Microvesicles and stem cell compositions for therapeutic use

Abstract

To provide stem cells and exosome compositions having therapeutic utility to treat a variety of diseases and disorders, e.g., cardiovascular disease, Duchenne muscular dystrophy, and fibrotic disease.SOLUTION: The invention provides an exosome or microvesicle selected from among an iPSC cell, an embryonic stem cell, and a stem cell that has been contacted with an effective amount of an isoxazole compound, a derivative or equivalent thereof, or Danazol, where the exosome or microvesicle overexpresses one or more of: a) a microRNA (miRNA) selected from among mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-21, mir-30c, mir-214 and mir-548q; and / or b) one or more proteins selected from among Tsg101, CD9, Hsp70, Flotillin-1 and GAPDH.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [References] This application is contained in 35 USC Section 120 and is a continuation-in-part application following U.S. Patent Application No. 14 / 255,789 filed on 17 April 2014, U.S. Patent Application No. 15 / 951,354 filed on 24 November 2015, and U.S. Patent Application No. 15 / 201,292 filed on 1 June 2016. The contents of each of these applications are incorporated herein by reference.

[0002] [Government support statement] This invention was partially supported by grants from the National Institutes of Health Sciences, RO1 HL126516, HL134354, and RO1 AR070029. Therefore, the U.S. government holds the rights to this invention.

[0003] [Refer to the sequence table submitted to EFS-WEB] This application was filed electronically via EFS-Web and includes one txt digital sequence table. This txt file is named [2018-06-12 ISPH 004 ST25.txt], was created on June 12, 2018, and contains a sequence table of 3,537 bytes. The txt file is part of this specification and is incorporated herein by reference. The contents of the sequence table recorded in computer-readable format are identical to the contents of the written sequence table and do not contain any new information. [Background technology]

[0004] This application includes references to various technical and patent publications to provide a more detailed explanation of the latest technologies relating to the present invention. All publications mentioned herein are incorporated herein by reference.

[0005] Induced pluripotent stem (iPS) cells can differentiate into cardiac progenitor cells and are an important cell source for the treatment of heart disease and drug screening. Furthermore, induced pluripotent stem cells have the potential to cause cancer, and when applying regenerative medicine using these cells to clinical applications, it is important to induce differentiation into cardiomyocyte progenitor cells that do not carry the risk of causing cancer. For this reason, efforts have been made to improve the efficiency of reprogramming, and methods for generating induced pluripotent stem cells without using viruses have been developed. Numerous growth factors and compounds have been discovered that improve induction efficiency, including DNA methyltransferase inhibitors (5'-azacitidine and RG108), histone deacetylase inhibitors (valproic acid), histone methyltransferase inhibitors (BIX_01294), and Wnt3A and ALK5 inhibitors. (1-6) Thanks to various experiments, methods have been shown to induce induced pluripotent stem cells into the cardiogenic cell spectrum before transplantation. However, such methods are complicated and costly, and require the administration of numerous growth factors and compounds, making it highly likely that sufficient uniformity and reproducibility necessary for drug development and clinical application cannot be maintained. Therefore, it is necessary to improve existing methods and techniques. This specification satisfies this requirement and, furthermore, provides the associated advantages. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This disclosure provides, in part, a method for using only one type of small molecule compound to apply the small molecule compound to induced pluripotent stem cells (IPSCs) to express the cells and pre-treat the cells propagated from the method (e.g., cardiac progenitor cells, endothelial cells, smooth muscle cells, myocytes, myofibrillator progenitor cells, cardiomyocytes and blood vessels, and exosomes or microvesicles produced by those cells). The cells and exosome or microvesicle compositions isolated from the cells activate endogenous stem cells, cardiomyocytes, myocytes and paracrine factors. Furthermore, given the inefficient existing methods for producing these cells and exosomes or microvesicles, the above one-step, commercial, scalable, and safe method may be the most economical way to produce countless cells, exosomes, or microvesicles for the treatment of myocardial infarction, Duchenne muscular dystrophy, myopathies and other diseases.

[0007] Accordingly, in one embodiment, an isolated population or composition comprising an isolated population of exosomes or microvesicles is provided herein. These are, on the one hand, isolated from cells selected from the following groups: iPS cells, embryonic stem cells, or stem cells, each previously contacted with an effective amount of an isoxazole compound, its derivative or equivalent isoxazole compound. The exosomes or microvesicles then overexpress a microRNA, miRNA or mir, selected from the group mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-30c, mir-214, or mir-5487, and one or more proteins selected from Tsg101, CD9, Hsp70, Flotilin-1, or GAPDH. Furthermore, the exosomes or microvesicles may further overexpress one or both of mir-30c and / or mir-21. In one embodiment, at least 70%, or 80%, or 85%, or 90%, or 95%, or 98% of the cells express the mir and / or protein described. [Means for solving the problem]

[0008] Furthermore, this specification also provides one or more cells or isolated cell populations of cardiomyocyte progenitor cells (CPCs), cardiomyocytes, muscle cells, endothelial cells, smooth muscle cells, and skeletal muscle cells generated from iPSCs, embryonic stem cells, and cells selected from stem cells. Herein, each cell is generated from iPSCs, embryonic stem cells, and cells selected from stem cells that have previously come into contact with isoxazole compounds, derivatives, or equivalents thereof, and the cells constituting the population overexpress one or more of mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-30c, mir-214, or mir-548q; and / or overexpress muscle genes selected from the group of paZ3, PAX7, MYF5, MYOD, MYOG, and dystrophin. Also provided are cardiomyocytes expressing one or more of cTnT, cTnl, cMLC2V, and VE-cadherin. In yet another aspect, provided herein are cells or cell populations of smooth muscle actin (SMA) and calponin. In one aspect, at least 70%, or 80%, or 85%, or 90%, or 95%, or 98% of cells overexpress the aforementioned mir and / or muscle genes and / or proteins. Exosomes or microvesicles may be isolated from any of the following: cardiomyocyte progenitor cells, cardiomyocytes, skeletal muscle, endothelial cells, myocytes, or smooth muscle cells.

[0009] In one embodiment, the population or cells further comprises a pharmaceutically acceptable carrier, which is a non-natural carrier. The composition also, in one embodiment, comprises preservatives and / or cryopreservatives to facilitate lyophilization and / or storage of the composition. Furthermore, the composition further comprises proteins and / or nucleic acids that code for improved tissue function or proteins to facilitate regeneration. Such non-limiting examples include TGF-B, WNT proteins, cytokines, or histone deacetylases.

[0010] Further provided are compositions for the repair or regeneration of damaged or affected cardiac tissue, comprising synthetic microRNA-146a. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier which is not naturally occurring. The composition also comprises preservatives and / or cryopreservatives to facilitate lyophilization and / or storage of the composition.

[0011] Further provided are compositions comprising one or more of synthetic microRNA-373, microMA373 mimetic, or microMA373 exosome. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier, which is a carrier that does not exist naturally. The composition also, in one embodiment, comprises a preservative and / or cryopreservative to facilitate lyophilization and / or storage of the composition. These compositions are useful for treating fibrotic diseases by administering an effective amount to a subject in need. A non-limiting example of a fibrotic disease is myocardial fibrosis.

[0012] Populations or cells of exosomes or microvesicles are isolated from populations of stem cells or progenitor cells cultured in the presence of an effective amount of isoxazole compound or a derivative thereof. In one embodiment, the isoxazole compound is selected from isoxazole-1 (isx-1), isoxazole-9 (isx-9), or danazole.

[0013] Methods for providing one or more of the following to subjects requiring: regeneration of damaged tissue; improvement of the vitality of damaged tissue; promotion of the formation of new heart, muscle, skeletal tissue, blood vessels, capillaries, and certain muscle cells; promotion of cardiac recovery; promotion of cardiac recovery in subjects with acute cardiac events; promotion of cardiac recovery in subjects with Duchenne muscular dystrophy and Duchenne muscular dystrophy-related cardiomyopathy; promotion of cardiac recovery in subjects with age-related diseases: HHS syndrome, pulmonary fibrosis, aplastic anemia, hepatic fibrosis, congenital keratosis disorders, lung diseases, endocrine disorders, bone marrow failure, polycystic ovary syndrome (PCOS), Cushing's syndrome, acromegaly, cerebrovascular disease (stroke), hypertension, Parkinson's disease, vascular dementia, dementia, macular degeneration, Alzheimer's disease, age-related hearing loss, celiac disease, COPD, bipolar disorder, hydroxyurea, sickle cell disease, hypertension, atherosclerosis Arthritis, osteoporosis, osteoarthritis, cancer, type 2 diabetes, or telomerase dysfunction associated with shortened telomeres; promoting the regeneration of damaged tissue in stroke, arthritis, Alzheimer's disease, memory loss, cystic fibrosis, inflammatory diseases, or cancer; reducing the thickness of the heart wall in damaged tissue in myocardial infarction; quinase C, iL-6, mmp This method involves genetically modifying one or more proteins, such as PDGF; reducing or inhibiting the expression of inflammatory proteins; potentially cytokines, chemokines, or macrophages; directly and indirectly promoting angiogenesis; directly and indirectly inhibiting cell replication; and managing effective amounts of exosomes and microvesicles, as well as isolated cells and their compositions as specified herein, to promote cardiac regeneration in subjects with the following diseases: coronary artery disease, myocardial infarction, heart failure, hypoplastic left heart syndrome, peripheral artery disease (PAD), cardiac hypertrophy, valvular heart disease (aortic stenosis), myocardial hypertrophy, and hypertrophic fibrosis. Furthermore, this method includes the management of non-embryonic stem cells or progenitor cells, which are the main components of the tissue in which the method is used. These cells are tissue-like cells of the tissue in need of repair. Usable stem cells are non-embryonic stem cells or autologous progenitor cells.

[0014] Also provided herein is a method for preparing the cell population described herein from a population of human induced pluripotent stem cells (hiPSCs), the method comprising contacting the human induced pluripotent stem cells (hiPSCs) with an effective amount of givinostat (GIV).

[0015] Further provided is a method for one or more of the following: providing antioxidant therapy; promoting activation of resident or local cardiomyocytes; promoting angiogenesis or release of paracrine factors; promoting activation of WNT, BMP, and cytoskeletal remodeling; promoting TGF-β-induced EMT signaling and cardiac differentiation; increasing expression of WNT5 and WNT11 or proteins of the BMP family and BMP4; increasing expression of cardiac transcription factors selected from the following population: nkx2.5, mef2c, gata4, and isl-1; promoting expression of genes required for developing the PIP3 signaling pathway to the cardiomyocyte, muscle contraction, and NF-AT hypertrophy signaling pathway; reducing fibrosis and apoptosis; promoting myogenic and muscle differentiation; promoting release of cytokines selected from the population comprising angiopoietin-2, Il-6nmp, pgfbb, timp1, or genes disclosed herein and in the figures; promoting upregulation of genes selected from the population of wnt3a, wnt5a, wnt11; and promoting cytoskeletal remodeling, the method comprising administering to a subject in need thereof an effective amount of a population of exosomes or microvesicles or cells, or a composition comprising the same.

[0016] Furthermore, in the present specification, there are provided cells or cell populations, as well as cells and populations prepared by the methods disclosed herein, wherein at least 80%, or 85%, or 90%, or 95%, or 97%, or 99%, or 100% of the cells overexpress one or more proteins selected from the following group: cTN1, MLC2v, TNT, VE-cadherin, CD31, a-SMA (actin), calponin, or Cx43. There are also provided cells or cell populations that overexpress skeletal muscle formation genes selected from the following group: Meox1, Meox2, Tcf15, Pax3, Pax7, MyoD1, MYF5, dystrophin, or desmin. The expression of these genes, mRNAs, and / or proteins is known in the art and can be determined by the methods briefly described herein. Cells, microvesicles, and / or exosomes isolated from the above cells are advantageously used in methods for regenerating skeletal muscle cells and in methods for treating DMD.

[0017] In addition, in the present specification, there are provided cell populations that overexpress xESI myogenic genes selected from the following group: Pitx2, IS11, Nkx2.5, Handi, GATA4, Tbx5, TnnT2, My17, MLC2v, Myf2e, Cdh4, or Lhx2. In one aspect, at least 80%, or 85%, or 90%, or 95%, or 97%, or 99%, or 100% of the cells in the above cell population overexpress xESI myogenic cells. The expression of these genes, mRNAs, and / or proteins is known in the art and can be determined by the methods briefly described herein. These cell populations and cells are advantageously used in methods for regenerating myocardial tissue and in methods for treating cardiac dysfunction associated with Duchenne muscular dystrophy (DMD). These methods consist essentially of or consist of administering an effective amount of the cell population described herein and / or administering an effective amount of a population of exosomes or microvesicles described herein to a subject that needs it.

[0018] Furthermore, provided herein are populations of exosomes and microvesicles isolated from these cell populations.

[0019] A method for regenerating cardiomyopathy is provided, comprising administering an effective amount of the cell population described herein to a subject in need thereof, and / or an effective amount of the exosome or microvesicle population described herein is further provided.

[0020] Methods for repairing or regenerating damaged or affected cardiac tissue in subjects requiring such repair are also provided herein.

[0021] Also offered are compositions for the repair or regeneration of damaged or affected cardiac tissue, or for the treatment of one or more of them: HHS syndrome, congenital keratosis, pulmonary fibrosis, aplastic anemia, hepatic fibrosis, bone marrow failure, lung disease, endocrine disorders, polycystic ovary syndrome (PCOS), Cushing's syndrome, and acromegaly, cerebrovascular disease (stroke), hypertension, Parkinson's disease, dementia, Alzheimer's disease, age-related hearing loss, celiac disease (CD), COPD, bipolar disorder, hydroxyurea, sickle cell disease, atherosclerosis, arthritis, osteoporosis, osteoarthritis, vascular dementia, or macular degeneration, cancer, type 2 diabetes, or telomerase dysfunction associated with shortened telomeres. The composition contains a MicroRNA-195 inhibitor. Non-limiting example: HSTUD0320 (SIGMAMISSION® synthetic microRNA inhibitor), human HSA-miR-195-5p and inhibitory hsa-miR 195-5p miRNA / microRNA lentictor (available at ABM Goods (abmgood.com)). When an effective amount of bone marrow stem cells or iPS cells are present, this composition promotes and cooperates with telomere elongation and regeneration of senescent stem cells. This method promotes cardiac regeneration in elderly patients with heart disease such as heart failure, and in patients such as those who have suffered a heart attack.

