treated tissue (FIG. 33D, E) 7 days' post CTX injury. In the muscle following 1M post CTX injury, transplantation of human myoblasts, control-MPC and Givi-MPC significantly decreased muscle necrosis compared to PBS treated collateral TA muscle (FIG. 34A-34D). No significant difference in muscle fiber necrosis was observed between human myoblasts and control-MPC treated TA muscle. However, Givi-MPC treatment resulted in reduced necrosis area compared to other MPCs treatment (FIG. 34E). Similarly, Givi-MPC transplantation reduced collagen deposits (red) compared to PBS, human myoblasts and control-MPC treated muscle (FIG. 34F, 34G-34I).

[0495] Givi-MPC Repopulated the Muscle Stem Cell Pool

[0496] A significant number of Givi-MPC were transformed into muscle stem cells and occupied their sites as evidenced by double positivity for Pax7 and HNA cell under basal lamina at 1M post-transplantation (FIG. 35A). A schematic outline of reinjury experiments with CTX is provided (FIG. 35B). Compared with contralateral PBS treated TA muscle, expression of dystrophin was detected in Givi-MPC treated TA muscle after reinjury (FIG. 35C). Furthermore, Givi-MPC treated TA muscle showed increased muscle regeneration and less infiltration of inflammatory cells compared with contralateral PBS treated muscle (FIG. 35D). These data indicated that the engrafted Pax7 positive cells responded to reinjury and formed new muscle fibers.

[0497] Extracellular Vesicles Derived from Givi-MPC Facilitated Angiogenesis in Muscle Following CTX Injury

[0498] Angiogenesis is critical for muscle regeneration (18,19). Givi-MPC treatment caused higher capillary density (CD31 positivity) in TA muscle 1M post CTX injury (FIGS. 36A and 36B). Next we tested whether increased angiogenesis was due to paracrine effects by EV released from MPCs. We isolated EV from Givi-MPC using size exclusion columns. Using tunable resistive pulse sensing (TRPS) technique, we measured the concentration and size of EV from Givi-MPC. The size of isolated EV was roughly 118 31.7 nm. In vitro tube formation assay indicated that EV from Givi-MPC promoted tube formation (FIG. 36C) and resulted in higher average tube length (FIG. 36D) compared to treatment with EV from human myoblasts or control-MPC. We further analyzed the miRNA cargo contents of EV from Givi-MPC. A heatmap of significantly upregulated and downregulated miRNAs in EV from Givi-MPC compared to EV from human myoblasts was shown in FIG. 36E. miR-210, miR-181a, miR-17 and miR-107 were enriched in EV from Givi-MPC.

[0499] Discussion

[0500] In the present study, we successfully generated highly proliferative and integration free MPC from multiple hiPS cell lines using CHIR99021 and Givi. These cells expressed myogenic markers including Pax7 and desmin, which were also capable to differentiate into muscle cells under specific differentiation medium in vitro. Of particular significance was the ability of these MPCs to differentiate in dystrophic mouse model, making them more suitable for therapeutic applications. These cells possess special properties which make them unique for therapeutic applications. Migration and engraftment of transplanted cells to the site of injury are crucial to initiate differentiation into skeletal muscle components in the dystrophic muscle(20,21). Limited cell migration hampers engraftment efficiency in skeletal muscle (22,23). In the present study, we found MPC induced by Givi exhibited superior migration and proliferation capabilities compared with human myoblasts and control MPC generated by CHIR99021 and FGF. Go analysis further showed upregulation of cell migration related genes enabling them to migrate to distant injured muscle (10). In our data, genes related to migration were significantly upregulated with Givi treatment. ITGA4 was the most upregulated gene with 25.61-fold change. Integrin subunit 4 (ITGA4) is a member of the integrin alpha chain family of proteins. Integrin a subunits which pair with 1 play a critical role during in vivo myogenesis. Integrin 4 subunit is expressed in the myotome and in early limb muscle masses during muscle development (24,25). Murine Lbax1.sup.+ embryonic muscle progenitors expressed ITGA4 (26). It has been reported that teratoma derived MPC possessed high engraftment efficiency in muscle dystrophy model (10). However, the mechanism of upregulation of ITGA4 by Givi and migration medicated by ITGA4 need further study. DMD is a disease with body-wide systemic and progressive skeletal muscle loss, thus further study for the role and mechanism of ITGA4 in MPC migration will move MPC-based therapy for DMD forward to clinical application. In agreement with in vitro observations, we also observed higher engraftment efficiency of Givi-MPC compared to human myoblasts and control MPC upon transplantation in muscle tissue from Mdx/SCID mice following CTX injury. The significant engraftment in muscles of Mdx/SCID mice by human iPS-derived skeletal myogenic progenitors resulted in more dystrophin expressing myofibers or human laminin positive myofibers. Besides dystrophin, presence of neuromuscular junctions in human myofibers using -BTX together with dystrophin in Mdx/SCID mice with Givi-MPC transplantation, suggest that formation of functional myofibers has occurred.