[0022] Furthermore, a method is provided which involves administering one or more microRNA fragments or derivatives thereof to a subject. After administration of one or more microRNA fragments, the one or more microRNA fragments alter gene expression in the damaged tissue, improve the viability of the damaged tissue, and promote the formation of new tissue in the subject. On the one hand, the microRNA fragments or derivatives are synthesized. On the other hand, the synthesis of the microRNA fragments or derivatives utilizes a sequence that mimics one or more endogenous microRNA molecules. On the other hand, the microRNA fragments or derivatives are modified to increase their stability. The microRNA fragments are administered by an appropriate method determined by a treating physician or specialist. A non-limiting example is the administration of multiple synthetic liposomes containing one or more of the described microRNA fragments or derivatives.

[0023] Further provided is a method for generating exosomes or microvesicles, comprising culturing a population of non-embryonic human regenerative cells in the presence of a hydrolase enzyme to induce cells to secrete exosomes or microvesicles, thereby generating exosomes or microvesicles. In a further embodiment, the method further comprises isolating the exosomes or microvesicles from the culture medium and / or cells.

[0024] In one respect, the hydrolytic enzymes include one of the DNAse I enzyme superfamilys. A non-limiting example is sphingomyelinase. On the one hand, the type of sphingomyelinase is selected from lysosomic acid sphingomyelinase, secreted zinc-dependent acid sphingomyelinase, neutral sphingomyelinase, and alkaline sphingomyelinase. Furthermore, the neutral sphingomyelinase includes one or more magnesium-dependent neutral sphingomyelinases and magnesium-independent neutral sphingomyelinases. Also, the neutral sphingomyelinase includes one or more neutral sphingomyelinase type I, neutral sphingomyelinase type 2, and neutral sphingomyelinase type 3. [Brief explanation of the drawing]

[0025] This patent and application must include at least one color drawing. Copies of the patent and application, including that color drawing, will be provided by the Secretariat upon request and for the required fee.

[0026] [Figure 1] Figure 1 shows the characterization of iPS cells to represent pluripotency markers. It is a representative micrograph showing iPS cell clones expressing embryonic stem cell-specific markers Oct3 / 4, Sox2, Nanog, and endogenous Oct3 / 4.

[0027] [Figure 2] Figure 2 shows the results of the DNA methyltransferase (DNMT) activity assay. DNA methylation analysis showed significant (95%) inhibition of DNA methyltransferase activity in iPS cells treated with small molecule compounds, isoxazoles, or isoxazole-like compounds compared to untreated iPS cells.

[0028] [Figure 3] Figures 3A-3B show that treatment with small molecule compounds, isoxazoles, or isoxazole-like compounds induces cytoprotection in vitro, and that small molecule compound treatment prevents oxidant-induced apoptosis and promotes proliferation. In Figure 3A, apoptosis was determined for each TUNEL assay. Compared to untreated iPS cells, fewer TUNEL-positive cells / microscopic fields were observed in iPS cells pre-treated with small molecule compounds, isoxazoles, or isoxazole-like compounds. In Figure 3B, a significantly increased pro-mitotic response was observed in iPS cells treated with small molecule compounds compared to untreated iPS cells. All values ​​are expressed as mean ± SEM, *p < 0.05 versus control.

[0029] [Figure 4]Figure 4 shows cytoplasmic translocation of cytochrome c (indicating cell damage). Immunostaining of cytochrome c in untreated iPS cells and iPS cells treated with small molecule compounds, isoxazoles, or isoxazol-like compounds after exposure to H and O, (100 μmol), (DAPI-bound image), (original magnification: 200x). Compared to untreated iPS cells, cytoplasmic translocation of cytochrome c in iPS cells treated with small molecule compounds, isoxazoles, or isoxazol-like compounds is significantly reduced.

[0030] [Figure 5] Figures 5A-5B show that small molecule compounds, isoxazoles, or isoxazol-like compounds can suppress cardiac differentiation of iPS cells in vitro. Figure 5A shows immunostaining of the cardiac-specific gene Nkx-2.5 and myomatous actin in iPS cells treated with small molecule compounds (DAPI-bound image), (original magnification: 400x). Figure 5B shows RT-PCR analysis of the cardiomyocyte-specific marker Nkx-2.5 in iPS cells treated with small molecule compounds compared to untreated iPS cells. Nkx-2.5 in iPS cells treated with small molecule compounds, isoxazoles, or isoxazol-like compounds was shown to be significantly upregulated compared to that in untreated iPS cells.

[0031] [Figure 6] Figure 6 shows affymetrix assay-based gene expression profiling of iPS cells treated with small molecule compounds and isoxazole-like compounds, and untreated cells, further demonstrating 2-3x downregulation of proteins related to Dnmt1, Dnmt3b, and the Max gene. This phenomenon is associated with DNA hypomethylation and myc-dependent cell transformation. Furthermore, there was 2-3x concomitant overregulation of CCL7, CXCR2, CXCR5, endogenous membrane protein 2A, or ephrin A3.

[0032] [Figure 7]Figures 7A-7 show miR-microassay analyses of iPS cells treated with small molecule compounds and isoxazole-like compounds, and untreated cells. The MIR expression profile of untreated iPS cells, as shown by microassay analysis, was completely different from that of iPS cells treated with small molecule compounds (Figures 7A-7D). miRs important for reprogramming and differentiation were observed in untreated iPS cells and iPS cells treated with small molecule compounds. Microassay analysis showed superior regulation of cardiac-specific miR-133 and miR-762 and downregulation of pluripotency involving the miR-290-295 population and the let-7 family in iPS cells treated with small molecule compounds. Figure 7 shows GPCR signaling in iPS cells treated with small molecule compounds and isoxazole-like compounds. Western blot analysis showed that GA(pan) in iPS cells treated with small molecule compounds was significantly upregulated compared to untreated iPS cells or those blocked by a combination of GPCR blockers and pertussis toxin.

[0033] [Figure 8] Figures 8A-8 show that transplantation of iPS cells treated with isoxazole and isoxazole-like compounds attenuated infarct size enlargement and regeneration of mature muscle fibers in vitro. One animal was injected with a total of 3.6 x 10⁵ cells into its infarct and surrounding area after coronary artery ligation. The animal was killed after 6 weeks and 7 days. Significant new fiber regeneration in the infarcted heart reduced infarct size (Figures 8C and 8D). The number of iPS-derived growing cells observed in heart transplants of untreated iPS cells was very limited (not shown). There was a significant increase in systolic left ventricular dimension (LVESD) and diastolic left ventricular dimension (LVEDD) after myocardial infarction, which was significantly reduced by iPS cells treated with isoxazole and isoxazole-like compounds (Figure 8E). Similarly, treatment increased heart rate reduction and ejection fraction (Figure 8F).

[0034] [Figure 9]Figures 9A-9 show the characteristics of small, immature stem cells (SJSCs) taken from xenomyelocyte-derived stem cells (BMSCs) of aged and young mice, as described in Experiment 2. Figures 9A and 9 show that SJSCs from both aged and young mice were positive for CD29, CD44, CD59, and CD90, but negative for CD45 and CD117 B. In Figure 9C, the cell proliferation curve shows that the cell proliferation rate of SJSCs was higher than that of BMSCs. Compared to BMSCs, aged SJSCs showed stronger expression of pluripotency markers, such as octamer-binding transcription factor 4 (Oct-4), Nanog, sex-determining region Y-box 2 (Sox-2), Kruppel-like factor 4 (Klf-4), and Rex1.G. Figure 9 shows cardiac differentiation markers such as Gata-4 and muscle cell-specific enhancer factor 2C (Mef2c). Figure 9 shows anti-aging markers such as sirtuin 1 (Sirt1) and telomerase reverse transcriptase (Tert). In Figure 9F, we found that SJSCs from aged bone marrow had less aging-related β-galactosidase (β-gal) than other cells from aged BM. Figure 9 shows fluorescence quantification of in situ hybridization (FISH) analysis, demonstrating that SJSCs retained longer telomeres than BMSCs. Telomere shortening observed in SJSCs was delayed in BMSCs.

[0035] [Figure 10]Figures 10A-10 show the characteristics of Sca-17 CSCS. Figure 10A is a brief description of the CSC isolation procedure, and Figure 10 shows that Sca-1+ expression in isolated CSCs was confirmed by immunocytochemistry and flow cytometry. (Light gray = Sca-1+; Dark gray = DAPI) In Figure 10C, positivity for discoidine domain receptor 2 (DDR2) and prolyl 4-hydroxylasebeta (P4HB) antibodies is rare in CSC cultures. Figure 10 shows the analytical representations of RT-PCR for hematopoietic progenitor cell markers (c-kit), pluripotency markers (October 4, Sox2, Nanog), stem cell population marker (Bcrpl), early cardiac lineage markers (Nkx-2.5, GATA4, MEF2C), and vascular progenitor cell marker (Fiki) in isolated Sca-1+ cell colonies. The notation C above the column indicates the culture colony and its identification number. They maintained approximately 80–90% of Sca-1+ positive cells up to passage 5–30, as measured by flow cytometry. All experiments were performed within this passage. Bar = 100 μm

[0036] [Figure 11] Figures 11A-11 show that connective tissue growth factor (CTGF) is a major factor in the reduction of the electrical stimulation-induced adhesion plaque kinase (FAK)-AKT (FAK / AKT) pathway. CTGF is a major factor in the reduction of the EleS-induced FAK / AKT pathway. Figure 11 shows the results of gene expression profiling based on real-time PCR taken for the extracellular matrix (ECM) and cell adhesion molecules. Four of each gene, which was hyperregulated and downregulated, were selected based on magnification changes. In Figures 11B and 11C, mRNA expression of connective tissue growth factor (CTGF) was confirmed by conventional RT-PCR and real-time PCR (n=5). Figure 11 shows that CTGF positivity by immunocytochemistry of electrical stimulation-induced cardiac stem cells (EleS CSCs) is higher than that of non-electrically stimulated cardiac stem cells (Non-EleS CSCs).

[0037] [Figure 12]Figure 12A-12 shows that Tert is a direct target of miR-195 in stem cell aging. Figure 12 shows that mTert, predicted by computational analysis, is a potential target of miR-195.

[0038] Figures 12B-12 show OMSC transfection with Tert mRNA expression restored to anti-mir-195, and the telomere-specific protein TRF2, as examined by reverse transcriptase polymerase chain reaction and Western blotting, respectively. As shown in Figure 12D, co-transfection with a PEZX-Luc vector containing mTert 3' UTR and plasmid-encoded miR-195 showed reduced luciferase activity (p < 0.01 vs. PEZX-miR-SC transfected cells). Transfection efficiency was normalized by sea urchin luciferase activity. Abbreviations: OMSCs, aged mesenchymal stem cells; Tert, telomerase reverse transcriptase; UTR, untranslated region.

[0039] [Figure 13]Figures 13A-13 show that deactivation of miR-195 by telomere extension and reactivation of anti-aging markers activates OMSCs. Figure 13 shows the structure of the Lenti-miR-195 inhibitor containing the mCherry reporter gene (red signal) and the Lenti-scramble vector. Figure 13 shows that deactivation of miR-195 significantly reduced aging-related b-gal expression in OSMCs compared to scramble-transfected OMSCs. Figure 13 shows that telomeres in OMSCs were significantly re-strengthened when transfected with the miR-195 inhibitor. As shown in Figure 13, the miR-195 inhibitor significantly reduced terminal deoxynucleotidyltransferase DUTP nickend-labeled-positive apoptosis cell death in OMSCs. As shown in Figure 13, transfection of OMSCs with miR-195 significantly increased TERT (non-expression), SIRT1, Bcl-2, and the phosphorylation of p-FOXO1 and p-Akt, but decreased the expression of p53 and cleaved caspase 3. Figures 13F and 13 show that the cochleading of miR-195 significantly restored the proliferative capacity of OMSCs, as investigated by cell proliferation assays and colony formation assays. Acronyms: OMSC, ancient mesenchymal stem cells; SIRT1, sirtuin (silent interlocking information regulation 2 homolog) 1; Bcl-2, B-cell lymphoma 2 protein; FOX01, rhabdomyosarcoma forkhead, also known as forkhead box protein 01, and p-FOX01 as phosphoforkhead box protein 01, also known as Akt, protein kinase B (PKB), Akt; p-Akt is phospho-Akt.

[0040] [Figure 14]Figures 14A-14 show the generation of cardiac progenitor cells in human induced pluripotent stem cells via isoxazole or isoxazole-like compounds in vitro. Figures 14A and 14 show the feature of human iPS cells by immunostaining for the discovery of pluripotency markers Oct4, Sox2, Tra-1-60, Tra-1-81 and SSEA4 (images bound with DAPI). Figure 14 shows a brief illustrative description of the procedure for generating cardiac progenitor cells from hiPS cells and the subsequent generation of cells of multiple cell lineages. Figure 14 shows the feature of cardiac progenitor cells and markers Nkx2.5 and GATA4 by immunostaining (images bound with DAPI). Figure 14 shows the differentiation of human iPS cells into beating cardiomyocytes. Figure 14 shows the differentiation of human induced pluripotent stem cells into beating cardiomyocytes in vitro, as shown by the discovery of VE-cadherin and CD31 by immunostaining analysis. Figure 14 shows angiogenesis by vascular progenitor cells labeled with calcein-AM dye. Figure 14 illustrates the differentiation of human iPS cells into smooth muscle progenitor cells in vitro using α-SMA and carppenin immunostaining.

[0041] [Figure 15] Figures 15A and 15B show the effect of CXCR4 expression on muscle progenitor cell (MPC) migration and schematic diagrams of chemotaxis experiments. Figure 15A shows a computational method for quantifying MPCs infiltrating a collagen gel with 30 fields per well. Figure 15B shows the evaluation results of MPC migration. MPCNull represents control MPCs expressing GFP / dystrophin; MPCCXCR4 represents MPCs expressing GFP / dystrophin and overexpressing CXCR4; and MPCsi-CXCR4 represents MPCs treated with CXCR4-targeting siRNA to knock down the CXCR4 gene. This data shows increased migratory activity in MPCs overexpressing CXCR4. n=4, *p < 0.05 vs control.

[0042] [Figure 16]Figures 16A-16D show that human iPS cell colonies derived from cardiac fibroblasts dissociate from acetase and adhere in the presence of Y27632. Figure 16A is a schematic diagram showing the cell culture conditions for generating cardiac fibroblasts from human iPS cells. Briefly, to generate muscle progenitor cells (MPCs) from hiPSCs, human induced pluripotent stem (iPS) cells (ATCC® ACS-1021™) induced from human cardiac fibroblasts were cultured using mTeSR™1 (STEMCELL Technologies Inc.) on 6-well plates coated with Vitronectin XF (STEMCELL Technologies Inc.). The iPS cells were passaged every 4-6 days using ReLeSR™ (STEMCELL Technologies Inc.). For differentiation from iPS cells to MPCs, iPS cells were dissociated into single cells using ACCUTASE™ (STEMCELL Technologies Inc.) and seeded at 1 x 10⁹ cells / cm² in mTeSRTM1 supplemented with a 5 UM RHO / ROCK pathway inhibitor (Y-27632, STEMCELL Technologies Inc.). After 24 hours, the medium was replaced with fresh mTeSRTM1. For the first three days, mTeSRTM1 was replaced daily. After that, the medium was replaced with mTeSRTM1 supplemented with 20 UM ISX-9 (MedChem Express), and the medium was changed 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 UM ISX-9, and the medium was changed every other day for the next 3-6 days. Small molecule compounds (Isx9 & GIV) were applied to initiate differentiation, and analysis was performed on day 9. Figure 16B shows the relative expression levels of skeletal muscle genes after treatment with Isx9 and Giv. Figures 16C and 16D show the overexpression dominance of muscle genes (PAX3, PAX7, MYF5, MYOG, MYOD), particularly ISX-9.

[0043] [Figure 17]Figures 17A-17F show the generation and characterization of cardiac progenitor cells (CPCs) derived from hiPSCs. Figure 17A is a schematic diagram of hiPSC-CPC generation. Figure 17B shows bright-field images of hiPSCs treated with DMSO or ISX-9 in RPMI / B27-negative insulin on days 1, 3, and 7. Figure 17C shows the time-dependent equations for Nkx2.5, GATA4, ISL-1, and Mef2c versus day 0, P < 0.05, and versus day 3, P < 0.05. Dependencies from three biological replicate experiments are shown. Figure 17D shows the finding of upregulation of Nkx2.5, GATA4, ISL-1, and Mef2c with ISX-9 on day 7 compared to the DMSO group. *vs. DMSO group, P < 0.05. Results from three biological replicate experiments are shown. Figure 17E shows fluorescence-activated cell sorting (FACS) analysis, which reveals 96.5 ± 2.3% Nkx2.5-positive cells after ISX-9 treatment. As shown by immunofluorescence staining in Figure 17F, transcription factors (Nkx2.5, GATA4, and ISL-1) were highly upregulated in hiPSCs treated with ISX-9. Scale bar = 200 μm.

[0044] [Figure 18] Figures 18A-18G: Differentiation of CPCs into three cardiac cell lineages in vitro. According to the treatment summary, CPCs discovered CM, EC, and SMC-specific proteins (Figure 18A). CM progenitor cells discovered α-myomatous actinin, cTni, MLC2v, cTnT, and Cx43. Scale bar = 50 μm. (Figure 18B) Under TEM, these cells were rich in ribosome-embedded endoplasmic reticulum, growing myofilaments, mitochondria, and glycogen particles. Chromatin material was uniformly distributed in the nucleoplasm. (Figure 18C) Endothelial progenitor cells expressed VE-cadherin and CD31, scale bar = 200 μm. LDL uptake by these cells was observed (Figure 18E), and they also formed tubular structures (Figure 18D). (Figure 18F) Smooth muscle progenitor cells expressed α-SMA and calponin, scale bar = 200 μm. (Figure 18G) FACS analysis revealed 95.2 + 2.1% CMS, 90.3 + 2.5% ECs, and 92.3 + 1.8% SMCs in basal differentiation medium.

[0045] [Figure 19] Figures 19A-19C: Expression profiles of whole mRNA and miRNA in CPCs. (Figure 19A) Heatmap of differentially expressed mRNA (Q < 10⁻¹⁵) detected by mRNA-seq in undifferentiated hiPSCs and hiPSCs treated with DMSO and ISX-9. (Figure 19B) Heatmap of miRNAs that showed significant increases or decreases individually between differentially expressed hiPSCs treated with DMSO and ISX-9. (Figure 19C) Pathway enrichment analysis of genes upregulated and downregulated in ISX-9-induced CPCs compared to hiPSCs treated with DMSO.

[0046] [Figure 20] Figures 20A-20G: Potentially important signaling pathways mediated by ISX-9 treatment. (Figure 20A) Time-dependent expressions of Wnt5, Wnt11, and Wnt3a in the group receiving ISX-9 treatment (vs. day 0, P < 0.05; vs. day 3, P < 0.05, n=6) from three biological replicate experiments. (Figure 20B) Increased expression of Wnt5, Wnt11, and Wnt3a in the group receiving ISX-9 treatment for 7 days compared to the DMSO group (vs. DMSO group, P < 0.05) from three biological replicate experiments. Discovery of increased expression of Wnt5 and Wnt11 (Figure 20C) and cardiac transcription factors (Nkx2.5, Mef2c, GATA4, and ISL1) in the group receiving ISX-9 treatment for 7 days. (Figure 20D) These findings are compared to the group receiving treatment only on day 3 from three biological replicate experiments. *Compared to the 3-day treatment group, P < 0.05. From three biological replicate experiments, (Figure 20E) outline of the study protocol for the signaling pathway of cardiac differentiation induced by ISX-9. Inhibition of the TGFB signaling pathway with LY2109761 in the early stages of differentiation, or inhibition of the standard Wnt signaling pathway with XAV939 and IWP2 (Figure 20F), or blockade of non-standard Wnt signaling with siWnt5, siWnt11 and WIF-1 in the late stages of differentiation (Figure 20G), together with ISX-9 treatment, significantly reduces the discovery of cardiac transcription factors. *Compared to the ISX-9 group, P < 0.05.

[0047] [Figure 21] Figures 21A-21G: Cytoprotective effect of ISX-9 on CPCs. (Figure 21A) Morphological changes of hiPSCs in RPMI / B 27 medium treated with DMSO or ISX-9 under 1% O2 for 12 and 24 hours. (Figure 21B) Representative TUNEL stained images of mocks treated with DMSO and ISX-9 after 24 hours of hypoxic stress. (Figure 21C) Semi-quantitative estimate of TUNEL-positive cells vs. DMSO group, P < 0.05. (Figure 21D) Heatmap comparing the concentrations of paracrine factors released from cells by different treatments. Rows show cytokines and columns show independent conditions. The color scale of the heatmap shows the minimum and maximum values ​​of all values ​​on the right, according to the absolute concentration of cytokines (log 10, pg / ml). (Figure 21E) TUNEL stained image 3 days post-Mi. Host cardiomyocytes were identified by α-sarcoma actinin. (Figure 21F) Quantification of TUNEL-positive cells at the border of infarct areas with different treatments. *vs. DPBS group, P < 0.05; #vs. hiPSC group, P < 0.05. (Figure 21G) Quantification of TUNEL-positive cardiomyocytes at the border of cardiac infarct areas in different groups of mice. *vs. DPBS group, P < 0.05; #vs. hiPSC group, P < 0.05.

[0048] [Figure 22] Figures 22A-22H: CPC transplantation reversed cardiac remodeling after MI. Time course of LVESD (Figure 22A), LVEDD (Figure 22B), FS (Figure 22C), and EF (Figure 22D) in mice after transplantation. * P < 0.05 vs. DPBS treatment group at the same point, # P < 0.05 vs. hiPSC treatment group at the same point. DPBS=13, hiPSC=16, and CPC=16. (Figure 22E) Representative echographs of M-mode and B-mode echocardiography at 3 months post-MI. (Figure 22F) Assessment of cardiac fibrosis at 7 levels by Masson's trichrome staining at 3 months post-MI. (Figure 22G) Representative cardiac sections. (Figure 22H) Quantification of scar tissue size. * P < 0.05 vs. DPBS treatment group at the same time point, # P < 0.05 vs. hiPSC treatment group at the same time point (n=5).

[0049] [Figure 23] Figures 23A-23J: Differentiation of CPCs transplanted into an infarcted heart into cardiac lineage cells. (Figures 23A-23B) Transplanted CPCs are identified by PKH-26 fluorescence (red fluorescence); muscle fibers were visualized at 3M post-MI via immunostaining with human-specific cTnT and α-sarcomeric actinin (green fluorescence). (Figures 23C-23D) Immunofluorescence images of PKH-26, EC and SMC makers in an infarcted heart at 2M post-MI. Scale bar = 50 μm. (Figures 23E-23G) Transplanted CPCs (Figure 23E) and hiPSCs (Figure 23F) in the infarct and border area at 72 hours post-MI are quantified by human mitochondrial antigen (human-mit) tracking, with representative immunofluorescence images. * P < 0.05 vs hiPSC treatment group, n=3. (Figures 23H-23J) Representative immunofluorescence images and quantification of differentiated CM in the infarct and border area of ​​mice treated with cTnl and human-mitt et al. King-based CPCs (FIG. 23H) and hiPSCs (FIG. 23I) three months after MI. * P < 0.05 vs. hiPSC-treated group, n=3.

[0050] [Figure 24] Figures 24A-24D show the characterization of exosomes from iPSCs and CPCISX-9. Figure 24A is the characterization of exosomes isolated from iPSCs and CPCISX-9, visualized by transmission electron microscopy (TEM). Cable bar = 200 μm. Figure 24B is a representative Western blot showing that exosomes from iPSCs and CPCISX-9 were enriched with the exosome-specific marker Tsg101. Other common exosome markers are: CD9, Hsp70, and Flotillin-1. Calnexin is absent in the exosomes. Figure 24C is a representative size distribution atlas of exosomes from iPSCs and CPCISX-9 detected by TRPS. Figure 24D shows the average size of exosomes measured by TRPS. No significant size difference was observed between exosomes derived from iPSCs and exosomes derived from CPCISX-9.

[0051] [Figure 25] Figures 25A-25B show profiling of miRNA expression and validation of microarray data. Figure 25A is a heatmap analysis of microarray data, showing significant upregulation of miRNAs in Exo-CPCISX-9 by miRNAs in Exo-iPSC, Exo-EB, and Exo-CPCcomm. Red and green distinguish up- or down-regulated miRNAs, respectively (P < 0.05). n=3. Figure 25B shows validation of microarray data using real-time PCR. Quantitative data shows significant overexpression of miR-373, miR-367, miR-520, miR-548ah, and miR-548q in Exo-CPCISX-9. RNA samples are from three biological replication experiments. *, P < 0.001.

[0052] [Figure 26]Figures 26A-26E show that exosomes derived from CPC ISX-9, which are rich in miR-373, reversed TGFB stimulation in fibroblasts. In Figure 26A, exosomes from CPC ISX-9 labeled with PKH26 (red) were incubated with fibroblasts and observed within the fibroblasts (green, calcein AM), mainly located in the perinuclear region. Figure 26B shows real-time PCR results, demonstrating that 25 nM and 50 nM concentrations of anti-miR-373 (miR-373 inhibitor) effectively downregulated miR-373 expression in CPC ISX-9. Figure 26C shows that miR-373 expression in CPC ISX-9 exosomes was significantly inhibited after treatment with 25 nM anti-miR-373. Figure 26D shows the expression of miR-373 in fibroblasts treated with Exo-CPC ISX-9 or anti-miR-373-Exo CPC ISX-9. Results from three independent experiments are shown in the table, n=3, P < 0.001. Figure 26E shows the expression of fibrosis genes in fibroblasts stimulated with TGFB after treatment with Exo-CPC ISX-9 or anti-miR-373-Exo-CPC ISX-9. *vs. Exo-CPC ISX-9 group, n=3, P < 0.05.

[0053] [Figure 27]Figures 27A-27D show that an exosome derived from CPC ISX-9 promoted the proliferation of cardiomyocytes in mice after myocardial infarction (MI). Figure 27A is a representative image of ki67-positive cardiomyocytes (cTnT-positive) 30 days after MI in mice treated with Exo-CPC ISX-9. Bar = 50 μm. Figure 27B is semi-quantitative data of cardiomyocyte proliferation in the periinfarct area 30 days after myocardial infarction, indicated by ki67 positivity. PBS group: n=940 cardiomyocytes from three hearts; Exo-iPSC group: n=950 cardiomyocytes from three hearts; Exo-CPC ISX-9 group: n=951 cardiomyocytes from three hearts. *vs. PBS group, P < 0.05; #vs. Exo-iPSC group, P < 0.05. Figure 27C is a representative image of arterioles in the periinfarct area of ​​mice 4 weeks after MI. In the vascular structure, arterioles were identified by Q-SMA positive staining (green). Bar = 100 μm. Figure 27D is a quantitative estimate of arterioles in MI mice treated with exosomes derived from each iPSC. *vs. PBS group, P < 0.05; #vs. Exo-iPSC group, P < 0.05, n=3.