[0501] Histological analysis showed that fewer muscle fibers had undergone necrosis and fibrosis in injured TA muscle of Mdx/SCID mice treated with Givi-MPC. Inflammatory cell infiltration in general contributes to myofiber necrosis (27,28). Although Mdx/SCID mice are immunodeficient, it has been reported that M1 macrophages participated in skeletal muscle regeneration in SCID mice (29), suggesting partial immune reactivity in these mice. It has been reported Givi has potential anti-inflammatory effects (30,31). For example, Givi decreased inflammation in a mouse model with myocardial infarction (31). With HE staining, we found infiltration of larger number of inflammatory cells in TA muscle from Mdx/SCID mice treated with PBS, or human myoblasts or control MPC treatments 7 days post-CTX injury. Negligible macrophage infiltration identified by CD68 staining was observed in Givi-MPC transplanted Mdx/SCID mice 7 day post-CTX injury. These observations support that Givi-MPC had anti-inflammatory effects upon transplantation in CTX injured muscle suggesting that properties of MPC depend on the source of reprogramming molecule. Besides immediate effects on engraftment and differentiation, the long-term maintenance of newly formed skeletal muscle is ultimately dependent on the ability of the transplanted MPCs to contribute to the skeletal muscle stem cell pool (10). Here we observed Givi-MPC derived Pax7 positive cells under basal lamina upon transplantation, and with subsequent reinjury the Givi-MPC contributed to secondary regeneration in the Mdx/SCID mice. This observation supported that a subpopulation of Givi-MPC can seed the stem cell pool.

[0502] Angiogenic impairment of the vascular endothelial cells (EC) isolated from mdx mice compared with wild type mice has been reported (32) causing a marked decrease in the vasculature in TA muscle of mdx mice (33). Local delivery of muscle-derived stem cells engineered to overexpress human VEGF into the gastrocnemius muscle of Mdx/SCID mice resulted in marked increase in angiogenesis accompanied by enhanced muscle regeneration and decreased fibrosis compared with mice treated with non-engineered cells (34). In addition, satellite cells isolated from mdx mice exhibited reduced capacity to promote angiogenesis, as demonstrated in a co-culture model of satellite cells of Mdx mice and microvascular fragments (35). Here, our study demonstrated that after Givi-MPC transplantation, an increase in capillary density was observed as evidenced by CD31 staining in CTX injured Mdx/SCID mice compared to treatment with other MPCs. These results enforce the idea that an interaction between EC and MPC was important for myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration (18). To further strengthen this observation, we found that EV from Givi-iMPC were enriched in several miRNAs including miR-181a, miR-17, miR-210 and miR-107, miR-19b compared with EV from human myoblasts. Due to role of EV in cell-to-cell communication, these enriched miRNAs have been demonstrated to participate in angiogenesis. Activation of miR-17-92 cluster promoted angiogenesis via PTEN signaling pathway, however, EC miR-17-92 cluster knockout impaired angiogenesis (36). miR-181a and miR-210 are also reported to promote angiogenesis (37-40). Thus it is very likely that Givi-MPC interacted with resident EC to initiate myogenesis and angiogenesis in Mdx/SCID mice after CTX injury.

Conclusion

[0503] We successfully generated highly expandable and integration free MPC from multiple hiPS cell lines using CHIR99021 and givinostat. Givinostat-induced MPC were highly proliferative and migratory and transplantation resulted in marked and impressive myoangiogenesis and restored dystrophin in injured TA muscle compared to control MPC and adult human myoblasts. It is concluded that hiPSCs pharmacologically reprogrammed into MPC with a small molecule, givinostat with anti-oxidative, anti-inflammatory and muscle gene promoting properties is an effective cellular source for treatment of muscle injury and restoration of dystrophin in DMD.

[0504] Funding

[0505] This study was supported by National Institutes of Health grants RO1 HL134354 & RO1 AR070029 (M Ashraf, Y Tang, and NL Weintraub).

[0506] Figure Legend

[0507] FIG. 29 Generation of muscle progenitor cell (MPC) from human iPSC using small molecules. (A) Schematic outline of generation of MPC from human PSC using combination of CHIR99021 and givinostat or CHIR99021 only. (B) Morphology of differentiating cells from 4 human iPSC lines (CF-iPSC, DF-iPSC-1, DF-iPSC-2 and DMD-iPSC) at 7 days. Bar=200 m. (C) Morphology of replated MPC and differentiated myotubes from 4 human iPSC lines at day 14. Bar=200 m

[0508] FIG. 30 Characterization of givinostat-induced MPC. (A) The treated hiPSC at day 14 expressed Pax7 and desmin. (B) The differentiated myotubes expressed MF20 as shown by immunostaining. Bar=50 m.