[0054] [Figure 28]Figures 28A-28G show that an exosome derived from CPC ISX-9 reverses cardiac remodeling in mice that have developed myocardial infarction. Figure 28A shows representative M-mode echocardiograms from three groups 30 days after MI. LVDs (28B) and LVDd (28C) were significantly reduced at 30 days post-MI in mice treated with Exo-CPC ISX-9. Exo-CPC ISX-9 treatment significantly increased EF (28D) and FS (28E). *vs. PBS group, P<0.05; #vs. Exo-iPSC group, P < 0.05, PBS group: n=10, Exo-iPSC group: n=9, Exo CPC ISX-9 group: n=11. EF, ejection fraction; FS, internal diameter shortening; LVDd, diastolic left ventricular dimension; LVDs, systolic left ventricular dimension. (28F) Representative Masson trichrome stained sections of the heart after different treatments. (28G) Quantitative estimation of fibrosis in mice after different treatments. * vs. PBS group, P < 0.05; # vs. Exo-iPSC group, P < 0.05, n=4.

[0055] [Figure 29] Figure 29 is a diagram illustrating a personalized cell-free therapy using exosomes derived from iPSC-derived cardiac progenitor cells.

[0056] [Figure 30] Figures 30A and 30B show the effects of three small molecule compounds (ISX9, Danzol, and Givinostat) on the expression of cardiac and skeletal muscle genes. Real-time PCR for dystrophin showing the effect of the small molecule compounds (ISX9 and GIV) in iPS cells is also shown. [Modes for carrying out the invention]

[0057] [Detailed description of the invention] definition The implementation of this invention, unless otherwise indicated, utilizes conventional tissue culture, immunology, molecular biology, microbiology, cell biology, and DNA recombination techniques within the scope of the art. For example: Sambrook, Fritsch and Maniatis (1989) Molecular Cloning: Laboratory Manual, 2nd Edition; FMAusubel et al. eds. (1987) Current Molecular Biology Protocols; Enzymology Manuscript Methods Series (Academic Press Co., Ltd.); PCR2: A Practical Approach (1995) (MJ MacPherson, B.D. Hames and G.T. Taylor eds.); Harlow and Lane, eds. (1998) Antibodies, Laboratory Manual; Harlow and Lane, eds. (1999) Use of Antibodies, Laboratory Manual; and R.Freshney, ed. (1987) Animal Cell Culture.

[0058] All numerical specifications, such as pH, temperature, time, concentration, and molecular weights including ranges, are approximations that vary appropriately in increments of 1 with (+) or (-) 1.0 or 0.1. It should be understood that, unless explicitly stated, all numerical representations are preceded by the term [approximately]. Also, unless explicitly stated, the reagents described herein are merely illustrative, and their equivalents are known in the art.

[0059] As used in the specification and claims, unless the context explicitly indicates otherwise, the singular form is one, and one and the word itself are included in plural references.

[0060] Terms such as autotransfer and autologous transplantation refer to treatments where the cell donor is also the recipient of cell replacement therapy. Terms such as allogeneic transplantation, allogeneic transplantation, and allogeneic graft refer to treatments where the cell donor is of the same species as the recipient of cell replacement therapy, but not the same individual. Cell transplantation in which the tissue compatibility between the donor cells and the recipient is matched is called syngeneic transplantation. Terms such as xenotransplantation, xenograft, and xenograft refer to treatments where the cell donor is of a different species than the recipient of cell replacement therapy.

[0061] Biocompatible scaffolds are tissue-engineering scaffolds or matrices that can function as substrates supporting appropriate cellular activity for generating the desired tissue. Their role is to facilitate molecular and mechanical signaling pathways without eliciting undesirable effects in those cells or inducing undesirable local or systemic responses in the final host. Furthermore, biocompatible scaffolds are precursors to implantable devices, which have the ability to perform their intended function with the desired degree of uptake in the host without eliciting undesirable local or systemic effects in the host. Biocompatible scaffolds are described in U.S. Patent 6638369.

[0062] As used here, cardiac patches and fibrin-embedded cardiac precursor patches, or epicardial patches, are bioengineered, 2D or 3D tissue patches containing iPS cells or iPS cell-derived organ lineages, or cardiac progenitor cells.

[0063] Cardiomyocytes are specialized muscle cells that primarily form the cardiac muscle of the heart. Cardiomyocytes consist of five main parts: 1. the cell membrane (myocyte membrane) and T-tubules for impulse conduction, 2. the sarcoplasmic reticulum, which is a calcium reservoir necessary for contraction, 3. the contractile element, 4. the mitochondria, and 5. the nucleus. Cardiomyocytes are subdivided into subtypes, which are not limited to these: atrial cardiomyocytes, ventricular cardiomyocytes, sarcoplasmic nodular cardiomyocytes, peripheral sarcoplasmic nodular cardiomyocytes, or central sarcoplasmic nodular cardiomyocytes. Stem cells proliferate to mimic the physiological functions of cardiomyocytes and / or differentiate into cardiomyocytes. This differentiation is detected by markers, which are not limited to these: myosin heavy chain, myosin light chain, actinin, troponin, tropomyosin, GATA4, Mef2c, and Nkx-2.5.100641. Cardiomyocyte markers [myosin heavy chain] and [myosin light chain] are part of a large family of motor proteins found in muscle cells that generate contractile force. These proteins have been sequenced and characterized. Examples: GenBank Accession Nos. AAD29948, CAC70714, CAC70712, CAA29119, P12883, NP 000248, P13533, CAA37068, ABR18779, AAA59895, AAA59891, AAA59855, AAB91993, AAH31006, NP _ 000423, and ABC84220. The genes for these proteins have also been sequenced and characterized. Examples: GenBank Accession Nos. NM _ 002472 and NM _ 000432.

[0064] The cardiomyocyte markers "myosin heavy chain" and "myosin light chain" are part of a large family of motor proteins found in muscle cells that generate contractile force. These proteins have been sequenced and characterized. For example, GenBank accessions Nos. AAD29948, CAC70714, CAC70712, CAA29119, P12883, NP _000248, P13533, CAA37068, ABR18779, AAA59895, AAA59891, AAA59855, AAB91993, AAH31006, NP _ _000423, and ABC84220. The genes for these proteins have also been sequenced and characterized. For example, GenBank accessions Nos. NM _ _002472 and NM _ _000432.

[0065] Cardiomyocyte marker actinin is a microfilament protein, the thinnest filament of the cytoskeleton found in the cytoplasm of all eukaryotic cells. The actin polymer plays a role in the actomyosin-driven contraction process and also acts as a platform for the ATP hydrolysis-dependent action of myosin in muscle contraction. This protein has been sequenced and characterized as follows: GenBank Accession Nos. NP_001093, NP_001095, NP_001094, NP_004915, P35609, NP_598917, NP112267, AAI07534, and NP_001029807. The gene for this protein has also been sequenced and characterized, for example: GenBank Accession Nos. NM_001102, NM_004924, and NM_001103.

[0066] Cardiac cell markers, troponins, are chemical compounds of three proteins essential for muscle contraction in skeletal and cardiac muscle. Troponins bind to the protein [tropomyosin] and are located in the grooves between actin filaments in muscle tissue. Tropomyosin can be used as a cardiomyocyte marker. These proteins have been sequenced and characterized. Examples: GenBank Accession Nos. NP 000354, NP 003272, P19429, NP 001001430, AAB59509, AAA36771, and NP 001018007. The genes for these proteins have also been sequenced and characterized. Examples: GenBank Accession Nos. NM 000363, NM 152263, and NM 001018007.

[0067] Clone proliferation is the growth of a cell population by causing a single cell to continuously divide into two identical daughter cells and a group of identical cells.

[0068] CTGF, also known as CCN2 or connective tissue growth factor, is a matrix cell protein belonging to the extracellular matrix-related, heparin-binding protein CCN family. (See also CCN intracellular signaling proteins.)

[0069] Telomerase reverse transcriptase (TERT) is the catalytic subunit of the enzyme telomerase and, together with the telomerase RNA component (TERC), constitutes the most important unit of the telomerase complex.

[0070] miR-290-295 cell clusters are members of a novel MicroRNA (miRNAs) family expressed during early embryogenesis and are specific to mouse embryonic stem cells (ESCs) and embryonic cancer cells (ECCs). Such clusters are known in the art and are described, for example, in Lichner et al. (2011) Differentiation, January 81 (1):11-24.

[0071] Chemokine (CC Motif) Ligand 7 (CCL7) is a small cytokine previously known as monocyte-specific chemokine 3. Its protein sequence is available under registration number NP_006264, while the mouse sequence is available under NP_038682 (also accessible at ncbi.nlm.nih.gov / gene / 6354, last accessed April 16, 2014).

[0072] The CXCR2 chemokine receptor 2 (CXCR2) is a protein encoded by this gene and is a member of the G protein-coupled receptor family. This protein is a receptor for interleukin-8 (ILS), binding to IL8 with high affinity and transmitting signals via the G protein-activated second messenger system. This receptor also binds to chemokine (CC motif) ligand 7 (CCL7). Information about this protein and gene can be found at nchbi.nlm.nih.gov / gene / 3579 (last accessed April 16, 2014).

[0073] Intrinsic membrane protein 2A is a stem cell marker. While the human gene sequence is reported in UniProtKB, the mouse sequence is reported in Q61500 (uniprot.org / uiprot, last accessed April 16, 2014).

[0074] DNA(cytosine-5)-methyltransferase 1 is an enzyme encoded by the DNMT1 gene. The complete sequence of this protein and its gene is available on the gene card: org / cgi-bin / carddisp.pl ? gene=DNMT1, last accessed April 16, 2014. Antibodies for detecting the protein are commercially available, e.g., from cell signaling technology (DNMT1 (D63A6) XP(registered trademark) Rabbit mAb # 5032). DNA(cytosine-5)-methyltransferase 3 is an enzyme encoded by the DNMT3 gene.

[0075] EFNA3, or ephrin A3, is a protein receptor. Human protein sequencing has been reported at ncbi.nlm.nih.gov / genes / 1944. Antibodies useful for protein detection and analysis are available from R&D Systems and Santa Cruz Biotechnology.

[0076] Let-7 is a family of microRNAs. Its sequence was reported on miRBase at mirbase.org / cgi bin / mirna_outline.pl ? fam=MIPF000002, last accessed on April 16, 2014. Such detection methods are publicly known in the art. For example, U.S. Patent Application Publication No. 2014 / 0005251.

[0077] MACS is a pluripotency marker that binds to MYC. Chappell et al. (2013) Genes & Dev. 27: 725-733.

[0078] The protein encoded by the Tsg101 gene is a member of a group of clearly inactive congeners of ubiquitin-coupled enzymes. Its gene product contains a coiled-coil domain that interacts with stasmin and a cytosolic lin protein involved in tumorigenesis. This protein may play a role in cell growth and differentiation, but may also act as a negative growth regulator. In vitro steady-state expression of this tumor susceptibility gene is important for maintaining genomic stability and cell cycle regulation. Mutations and alternative splicing of this gene occur frequently in breast cancer, and we propose that defects occur during tumorigenesis and progression in breast cancer.

[0079] CD9 encodes members of the transmembrane 4 superfamily, also known as the tetraspanin family. Tetraspanins are cell surface glycoproteins with four transmembrane domains, which form multimeric complexes with other cell surface proteins. The encoded protein functions in many cellular processes, including differentiation, adhesion, and signaling. Furthermore, the discovery of this gene plays a crucial role in suppressing cancer cell motility and metastasis.

[0080] As used here, microRNAs and miRNAs are post-transcriptional regulators. These post-transcriptional regulators typically bind to complementary sequences of three major untranslated regions (3' UTRs) in a target messenger RNA transcript (mRNA), usually causing gene silencing. Generally speaking, miRNAs are short non-coded ribonucleic acid (RNA) molecules, e.g., 21 or 22 nucleotides in length. MicroRNAs and miRNAs are used interchangeably.

[0081] mir-373 is annotated as ENSG00000199143 and mirBase:has-mir-373.

[0082] mir-210 is annotated as ENSG00000199038 and mirBase:has-mir-210, and is associated with susceptibility to sudden infant death syndrome.

[0083] Tcf15 codes for a fundamental helix-loop-helix transcription factor discovered early in development and may participate in the patterning of mesoderm and its derived cell types. This gene is annotated as Ensemble: ENSG00000125878 and Uniprot Q12870.

[0084] mir-377 is annotated as ENSG00000199015 and mirBase:has-mir-377.

[0085] mir-367 is annotated as ENSG00000199169 and mirBase:has-mir-367.

[0086] mir-520C is annotated as ENSG00000207738 and mirBase:has-mir-520c.

[0087] mir-548ah is annotated as ENSG00000283682 and mirBase:has-mir-548ah.

[0088] Dystrophin refers to the protein coded by the gene Dmd, annotated Ensembl:ENSG00000198947 and Uniprot: P11523. This protein is a component of the dystrophin-glycoprotein complex, which binds the cytoskeleton to the extracellular matrix. Mutations are associated with Duchenne muscular dystrophy, Becker muscular dystrophy cardiomyopathy, and their equivalents.

[0089] mir-548q is annotated as ENSG00000221331 and mirBase:has-mir-548q.

[0090] mir-548q is annotated as ENSG00000221331 and mirBase:has-mir-548q.

[0091] mir-335 codes for MicroRNA-335 and is annotated as ENSG00000199043 and miRBase: has-mir-335.

[0092] mir-21 encodes MicroRNA-21 and is annotated as ENSG00000284190 and miRBase: has-mir-21. This miRNA is expressed in stem cells and is involved in cancer.

[0093] mir-30c1 encodes a microRNA annotated as Ensemble: ENSG00000207962 and miRBase: has-mir-30c-1, which may be involved in ECM maintenance and cancer.

[0094] mir-30c2 encodes a microRNA annotated with Ensemble: ENSG00000199094 and miRBase: has-mir-30c-2. Similar to miR-30c1, mir-30c2 may be involved in ECM maintenance and cancer.

[0095] Meox1 encodes mesenchymal homeobox protein 1, annotated as Ensemble: ENSG00000005102 and Uniprot: P50221. This protein plays a role in somite development, and genetic mutations in it are associated with Klippel-Feyll syndrome.

[0096] Meox2 encodes mesenchymal homeobox protein 2 and is annotated as Ensemble:ENSG00000106511 and Uniprot:P50222. Based on homology with mouse proteins, this protein is thought to be involved in muscle formation and limb development. Mutations are associated with craniofacial and skeletal abnormalities, as well as Alzheimer's disease.

[0097] Pax3 is annotated as Ensembl:ENSG00000135903 and Uniprot:P23760. This gene encodes a member of the paired-box gene (PAX gene) family of transcription factors that regulate proliferation and migration during neurodevelopment and myogenesis. Mutations in this gene are associated with craniofacial-hearing-hand syndrome, rhabdomyosarcoma, and Waardenburg syndrome.

[0098] Pax7 is annotated as Ensemble:ENSG00000009709 and Uniprot:P23759. This gene encodes a member of the paired-box gene (PAX gene) family of transcription factors that regulate the proliferation of muscle progenitor cells. It is essential for embryonic development and is involved in cancers, including rhabdomyosarcoma.

[0099] MyoD1 is annotated as Ensemble:ENSG00000129152 and Uniprot:P15172. This gene encodes a myogenic helix-loop-helix transcription factor that regulates myocyte differentiation through cell cycle repression. This protein is known to interact with other major muscle factors such as Myf5, Myf6, and MyoG.

[0100] Myog codes for myogenin, a muscle-specific basic helix-loop-helix type transcription factor.

[0101] Myh2 codes for class II or normal myosin heavy chains and functions as a motor protein in skeletal muscle contraction. Mutations in this protein are associated with inclusion body myositis. Myh2 is annotated as Ensemble:ENSG00000125414 and Uniprot:Q9UKX2. Numerous splice variants have been reported.

[0102] Myh▲a▼ codes for the motor protein that forms the α-heavy chain subunit of cardiac myosin. This gene is annotated as Ensemble: ENSG00000197616 and Uniprot: P13533. Mutations in this gene are associated with atrial septal defect and hypertrophic cardiomyopathy.

[0103] Tbxl codes for a member of the growth-critical T-box transcription factor family and is annotated as Ensemble: ENSG00000184058 and Uniprot: Q43435. Mutations in this gene are associated with neural crest defects, DiGeorge syndrome, and epidemic facial syndrome.

[0104] Mespl codes for a fundamental helix-loop-helix transcription factor involved in the development of somatic and cardiac mesoderm, as well as the formation of the trunk pattern of somites. Mesp1 is annotated as Ensemble: ENSG00000166823 and Uniprot: QOBRJ9.

[0105] Des codes for the protein desmin, annotated as Ensemble: ENSG00000175084 and Uniprot: P17661. Desmin is a muscle-specific class III intermediate filament that forms the fibrous network of myofibrils. Mutations in Des are associated with myopathy of cardiac and skeletal muscle.

[0106] Cnntbl is a well-known gene that codes for β-catenin, a protein that is a key component of the standard Wnt signaling pathway. In the presence of Wnt, β-catenin trans is located in the nucleus and acts as a transcriptional regulator. This protein is also involved in regulating contact inhibition. Mutations in this gene are associated with intellectual disability and colorectal cancer. The Cnntbl gene is annotated as Ensemble: ENSG00000168036 and Uniprot: P35222.

[0107] Pax7 is annotated as Ensemble:ENSG00000009709 and Uniprot:P23759. This gene codes for a member of the paired-box gene (PAX gene) family of transcription factors that regulate the proliferation of muscle progenitor cells. It is essential for embryonic development and is associated with cancers, including rhabdomyosarcoma.

[0108] The Myf5 gene, Ensemble:ENSG00000111049, codes for a master transcriptional regulator of muscle differentiation that binds to and promotes the transcription of numerous myogenic factors (Uniprot:P13349). Mutations in Myf5 are associated with skeletal muscle cancer and rhabdomyosarcoma.

[0109] MyoD1 is annotated as Ensemble:ENSG00000129152 and Uniprot:P15172. This gene codes for a myogenic helix-loop-helix transcription factor that regulates myocyte differentiation through cell cycle repression. This protein is known to interact with other major muscle factors such as Myf5, Myf6, and MyoG.

[0110] XESI myogenic genes refer to myogenic regulators (MRFs), which are basic helix-loop-helix (bHLH) transcription factors MyoD, Myf5, myogenin, and MRF4 that regulate myogenesis. These proteins contain conserved basic DNA-binding domains that bind to E-box sequence DNA motifs. [2] They dimerize with other HLH-containing proteins via HLH-HLH interactions.

[0111] Pitx2 is annotated as ENSG00000164093 and Uniprot: Q99697. This gene codes for pairlike homeodomain transcription factor 2, which belongs to the bicoid family of homeodomain proteins. This protein regulates the hormone prolactin and is important for the development of the eyes, teeth, and abdominal organs. Mutations in this gene are associated with Axenfeld-Rieger syndrome.

[0112] ISL1 is a gene (Ensemble: ENSG00000016082) that codes for the transcription factor ISL LIM homeobox 1 (Uniprot: P61371). This protein regulates insulin gene expression in relation to the use of motor neurons and retinal ganglion cells. Mutations in ISL1 are associated with mature-onset diabetes and bladder atrophy.

[0113] Nkx2.5 codes for a master transcription factor involved in cardiac development, annotated as Ensemble: ENSG00000183072 and Uniprot P52952. Mutations in this gene result in atrial septal defects and congenital hypothyroidism.

[0114] Handl is a basic helix-loop-helix transcription factor annotated as Ensemble: ENSG00000113196 and Uniprot: 096004. During cardiac development, Handl is expressed asymmetrically with other Hand proteins to direct cardiac morphogenesis and the formation of the right ventricle and aortic arch. Mutations in the gene encoding the Hand protein are associated with congenital heart defects.

[0115] GATA4, annotated as Ensemble: ENSG000001366574, codes for a member of the gata family of zinc-finger transcription factors. This protein, Uniprot: P43694, is important for embryogenesis, cardiac development, and myocardial function. Mutations in it are associated with septal defects and various types of cancer.

[0116] Tbx5 is a member of the T-box gene family and contains a conserved DNA-binding domain. Numerous transcripts of Tbx5 have been curated by RefSeq, and its Ensemble gene identifier is ENSG00000089225. Its protein product, Uniprot: Q99593, is important for the development of the heart and limbs. Mutations in this gene are associated with Holt-Oram syndrome.

[0117] TnnT2 is a gene that codes for the binding unit of the cardiac troponin T2 (Uniprot: P45379)-tropomyosin-troponin complex. In response to changes in intracellular calcium concentration, TnnT2 regulates muscle contraction. This gene is annotated as Ensemble: ENSG00000118194 and is associated with familial hypertrophic cardiomyopathy and dilated cardiomyopathy.

[0118] My17 is the gene that codes for the calcium-binding motor protein myosin light chain 7. This gene is annotated as Ensemble: ENSG00000106631 and UniProt: 201449. Spontaneous mutations at this locus are associated with Fechtner syndrome and familial atrial fibrillation.

[0119] The MLC2v gene (more commonly represented as My12 in humans), curated as Refseq: NM_00432 and Uniprot: P10916, codes for motor protein myosin light chain 2. Calcium-dependent phosphorylation of this protein leads to the generation of contractility. This protein functions in cardiac development and cardiac contractility, and mutations in it are associated with left and right ventricular hypertrophic cardiomyopathy. Antibodies are available through Invitrogen and Santa Cruz Biotechnology.

[0120] Mef2c (Ensemble ID ENSG00000081189) is a gene that generates more than eight alternative splice transcripts, curated by RefSeq. Its protein product (Uniprot: Q06413) is a member of the MADS-box transcription enhancer-factor 2 family and plays a role in angiogenesis, cardiac morphogenesis, myogenesis, and the maintenance of different states. Genomic abnormalities within this locus are associated with intellectual disability, brain malformations, epilepsy, and proarrhythmic right ventricular dysplasia5. Cell Signaling Technologies, Novus Biologicals, and Invitrogen all offer products for the detection and study of this protein.

[0121] The Cdh4 gene generates three transcriptional mutants that code for the protein cadherin 4. As a member of the cadherin-per-family, Cdh4 functions as a calcium-dependent cell adhesion molecule crucial for brain segmentation and neuronal growth. This protein is also involved in kidney and muscle development. Cdh4 is annotated as Refseq: NM_001252399 and Uniprot: P55283. Purified proteins, antibodies, and other detection kits are widely available from suppliers including Invitrogen, Abcam, and R&D Systems.

[0122] Lhx2 codes for the Lim homeobox 2 protein, a member of the LIM domain family which has a cysteine-rich zinc-binding domain. Lhx2 has been curated by Refseq: NM 004789 and UniProt: P50458 and is thought to function as a transcriptional activator involved in cell differentiation and the development of the lymphoid and nervous systems. Antibodies and ELISA detection kits for Lhx2 are commercially available from Origene, Santa Cruz Biotechnology, and Invitrogen.

[0123] Gai is a heterotrimeric G protein subunit that inhibits the production of cAMP from ATP. Representative sequences are provided under GenBank, Ref.: NM_002069 and UnProt P63096. Antibodies that recognize this marker are commercially available from Santa Cruz Biotechnology.

[0124] Cytoskeletal remodeling refers to the dynamic rearrangement of microfilaments (actin), microtubules (tubulin), and intermediate filaments (i.e., vimentin, keratin, desmin), including the eukaryotic cytoskeleton. Although complex, this process occurs within minutes and facilitates biological functions such as cell migration, cytokinesis, and muscle contraction.

[0125] The enhancement of TGF-β-induced EMT (epithelial-mesenchymal transition) signaling means the differentiation of cells with epithelial-like characteristics into cells with mesenchymal-like characteristics, mediated by the signaling molecule TGF-β. The non-limited biological roles of this process, known as EMT, include cancer, fibrosis, cardiac development, and cardiac differentiation. Transforming growth factor-β (TGF-β) is a potent inducer of EMT both during development and in cancer. In TGF-β-induced EMT, activation of Smad proteins results in their nuclear translocation, DNA binding, and upregulation of EMT transcription factors. Non-limited examples of EMT transcription factors include Snail, Twist, and Zeb. EMT requires cytoskeletal remodeling, and cardiac differentiation means the efficient differentiation of human pluripotent stem cells (PSCs), such as iPS cells, into contractile cardiomyocytes.

[0126] Promoting gene expression for the generation of PIP3 signaling in cardiomyocytes, muscle contraction, and NF in the hypertrophic signaling pathway is intended to activate downstream signaling components of protein kinase AKT, which activates downstream anabolic signaling pathways necessary for cell proliferation and survival. Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), abbreviated as PIP3, is the product of phosphorylation of phosphatidylinositol (4,5)-bisphosphate (PIP2) of class I phosphoinositide 3-quinase (PI 3-quinase). It is a phospholipid present in the plasma membrane and transmits PIP3 signaling in cardiomyocytes.

[0127] Phosphoinositide 3-kinase (PI3K) is activated in cardiomyocytes by receptors with intrinsic tyrosine kinase activity, such as the insulin receptor (INSR), growth factor receptors (IGF1 receptor and HGF receptor), and G protein-coupled receptors (GPCRs). The involvement of INSR and IGF1 receptors induces receptor activation and autophosphorylation. The activated receptor then phosphorylates several intracellular protein substrates, most notably insulin receptor substrate (IRS1-4) proteins. Tyrosine-phosphorylated IRS1 recruits and activates the downstream effector PI3K, which uses inositol-containing phospholipids present in the plasma membrane as a substrate to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3). IRS proteins also recruit the adapters Shc and Grb-2. The protein tyrosine phosphatase PTP1B is associated with negatively regulating INSR signaling by dephosphorylating the phosphotyrosine residue of this receptor. The hepatocyte growth factor receptor (HGF receptor) induces tyrosine phosphorylation of GAB1 and its association with PI3K via the recruitment of a regulatory subunit (PI3KR class 1A) that stimulates a catalytic subunit (PI3KC class 1A). Activated adapters Shc and GRB-2 recruit the recruit exchange factor SOS-RAS, which activates H-RAS [4]. H-RAS directly stimulates the PI3K catalytic subunit (PI3KC class 1A). PI3K converts phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to PIP3 [6]. PIP3 is a second messenger that activates a diverse signaling cascade, including the PDK and AKT pathways. The phosphatase PTEN acts as a negative regulator of the PI3K / AKT signaling pathway, converting PI(3,4,5)P3 to PI(4,5)P2. AKT and PDK phosphorylate a variety of proteins that mediate various insulin and growth factor-induced cellular responses, such as glycogen synthesis, protein synthesis, cell cycle initiation, and cell survival through the regulation of apoptosis factors such as BAD and Bcl-x (L).

[0128] As used herein, MicroRNA or miRNAs are typically post-transcriptional regulators that tail complementary sequences in the three major untranslated regions (3'UTR) of a target messenger RNA transcript (mRNA), commonly causing gene silencing. miRNAs are typically short, non-coded ribonucleic acid (RNA) molecules, e.g., 21 or 22 nucleotides long. The terms MicroRNA and miRNA are used interchangeably.

[0129] miR-133 refers to a microRNA associated with immature or undifferentiated phenotypes. Methods for detecting it include, for example, microarray-RT-PCR and RNA-seq. Commercial kits for miR-133 are available from EMD Millipore (SmartFlare™ Detection Probes), enabling the detection of miRNA in living cells.

[0130] miR-762 is a non-coder RNA involved in post-transcriptional regulation of gene expression in multicellular organisms. The human sequence of miR-762 is reported under registry number MI0003892 (last accessed April 16, 2014). The mouse sequence is reported under NR_030428.1 (referenced at ncbi.nlm.nih.gov / gene / 79103, last accessed April 16, 2014). Such detection methods are publicly known in the art, and kits for them are commercially available, for example, from Origene (referenced at miR-762, origene.com, last accessed April 16, 2014).

[0131] miR-133 is a microRNA associated with immature or undifferentiated phenotypes. Detection methods include, for example, microarray-RT-PCR and RNA-seq. Commercially available kits for miR-133 detection are available from EMD Millipore (SmartFlare™ Detection Probes), enabling the detection of miRNA in living cells.