[0509] FIG. 31 Givi-MPC exhibit superior proliferation and migration capacity. (A) Representative images and quantitative estimate (b) of cell migration by adult human myoblasts, and control-MPC, Givi-MPC (arrow). Cells were stained with Calcein AM (green). Bar=1 mm. (A) Quantitative estimate of migrated cells. Givi MPC showed highest number of cells migrated compared with human myoblasts (P<0.0001) or CHIR99021 induced MPC (P<0.0001). No significant difference was observed between human myoblasts and control-MPC. (C) Heat map of the Human RT.sup.2 motility PCR Array. (D) Upregulated migration related genes in Givi-MPC vs. control-MPC using human cell motility PCR array. (E) The proliferation curves of human myoblasts vs MPC using CCK-8 assay. *P<0.05; .sup.#P<0.05 vs control-MPC. n=6. (F) Morphology of MPC colony. Bar=500 m. Number of colonies (G) and percentage of colonies with different cell number (H). control-MPC: CHIR99021 induced MPC; Givi-MPC: CHIR99021 and Givinostat induced MPC.

[0510] FIG. 32 In vivo myogenic potential of different MPC and myoblast in Mdx/SCID mice with CTX injury. (A) Dystrophin restoration in Mdx/SCID mice by MPC transplantation at 1M after CTX injury. Bar=50 m. (B). Transplanted cells were labeled with GFP (Green) and identified with human laminin staining (Red). Quantitation of engrafted fibers at 1M: Dystrophin+fibers (n=6) (C) and human laminin and GFP double positive fibers (n=3) (D). (E) Cross-section showing pre-synaptic staining with -bungarotoxin in dystrophin positive fibers (n=3). Bar=20 m.

[0511] FIG. 33 In vivo myogenic potential of different MPC and myoblast in Mdx/SCID mice with CTX injury. (A) Representative images of HE and Trichrome Masson staining in Mdx/SCID mice with human myoblasts or control-MPC or Givi-MPC transplantation 7 days after CTX injury. Black arrows indicate infiltrated inflammatory cells. (B) Quantification of muscle fiber necrosis between PBS treated collateral TA muscle or Givi-MPC treated TA muscle 7 days after CTX injury. (C) Quantification of muscle fiber necrosis of TA muscle among human myoblast or control-MPC or Givi-MPC transplantation mice 7 days after CTX injury. (D) Quantification of CD68 positive cells in TA muscle following MPC transplantation 7 days after CTX injury. (E) Representative images of macrophages (red, CD68) and human cells in TA muscle of Mdx/SCID mice with CTX injury following MPC transplantation.

[0512] FIG. 34 Givi-MPC decrease muscle necrosis and fibrosis in Mdx/SCID mice 1M after CTX injury. (A) Representative images of HE and Trichrome Masson staining in Mdx/SCID mice after transplantation with human myoblasts or control-MPC or Givi-MPC 1M after CTX injury. Bar=500 m (4) and Bar=100 m (20). Quantification of necrotic muscle fibers after treatment with human myoblasts (B), control-MPC (C) and Givi-MPC (D) 1M after CTX injury. (E) Comparison of muscle necrosis among human myoblasts or control-MPC or Givi-MPC transplantation mdx/SCID mice. (F) Representative images of tissue stained with Sirius red from Mdx/SCID mice. Bar=100 m. Quantification of muscle fiber fibrosis in collateral TA muscle treated with human myoblasts (G), control-MPC (H) and Givi-MPC (I) 1M after CTX injury. (J) Muscle fibrosis after MPC transplantation in mdx/SCID mice.

[0513] FIG. 35 Givi-MPC repopulated the muscle stem cell pool. (A) Muscle cells positive for Pax7 (green) and human nuclear antigen (red) cell under the basal lamina from Mdx/SCID mice after 1M of Givi-MPC transplantation. Bar=20 m. (B) Schematic of reinjury experiment. (C) 1M after reinjury, expression of dystrophin in Givi-MPC treated TA muscle tissue and contralateral PBS treated TA muscle tissue. Bar=50 m. (D) Representative HE stained images of Givi-MPC treated TA muscle tissue and contralateral PBS treated TA muscle tissue. Bar=50 m.

[0514] FIG. 36 Extracellular vesicles derived from Givi-MPC promoted angiogenesis. (A) Representative images of CD31 (Red) and laminin (Green) staining in Mdx/SCID mice 1M post injury. Bar=50 m. (B) Quantification of capillary density (CD31 positive capillaries). (C) Representative images of tube formation by human aortic endothelia cells (HAECs) following EV treatment from human myoblasts, or control-MPC or Givi-MPC (1 g/well, 24 well plate). HAECs were labeled with Calcein AM (Green). Bar=500 m. (D) Tube formation assay. Average tube length was analyzed from 3 biological repeated experiments. (E) Heatmap showing significant upregulation of miRs in EV derived from Givi-MPC compared to EV-human myoblasts.

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