[0132] miR-762 is a non-coding RNA associated with post-transcriptional gene expression regulation in multicellular organisms. The human sequence of miR-762 is reported under registry number MI0003892 (last accessed April 16, 2014). The mouse sequence is reported under NR _ 030428.1 (referenced at ncbi.nlm.nih.gov / gene / 79103, last accessed April 16, 2014). Such detection methods are publicly known in the art, and kits for them are commercially available, for example, from Origene (referenced at miR-762, origene.com, last accessed April 16, 2014).

[0133] miR-195 is an RNA gene and is reported to belong to the miRNA class. Diseases associated with miR-195 include squamous cell carcinoma of the tongue and primary peritoneal cancer. Its associated pathways include microRNA in cancer and microRNA in cardiomyocyte hypertrophy. miR-195 is also known as MIRN195, Has-MIR-195, and MiRNA 195. Its sequence and homologs are reported on the gene card webpage. The nucleic acid sequence was reported under GenBank registry number AK098506 and was last accessed on November 18, 2015.

[0134] ISL1 refers to an insulin gene enhancer protein that plays a crucial role in regulating insulin gene expression. ISL1 is also known to be central to the development of pancreatic cell lineages and is required for the generation of motor neurons. ISL1 has been identified as a marker in cardiac progenitor cells.

[0135] Tbx-5 is a cardiac transcription factor, also known as the T-box transcription factor (TBX5). In humans, Tbx-5 is a protein coded by the TBX5 gene. As shown in the GeneCards human gene database, this gene is a member of a gene family that shares a phylogenetically conserved common DNA-binding domain, the T-box. T-box genes code transcription factors involved in regulating developmental processes. This gene is closely related to the related family member T-box 3 (ulnar breast syndrome) on human chromosome 12. The coded protein may play a role in cardiac development and limb identity determination. Mutations in this gene are associated with Holt-Oram syndrome, a developmental disorder affecting the heart and upper limbs. Several transcript variants coding different isoforms of this gene have been described. The accession numbers for this protein are 099593 or A6ND77, or 015301, or Q96TBO. Antibodies against this protein are commercially available from R&D Systems, Browse EMD, OriGene Antibodies, and Novus Biologicals.

[0136] As used here, the term “proteins that facilitate tissue regeneration and / or improve tissue function” means proteins that can regenerate, restore, or improve tissue function or bone function. These proteins are potential cytokines that improve tissue regeneration or bone function. Due to their gel-forming properties, certain protein polymers can be very suitable for biomedical applications, such as tissue regeneration in surgery and wounds. Such non-limiting examples include the IGFBP5 protein, which enhances periodontal tissue, and PPARs, which activate liver regeneration.

[0137] As used here, "freeze-drying" means low-temperature drying or freeze-drying.

[0138] Cell-derived exosomes or microvesicles, also called extracellular exosomes or microvesicles, are membrane-bound structures released by cells in vitro and in vivo. Because extracellular exosomes or microvesicles can contain proteins, lipids, and nucleic acids, they can mediate intercellular communication between different cells, including different cell types, within the body. Two types of extracellular exosomes or microvesicles are exosomes or microvesicles, and microvesicles. Exosomes or microvesicles are small lipid-binding structures that mediate intercellular communication through the intercellular transport of proteins and RNA secreted extracellularly (El Andaloussi, S et al. (2013) Nature Reviews: Drug Discovery 12(5): 347-357). The size of exosomes or microvesicles ranges from approximately 30 nm to approximately 200 nm. Exosomes or microvesicles are released from cells by the fusion of multivesicular endosomes (MVEs) with the plasma membrane, while microvesicles are released from cells during direct budding from the plasma membrane (PM) and packaged with different factors. Microvesicles are generally larger than exosomes or microvesicles, ranging from approximately 200 nm to 1 μm, and have a variety of functions.

[0139] Cell-derived exosomes or microvesicles can be isolated from eukaryotic cells using commercially available kits from biovision.com and novusbio.com, or using the methods described herein. A non-limiting example of cells from which cell-derived exosomes or microvesicles can be isolated includes stem cells. Such non-limiting examples of stem cells include adult stem cells, embryonic stem cells, embryoid-like stem cells, non-embryonic stem cells, or induced pluripotent stem cells.

[0140] As used herein, the terms “overexpression,” “overexpression,” etc., are intended to encompass increasing the expression of nucleic acids or proteins to levels higher than those naturally present in exosomes or microvesicles. This term is intended to encompass the overexpression of endogenous and heterologous nucleic acids and proteins.

[0141] As used herein, the term “homogeneous” with respect to a population of E-cell-derived exosomes or microvesicles refers to a population of exosomes or microvesicles of similar size, or a combination thereof, derived from cells having equal amounts of exogenous nucleic acids and equal amounts of exogenous proteins. A homogeneous population is a population of exosomes or microvesicles derived from cells in which approximately 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% are cell-derived and share at least one characteristic. Another example of a homogeneous population is a population in which approximately 90% of the exosomes or microvesicles have a diameter of less than 50 nm.

[0142] As used herein, the term “heterogeneous” refers to a population of cell-derived exosomes or microvesicles having different amounts of exogenous nucleic acids, different amounts of exogenous proteins, different sizes, or combinations thereof.

[0143] The term "substantially" refers to the complete or nearly complete degree or extent of a characteristic, and on the one hand, it defines the purity of a population of exosomes or microvesicles that have been isolated or purified.

[0144] The term “purified population” for cell populations, cell-derived exosomes, microvesicles, or miRNAs refers to a plurality that has undergone one or more selection processes for the enrichment or miRNA population of a desired exosome or microvesicle compared to some or all of several other components normally found in culture media, as disclosed herein. Alternatively, “purified” may refer to the removal or reduction of undesirable residual components found in the conditioned medium (e.g., cell debris, soluble proteins, and others). As used herein, a “highly purified population” refers to a population of cell-derived exosomes or microvesicles from which cell debris and soluble proteins have been removed along with the cell-derived exosomes or microvesicles by at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%. The mirNAs described herein may be provided in isolated, purified, highly purified, homogeneous, substantially homogeneous, and heterogeneous forms.

[0145] As used herein, the terms “culture medium” and “culture medium” are interchangeable and refer to a solid or liquid substance used to support the proliferation of cells (e.g., stem cells). Preferably, as used herein, culture medium refers to a liquid substance capable of maintaining stem cells in an undifferentiated state. The medium may be an aqueous medium containing a combination of components such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors, and hormones. All of these are necessary for cell proliferation and can maintain stem cells in an undifferentiated state. For example, the culture medium may be a synthetic medium such as: minimum required media (MEM-a) (HyClone Thermo Scientific, Waltham, Mass., USA), DMEM / F12, GlutaMAX (Life Technologies, Carlsbad, Calif., USA), Neurobasal Medium (Life Technologies, Carlsbad, Calif., USA), KO-DMEM (Life Technologies, Carlsbad, Calif., USA), DMEM / F12 (Life Technologies, Carlsbad, Calif., USA), and supplemented with necessary additives as further described herein. In some embodiments, the cell culture medium may be a mixture of culture media. Preferably, all components contained in the medium of this disclosure are substantially pure and tissue culture grade. "Acclimatization medium" and "conditioning medium" are used interchangeably and refer to a culture medium in which cells are cultured for a period of time and in which the cells release / secrete components (e.g., proteins, cytokines, chemicals, etc.) into the medium.

[0146] "Composition" is also intended to encompass combinations of cells, cell populations, exosomes or microvesicles, mirNA, or such populations, or activators and other carriers. Examples: compounds or compositions, inactive (e.g., detectable substances or labels) or active, e.g., adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, etc. Carriers include: biocompatible scaffolds, pharmaceutical excipients and additives; proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars; polysaccharides or sugar polymers), which may exist alone or in combination, and may contain 1 to 99% by weight or volume, either alone or in combination. Exemplary protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (PHA), gelatin, and casein. Representative amino acid / antibody components that can also function in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame. Carbohydrate excipients are also intended to be within the scope of the present invention. Examples include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melegitose, maltodextrin, dextran, and starch; and argitols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myo-inositol.

[0147] Preservatives are intended to enhance the viability of the drugs in a composition. Non-limiting examples include benzoates (such as sodium benzoate and benzoic acid), nitrites (such as sodium nitrite), and sulfites (such as sulfur dioxide).

[0148] Antifreezing agents are compounds that protect drugs during freezing and thawing procedures. Non-exclusive examples of such agents include DMSO, glycerol, and PEG.

[0149] A "control" is an alternative subject or sample used in an experiment for comparative purposes. A control can be "positive" or "negative." For example, if the objective of the experiment is to determine the correlation between altered gene expression levels and a specific phenotype, it is generally preferable to use one positive control (a sample from the subject that has such alteration and exhibits the desired phenotype) and one negative control (a subject or sample from the subject that lacks the altered expression or phenotype). Furthermore, if the objective of the experiment is to determine whether a drug affects stem cell differentiation or the expression of exosomes, microvesicles, or miRNAs, it is preferable to use a positive control (a sample with aspects known to affect differentiation or expression changes) and a negative control (a drug known to have no effect, or a sample without the drug).

[0150] The term "cultivate" refers to the growth of cells or organisms in a test tube on or in various types of culture media. It is understood that offspring of cells grown in culture may not be exactly identical (i.e., morphologically, genetically, or phenotypic) to the parent cells. "Expanded" means the proliferation or division of cells.

[0151] As used herein, the term “detectably labeled” means that the active substance (biological molecule or small molecule compound) is bound to another molecule, compound, or polymer that facilitates the detection of the presence of the active substance in vitro or in vivo.

[0152] A “detectable label” is intended to be a directly or indirectly detectable compound or composition that is directly or indirectly conjugated to the composition to be detected. Examples include N-terminal histidine tags (AND-His), magnetically active isotopes (115Sn, 117Sn, and 119Sn), non-radioactive isotopes (13C and SN), and polynucleotides or proteins such as antibodies for generating a “labeled” composition. The term also includes sequences conjugated to polynucleotides that will provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP). A label may be detectable on its own (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze a chemical change in the detectable substrate compound or composition. Labels may be suitable for small-scale detection or even more suitable for high-throughput screening. Appropriate labels, as such, include, but are not limited to, the following items: magnetically active isotopes, non-radioactive isotopes, radioactive isotopes, fluorescent dyes, luminescent compounds, dyes, and proteins, including enzymes. Labels may be simply detected or quantified. Simply detected responses generally involve responses that merely confirm the presence of the substance, while quantified responses generally involve responses that have quantifiable (e.g., numerically reportable) values ​​such as intensity, polarization, and / or other properties. In luminescence assays or fluorescent sequences, detectable responses may be generated directly using a luminescent or fluorophore associated with the sequence component actually involved in binding, or indirectly using a luminescent or fluorophore associated with other (e.g., reporter or indicator) components.

[0153] Examples of luminescence labels that generate signals include, but are not limited to, bioluminescence and chemiluminescence. Detectable luminescence responses generally involve changes in or the generation of a luminescence signal. Suitable methods and luminescent phosphonates for luminescent labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Chemicals (6th edition). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0154] Examples of suitable fluorescent labels include, but are not limited to, the following items: fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malacite green, stilbene, lucifer yellow, cascade blue, and Texas red. Other suitable optical dyes are listed in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Chemicals (6th edition).

[0155] As used herein, “expression” refers to the process and / or the process by which polynucleotides are transcribed into mRNA. In that process, the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0156] "Differentially expressed" refers to the upward or downward expression of a gene, exosome or microvesicle, or marker, for example, compared to a control. The control, in this case, is a differentiated cell compared to a pluripotent cell or stem cell. When applied to genes, proteins, cells, populations, exosomes or microvesicles, miRNAs, or markers, "differential expression" refers to the differential production of a product compared to a control, such as expression levels found in its natural environment. Differentially expressed products are mRNA transcribed from a gene, or protein products coded by a gene. A differentially expressed gene may be overexpressed or underexpressed (aka inhibited) compared to the expression levels of normal, untreated, undenatured, or control cells. In one aspect ,it is, From the expression levels detected in the control sample, 1.5 times, 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, and at least 10 times expensive (In other words, it is overexpressed) ) or, It means low. The term "differentially expressed" also refers to a nucleotide sequence in a cell or tissue that is expressed when it is silent in control cells, or not expressed when it is expressed in control cells.

[0157] The term “stem cell” refers to a cell that is undifferentiated or partially differentiated and has the ability to self-replicate and produce differentiated offspring. Self-renewal is defined as the ability of a stem cell to proliferate and produce more such stem cells while maintaining its developmental potential (i.e., totipotency, pluripotency, etc.). The term “somatic stem cell” is used herein to refer to any stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissues. Natural somatic stem cells are isolated from a variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Exemplary natural somatic stem cells include, but are not limited to, mesenchymal stem cells (MSCs) and neural stem cells (NSCs). In some embodiments, a stem cell or progenitor cell may be an embryonic stem cell. As used herein, “embryonic stem cell” means a stem cell derived from tissue formed after fertilization but before the end of pregnancy. For example: pre-pregnancy tissue (such as a blastocyst), embryonic tissue, or fetal tissue collected at any time during pregnancy, usually about 10-12 weeks before conception. Most frequently, embryonic stem cells are pluripotent cells derived from an early embryo or blastocyst. Embryonic stem cells can be obtained from suitable tissues, including but not limited to human tissue, or directly from established embryonic cell lines. "Embryo-like stem cells" refer to cells that share one or more characteristics with embryonic stem cells, but not all of them.

[0158] "Differentiation" describes the process by which unspecialized cells acquire the characteristics of specialized cells, such as heart, liver, or muscle cells. "Oriented differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a specific cell type. "Dedifferentiation" defines cells that return to a less committed position within a cell lineage. As used herein, the terms "differentiated" or "differentiated" define cells that occupy a more established ("differentiated") position within a cell lineage. As used herein, "cells that differentiate into a mesodermal (or ectoderm or endoderm) lineage" define cells that become involved in a specific mesoderm, ectoderm, or endoderm lineage, respectively. Examples of cells that differentiate into a mesoderm lineage or give rise to a specific mesoderm cell include, but are not limited to, adipogenic, muscular, chondroplastic, cardiac, dermatoplastic, hematopoietic, angiogenic, myoplastic, nephroplastic, urogenogenic, osteogenic, pericardiogenic, or stromal cells.

[0159] As used herein, the terms “differentiated” or “differentiated” define cells that occupy a more established (“differentiated”) position within a cell lineage. “Dedifferentiated” defines cells that return to a less committed position within a cell lineage. Induced pluripotent stem cells are an example of dedifferentiated cells.

[0160] As used herein, the “lineage” of a cell defines the heredity of a cell, for example, its predecessors and progeny. The lineage of a cell places the cell within the genetic scheme of development and differentiation.

[0161] A “multipotent stem cell” or “multipotent stem cell” refers to a stem cell that regenerates at least two further differentiated progeny cells from itself and from distinct developmental lineages. The lineages may originate from the same germ layer (i.e., mesoderm, ectoderm, or endoderm) or from different germ layers. An example of two progeny cells with different developmental lineages from the differentiation of a multipotent stem cell is myogenic cells and adipogenic cells (both of mesoderm origin, yet still giving rise to different tissues). Another example is neurogenic cells (of ectoderm origin) and adipogenic cells (of mesoderm origin).

[0162] The term "precursor" or "progenitor cell" is intended to mean a cell that has the ability to differentiate into a specific type of cell. Progenitor cells can be stem cells. Progenitor cells can also be more specific than stem cells. Progenitor cells can be unipotent or pluripotent. Compared to adult stem cells, progenitor cells can be in a later stage of cell differentiation. Examples of progenitor cells, though not limited to them, include progenitor neurons.

[0163] "Parthenogenetic stem cells" refer to stem cells that arise from the activation of parthenogenesis in eggs. Methods for producing parthenogenetic stem cells are publicly known in this field. For example, Cibelli et al. (2002) Science 295 (5556): 819, and Vrana et al. (2003) Proc. Natl. Acad. Sci. USA 100 (Suppl. 1) 11911-6.

[0164] As used herein, “pluripotent cell” defines a less differentiated cell capable of producing at least two distinct (genotypic and / or phenotypic) further differentiated progeny cells. In another context, “pluripotent cell” includes non-pluripotent cells, typically induced pluripotent stem cells (iPSCs), which are artificially induced stem cells derived from adult somatic cells produced by historically inducing the expression of one or more stem cell-specific genes. Such stem cell-specific genes include, but are not limited to, families of octameric transcription factors; for example, Oct-3 / 4; Sox gene family (Sox1, Sox2, Sox3, Sox 15 and Sox 18); Klf gene family (Klf1, Klf2, K1f4 and K1f5); Myc gene family (c-myc and L-myc); Nanog gene family (OCT4, NANOG and REX1) or LIN28. Examples of iPSCs are listed below: Takahashi et al. (2007) Cell Advance online publication 20 Nov. 2007; Takahashi & Yamanaka (2006) Cell 126: 663-76; Okita et al. (2007) Nature 448: 260-262; Yu et al. (2007) Science Advance online publication 20 Nov. 2007; and Nakagawa et al. (2007) Nat. Biotechnol. Advance online publication 30 Nov. 2007.

[0165] Embryoid bodies, or EBs, are three-dimensional (3D) aggregates of embryonic stem cells that form during culture and facilitate subsequent differentiation. When grown in suspension culture, EB cells form small aggregates of cells surrounded by an outer layer of visceral endoderm. As they grow and differentiate, EBs develop into cystic embryoid bodies with a fluid-filled cavity and an inner layer of ectoderm-like cells.

[0166] "Induced pluripotent cells" refer to embryo-like cells reprogrammed from adult cells into an immature phenotype. Various methods are known in the art. For example, "A simple method for inducing pluripotency: acid." Nature, 29 Jan. 2014, available at sciencedaily.com / releases / 2014 / 01 / 140129184445, last accessed February 5, 2014; also U.S. Patent Application Publication No. 2010 / 0041054. Human iPS cells also express stem cell markers and can generate cells characteristic of all three germ layers.

[0167] As used herein, the term “cardiac precursor” means a dynamic precursor capable of differentiating into the cardiac cell type from which it ultimately derives. Cardiac progenitor cells (CPCs) represent the early stages of mesoderm involvement in the cardiac lineage and exhibit the classic CPC marker KDR / PDGFR-apos / CKITneg, responding to tolerant conditions for proliferation as a progenitor cell population and / or differentiation into terminal cardiac cells.

[0168] As used herein, the term “skeletal myogenic precursor” refers to cells characterized by the expression of Pax3 and Pax7, and which give rise to postnatal muscle satellite cells.

[0169] As used herein, “fibroblasts” refers to cells expressing the following markers: markers,

[0170] As used herein, “skeletal myoblast” refers to a cell expressing the following markers: MyOG, desmin, local pain, and human α-skeletal actin.

[0171] As used herein, “pluripotent cell” refers to a poorly differentiated cell capable of producing at least two different (genotypic and / or phenotypic) further differentiated progeny cells.

[0172] Young or juvenile stem cells are derived from mice aged 2 months or younger that possess anti-aging genes. In vitro, the cells average approximately 3–5 μm in size and express embryonic stem cell markers, OCT4 and surface markers, CD29, CD44, and CD90.

[0173] As used herein, “fibroblasts” refers to cells expressing the following markers: markers,

[0174] As used herein, “skeletal myoblast” refers to a cell expressing the following markers: Myog, Desmin, local pain, and human α-skeletal actin.

[0175] As used herein, the term “pluripotency gene or marker” refers to an expressed gene or protein associated with immature or undifferentiated phenotypes, such as Oct 3 / 4, Sox 2, Nanog, c-Myc, and LIN-28. Methods for identifying such are known in the art, and systems for identifying such are commercially available, for example, from EMD Millipore (MILLIPLEX® Map Kit).

[0176] Skeletal myoblasts (SMs) are immature cells that can be isolated from between the basement plate and the myocyte membrane. They make up 2-5% of the intralaminar nuclei of mature skeletal muscle. Skeletal myoblasts are activated in response to muscle injury or muscle degeneration due to disease. Skeletal myoblasts discover desmin, CD56, Pax3, Pax7, c-met, myocyte nuclear factor, M-cadherin, VCAM1, N-CAM, CD34, Leu-19, and syndecans 3 and 4. As the cells differentiate into multinucleated myotubules, activated skeletal myoblasts initially express Myf-5 and / or MyoD, and finally express myogenin and MRF4.

[0177] As used herein, the term “immature stem cells (SJSCs)” refers to stem cells isolated from aged bone marrow-derived stem cells (BMSCs) that possess high proliferative and differentiation potential. Igura et al. (2013) 305(8): H1354-62. Flow cytometry analysis of SJSCs revealed the presence of CD29(+) / CD44(+) / CD59(+) / CD90(+) mesenchymal stem cell markers, but negative for CD45(-) / CD117(-). SJSCs exhibit higher proliferative, colonization, and differentiation potential compared to BMSCs. These cells have also been reported to significantly express cardiac lineage markers (Gata-4 and muscle cell-specific enhancer factor 2C), as well as pluripotency markers (octamer-binding transcription factor 4, sex-determining region Y-box 2, stage-specific embryonic antigen 1, and Nanog), and anti-aging factors such as telomerase reverse transcriptase and sirtuin 1.

[0178] Bone marrow stromal cells, also known as mesenchymal stromal cells, are pluripotent stem cells capable of differentiating into various cell types. Cell types to which MSCs have been shown to differentiate in vitro or in vivo include osteoblasts, chondrocytes, myocytes, and adipocytes. Mesenchyme is the embryonic connective tissue derived from the mesoderm, which differentiates into hematopoietic and connective tissues; MSCs do not differentiate into hematopoietic cells. Stromal cells are connective tissue cells that form the supporting structure in which functional tissue cells reside. While this is an accurate description of one function of MSCs, the term fails to convey the relatively recently discovered role of MSCs in tissue repair. Methods for isolating, growing, and differentiating such cells are known in technical and patent literature. For example, U.S. Patent Application Publications 2007 / 0224171, 2007 / 0054399, and 2009 / 0010895. These are incorporated by reference.

[0179] Adipose-derived stem cells (ASCs), also known as adipose tissue-derived stem cells (ADSCs), are routinely isolated from the stromal vascular fraction (SVF) of homogenized adipose tissue. Like other types of mesenchymal stem cells (MSCs), ADSCs remain difficult to define due to the lack of definitive cellular markers. Adipose-derived stem cells (ASCs) are a source of mesenchymal stem cells possessing self-renewal properties and pluripotent differentiation.

[0180] Hematopoietic stem cells are defined as stem cells that give rise to all red blood cells, white blood cells, and platelets. They are generally isolated using the marker CD34+. On the one hand, hematopoietic stem cells are adult stem cells that include the following marker profile: CD34+ and / or CD34+ / Thy-1-HSC. Referenced by Andrews, RG et al. (1990) J. Exp. Med. 172(1): 355-358, incorporated herein by reference.

[0181] Mesenchymal stem cells (MSCs) are defined as pluripotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (osteocytes), chondrocytes (chondrocytes), myocytes (muscle cells), and adipocytes (adipocytes).

[0182] As used herein, the term chemically derived or chemically modified pluripotent stem cells (iPSCs) is intended to include iPSCs treated with low-molecular-weight compounds such as isoxazoles or their derivatives, or isoxazole-like molecules.

[0183] Isoxazoles are a type of compound found in several natural products, such as ibotenic acid, or many drugs containing COX-2 inhibitors and fluoroxanes (nitric oxide donors). Isooxazoles are useful isomers of pyridines and have been shown to treat depression, enabling the construction of tetracycline antibiotic derivatives that inhibit voltage-gated sodium channels to control pi. Compounds of this class are available from Sigma-Aldrich, and methods for synthesizing them are known in the art, as described, for example, in US Pat. Nos. 5,059,614 and 8,318,951 and International Publication 1999 / 002507. Structurally, isoxazoles are five-membered heterocyclic compounds with oxygen and nitrogen atoms at the 1 and 2 positions. Their partially saturated analogs are called isoxazolines, and their fully saturated analogs are called isoxazolidines. Examples of isoxazol-like compounds include derivatives, and such non-limiting examples include sulfamethoxasol, sulfisoxasol, oxacillin, cycloserine, and asibicin. Isoxazol, isoxazoline, and isoxazolidine can be considered useful synthons in organic synthesis. Isoxazol can be efficiently converted into various classes of medically important molecules. For example, anthracene-9-ylmethylene-(3,4-dimethylisoxazol5-yl)amine can be synthesized in high yield by reacting anthracene-9-carbaldehyde with 5-amino-3,4-dimethylisoxazol in ethanol. In one embodiment, all derivatives of isoxazol are considered “isoxazol-like compounds” or “analogous compounds.” In one embodiment, isoxazol derivatives such as 5-amino-3-methyl-4-isoxazolcarboxylic acid semicarbazide and thiosemicarbazide can be synthesized. Reactions between 5-amino-3-methyl-4-isoxazolic acid hydrazides and isocyanates and isothiocyanates can also be designed and carried out. In nucleophilic addition reactions with compounds containing a primary amino group, isocyanates form urea derivatives, and isothiocyanates form thiourea derivatives. Only the hydrazide terminal group (NH₂) is involved in this reaction.The amino group at position 5 of the isoxazole ring remains unreacted under reaction conditions. The reaction mechanism involves nucleophilic attack of the nitrogen atom in the hydrazide group (-NH₂) on the carbon atom of the isocyanate or isothiocyanate. Intermediates appear that form substituted 5-amino-3-methyl-4-isoxazole carboxylic acid semicarbazides and thiosemicarbazides via amidoiminol tautomerization. In one embodiment, an example of an isoxazole derivative may include 5-sulfanilamide-isoxazole of the general formula where R and R are lower alkyl and / or lower alkoxyalkyl groups. Sulfanilamide derivatives can be produced in which the isoxazole ring is bonded to the N position of a sulfanilamide molecule, for example, a sulfanilamide radical at position 4 of the isoxazole ring. Furthermore, sulfanillyl derivatives of 5-amino-isoxazol, namely 5-sulfanilamide-3-methyl-isoxazol, can also be considered isoxazol derivatives. In one embodiment, both the 3rd and 4th positions of the isoxazol ring of the sulfanilamide derivative can be substituted with alkyl and / or corresponding alkoxyalkyl groups. Non-limiting examples of “isoxazol-like compounds” or “similar compounds” include the following items: 1,2-oxazol, 4-deuterio-1,2-oxazol, 1,2-oxazol potassium, hydrolon; 1,2-oxazol, 1-oxide-1,2-oxazol-1-ium, 1,2-oxazol; hydrobromide, 1,2-oxazol; hydrochloride, ethane; 1,2-oxazol potassium. 1,2-oxazole; hydroxide, 1,2-oxazole; hydrate, hydrochloride, ethane. 1,2-oxazole, 1,2-oxazole. Cyanate, 2-oxide-1,2-oxazole-2-ium, carbon monoxide.Chromium; 1,2-oxazole, ethane; 1,2-oxazole, ethane; 1,2-oxazole; propane, 1,2-oxazole-2-ium-2-sulfonate, carbonyl dichloride; 1,2-oxazole, isocyanate; 1,2-oxazole, ethoxyethane; 1,2-oxazole, 2,2-dimethylpropane; ethane; 1,2-oxazole, ethane; methoxyethane; 1,2-oxazole, ethane; 2-methylpropane; 1,2-oxazole, 1,2-oxazole; urea, ethanol; 1,2-oxazole, carbonic acid; 1,2-oxazole, 1,2-oxazole-1-ium-1-sulfonic acid, 1,2-oxazole-2μm; iodide.

[0184] Isoxazol-9 (ISX-9) is a small molecule compound inducer of adult neural stem cell differentiation both in vitro and in vivo (Schneider et al.). It has been shown to act via a calcium-activated signaling pathway dependent on muscle cell enhancer factor 2 (MEF2)-dependent gene expression (Schneider et al., Petrik et al.). This compound is also available from Sigma-Aldrich or StemCell Technologies. Its molecular formula is CuH₂N₂O₃S, and its chemical name is N-cyclopropyl-5-thiophene-2-yl-1,2-oxazol-3-carboxamide. Its two-dimensional structure is: TIFF0007837302000001.tif62163

[0185] [Isoxazole 1](ISX-1) is a small molecule compound with the following structure: TIFF0007837302000002.tif61164

[0186] As used herein, isoxazol-like compounds and isoxazol-like compounds refer to drugs or small molecule compounds having the same functional properties as isoxazol as described herein. Non-limiting examples include, for example, cardioneogens; CDNG1 / vuc230, CDNG2 / vuc198, and CDNG3 / vuc247 (see Terri et al. (2011) Chem Biol., December 23 18(12):1658-1668), as well as sulfisoxazols as described below herein, and leflunomide (Arava), also known as 5-methyl-N-[4-(trifluoromethyl)phenyl]-1,2-oxazol-4-carboxamide.

[0187] Isooxazolic compounds and derivatives therefore have the following formula: TIFF0007837302000003.tif75154 Here, both R and R2 are hydrogen, or R is hydrogen and R2 is one selected from the group consisting of C.-C. alkyl, C2-C6 cycloalkyl, C2-C4 alkenyl, C2-C6, alkynyl, benzyl substituted or unsubstituted, or R and R2 are bonded to each other to form a ring selected from azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl; R2', R3 and R4 are one selected from the group consisting of hydrogen, halogen, C.-C. alkyl, C3-C6 cycloalkyl, substituted or unsubstituted aromatic or teloaromatic ring, cyano, nitro, and acyl; Y is 0, NH or S.

[0188] On the other hand, isoxazolic compounds have the formula: TIFF0007837302000004.tif77127 Here, R1 and R2 are selected from C1-C4 alkyl, phenyl, benzyl, trifluoromethyl, or halogen, respectively, and R3 is selected from hydrogen, hydroxy, C1-C4 alkyl, or alkoxy. R4 is selected from hydrogen, trifluoromethyl, C1-C4 alkoxy, C1-C4 alkyl, or C1-C4 hydroxyalkyl when it is at position 3 or 5, and R5 is selected from hydrogen or C4-C4 alkyl, and R4 and R5 together form a tetramethylene group. Z at position 3 or 5 in the heterocycle is selected from -N(R6)-CO-, -CO-N(R6)-, -N(R6)CO-N(R6)-, -CH(R6)-NH-CO-, or -NH-CO-CH(R6). Here, R^ is selected from hydrogen or C1-C4 alkyl. Non-restrictive examples include, for example: 5-(trifluoromethyl)-3-(4-methoxyphenyl)isoxazo-l-4-carboxylic acid, 5-(trifluoromethyl)-3-(4-fluorophenyl)isoxazo-l-4-carboxylic acid, 5-(thiophen-2-yl), isoxazo-l-3-carboxyaldehyde, 5,6,7,8-tetrahydro-4h-cyclohepta[d]isoxazo-l-3-carboxylic acid, 4,5,6,7-tetrahydro-benzo[d]isoxazo-l-3-carboxylic acid, 3-amino-5-methylisoxazol, 4-amino-n-(5-methyl-3-isoxazolyl)benzenesulfonamide, 3-phenylisoxazol-5-boronic acid pinacol ester, 5-phenylisoxazol, 1-phenyl-1-cyclopentanecarboxylic acid, 3-phenyl-benzo[c]isoxazol-5-carboxylic acid, 5-methyl-3-phenylisoxazol-4-carboxylic acid, 3a, 4, 5, 6, 7, 8, 9, 9a-octahydrocyclooctadi[d]]isoxazol-3-carboxylic acid, 5-(3-nitrophenyl)isoxazol, 3-(4-nitrophenyl)isoxazol, 3-hydroxy-5-aminomethylisoxazol, 5-morpholinomethyl)isoxazol-3ca-carboxylic acid hydrochloride, 5-(morpholinomethyl)isoxazol-3-carbaldehyde, 3-methyl-5-(trifluoromethyl)isoxazol-4-carboxylic acid, methyl-5-(thiophen-2-yl)isoxazol-3-carboxylic acid, 3-(methylsulfonyl)-5-(2-thienyl)isoxazol-4-carbonitrile, 5-methyl-3-(2-pyrrolidinyl)isoxazol, 3-methyl-5-(2-pyrrolidinyl)isoxazol Loridinyl)isoxazol, 3-(1-methyl-1h-)pyrazole-4-yl)-isoxazol-5-carboxylic acid, 3-(1-methyl-1h-pyrazole-4-yl)-4,5-dihydro-isoxazol-5-carboxylic acid, 5-(4-methylphenyl)isoxazol-3-carboxylic acid, 5-methyl-3-phenylisoxazol-4-carboxylic acid, 5-(4-methylphenyl)isoxazol-3-carboxylate Aldehydes, 5-methyl-3-(4-phenoxyphenyl)isoxazo-l-4-carboxylic acid, 3-methyl-5-(4-methyl-1,2,3-thiadiazo-l-5-yl)isoxazo-l-4-carboxylic acid, 3-methyl-5-(5-methylisoxazo-l-3-yl)isoxazo-l-4-carboxylic acid, methyl 5-(4-methoxyphenyl)isoxazo-l-4-carboxylate, methyl 5-(4-methoxyphenyl)isoxazo-l-3-carboxylate, 5-methylisoxazole, methyl 5-(4-fluorophenyl)isoxazo-l-4-carboxylate, methyl 5-(4-fluorophenyl)isoxazo-l-3-carboxylate, methyl 5-(4-chlorophenyl)isoxazo-l-4-carboxylate, methyl 5-(4-bromophenyl)isoxazo-l-4-carboxylate, 5-(4-methoxyphenyl)) isoxazol-3-carboxylic acid, 5-(3-methoxyphenyl)-isoxazol-3-carboxylic acid, 3-(2-methoxyphenyl)isoxazol-5-carboxylic acid d, 5-(4-methoxyphenyl)isoxazol-3-carboxyaldehyde, 3-(4-methoxyphenyl)isoxazol-5-carbaldehyde, 3-(2-methoxyphenyl)isoxazol-5-carbaldehyde, 5-(4-methoxyphenyl)isoxazol, 3-(4-methoxyphenyl)isoxazol, 3-(2-methoxyphenyl)-4,5-dihydroisoxazol -5-carboxylic acid, 3-methoxyisoxazol-5-carboxylic acid, isoxazol-5-carboxylic acid, isoxazol-4-carboxylic acid, isoxazol-5-carbothioamide, isoxazol-5-carbonyl chloride, isoxazol-3-carbonitrile, isoxazol-3-carbaldehyde, isoxazol-4-boronic acid, isoxazol, 5-cyclopropyl-4-[2-(methylsulfonyl)-4-(trifluoromethyl)benzoyl]isoxazol, 6-(5-(thiophen-2-yl)isoxazol-3-carboxamide)hexyl 5-((Z as, 4s, bar-))-2-oxohexahydro-1H-thieno[3,4-diimidazol-4-yl)pentanoate, isocarboxazide 5-methyl-3-isoxazol-carboxylic acid 2-benzylhydrazide, 5-isobutylisoxazol-3-carboxylic acid, 4-iodo-5-methylisoxazol, 3,3'-iminobis(n,n-dimethylpropylamine), 3-(3-hydroxyphenyl)-isoxazol-5-carboxylic acid methyl ester, 5-(4-hydroxyphenyl)-isoxazol-3-carboxylic acid, 5-(3-hydroxyphenyl)-isoxazol-3-carboxylic acid, 5-hydroxymethyl)-3-methylisoxazol, 3-hydroxy-5-methylisoxazol, 5-(1-hydroxyethyl)-3-(4-trifluoromethyl Phenyl)isoxazol, 3a,4,5,6,7,7a-hexahydro-benzo[d]isoxazol-3-carboxylic acid, 5-(2-furyl)isoxazol-3-carbaldehyde, 5-furan-2-ylisoxazol-3-carboxylic acid, 6-fluoro-3-(4-piperidinyl)benzisoxazol, 5-(4-fluorophenyl)isoxazol-3-methol, 3-(2-fluorophenyl)isoxazol-5-carboxylic acid, 5-(4-fluorophenyl)isoxazol-3-carboxyaldehyde, 3-(4-fluorophenyl)isoxazol-5-carbaldehyde, 3-(3-fluorophenyl)isoxazol-5-carbaldehyde, 3-(2-fluorophenyl)isoxazol-5-carbaldehyde, 5-(4-fluorophenyl)isoxazol, 3-(4-fluorophenyl)isoxazol, 5-(3-fluoro-4-methoxyphenyl)isoxazol-3-carboxylic acid, ethyl 5-(trifluoromethyl)-3-(4-methoxyphenyl)isoxazol-4-carboxylate, ethyl-5-(tributylstanyl)isoxazol-3-carboxylate, ethyl 5-(thiophene 2-yl)isoxazol-3-carboxylate, 5-ethyl-isoxazol-4-carboxylic acid, 5-ethyl-isoxazol-3-carboxylic acid, 5-(4-fluorophenyl)isoxazol-4-carboxylate ethyl, 5-(4-fluorophenyl)isoxazol-3-carboxylate ethyl, 5-(ethyl) 2,3-Dihydrobenzo[b][1,4]dioxin-7-yl)isoxazole-3-carboxylate, ethyl 3-(4-chlorophenyl)-5-(trifluoromethyl)isoxazole-4-carboxylate, ethyl 5-(4-chlorophenyl)isoxazole-3-carboxylate, ethyl 3-(4-bromophenyl)-5-(trifluoromethyl)isoxazole-4-carboxylate, 5-(4-bromophenyl)isoxazole-3-carboxylate ethyl, 5-amino-4-(4-chlorophenyl)isoxazole-3-carboxylate ethyl, 5-amino-4-(4-bromophenyl)isoxazole-3-ethyl. Carboxylate, ethyl 6b-acetyl-2-(acetyloxy)-4 a,6 a-dimethyl 2,3,4,4 a,4b,5,6,6 a,6b,9 a,10,10 a,10b,11-tetradecahydro-1h Naphtho[2',1:4,5]indeno[2,1-d]isoxazol-9-carboxylate, 3,5-dimethyl-4-(tributylstanyl)isoxazol, 5-(1,5-dimethyl-1h-pyrazole)-(4-yl)-isoxazol-3-carboxylic acid, 5-(1,3-dimethyl-1h-pyrazole-4-yl)isoxazol-3-carboxylic acid, 5-(1,5-dimethyl-1h-pyrazole)4-yl)-isoxazol, dimethylisoxazol-4-boronic acid pinacol ester, 3,5-dimethylisoxazol, 3-(dimethylamino)-1-(2-pyridyl)-2-propen-1-one, 5-(3,5-difluorophenyl))Isoxazol, [2,6-dichloro-4-(trifluoromethyl)phenyl]hydrazine, 5-(2,5-dichlorophenyl)isoxazol-3-carboxylic acid, danazol, 3-(4-chlorophenyl)-5-(trifluoromethyl)isoxazol-4-carboxylic acid, 5-(4-chlorophenyl)isoxazol-3-propionic acid, 5-(4-chlorophenyl)isoxazol-4-carboxylic acid, 5-(4-chlorophenyl)isoxazol-3-carboxylic acid, 3-(4-)chlorophenyl)isoxazol- 5-Carboxylic acid, 3-(3-chlorophenyl)isoxazol-5 carboxylic acid, 5-(4-chlorophenyl)isoxazol-3 carboxyaldehyde, 3-(4-chlorophenyl)isoxazol-5 carbaldehyde, 3-(3-chlorophenyl)isoxazol-5 carbaldehyde, 3-(2-chlorophenyl)isoxazol-5 carbaldehyde, 5-(4-chlorophenyl)isoxazol, 3-(4-chlorophenyl)isoxazol, 5-(chloromethyl)isoxazol-4 carboxylic acid, 3- (Chloromethyl)-5-(2-furyl)isoxazol, 4-chloromethyl-3,5-dimethylisoxazol, 5-(chloromethyl)-3-(4-chlorophenyl)isoxazol, 5-(3-chloro-4-methoxyphenyl)isoxazol-3-carboxylic acid, 3-chloro-4-fluorobenzaldehyde, 3-(5-chloro-2,4-dimethoxy-)phenyl)4,5-dihydroisoxazol-5-carboxylic acid, 5-tert-butyl-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-carboxylic acid 5-(4-bromophenyl)-5-(trifluoromethyl)isoxazo-l-4-carboxylic acid, 5-(4-bromophenyl)isoxazo-l-3-propionic acid, 5-(4-bromophenyl)isoxazo-l-3-carboxylic acid hydrazide, 5-(4-bromophenyl)isoxazo-l-4-carboxylic acid, 5-(4-bromophenyl)isoxazo-l-3-carboxylic acid, 3-(4-bromophenyl)isoxazo-l-5-carboxylic acid, 3-(4-bromophenyl)isoxazo-l-5-carboxyaldehyde, 5-(4-bromophenyl)isoxazol, 5-(3-bromophenyl)isoxazol, 3-(4-bromophenyl)isoxazol, 5-(bromomethyl)-3-(4-methoxyphenyl)isoxazol, 4-(bromomethyl)isoxazol, 5-bromomethyl)-3-(4-fluorophenyl)isoxazol, 5-(bromomethyl)-3-(4-chlorophenyl)isoxazol, 5-(bromomethyl)-3-(4-bromophenyl)isoxazol, 6-bromo-3methylbenzo[d]isoxazol, 5-bromo-3-methylbenzo[d]isoxazol, 4-bromo-5-(4-methoxyphenyl)isoxazol, 3-bromoisoxazol, 3-bromo-5-(2-hydroxyethyl)isoxazol, 4-bromo-5-(4)-fluorophenyl)isoxazol, 3-bromo-5-(4-fluorophenyl)isoxazol, 4-bromo-5-(4-chlorophenyl)isoxazol, 4-bromo-5-(4-bromophenyl)isoxazol, 6-bromo-benzo[d]isoxazol 3-carboxylic acid, benzo[d]isoxazol-3-carboxylic acid, 3-amino-5-methylisoxazol, 5-amino-3-(4-methoxyphenyl)isoxazol, 3-aminoisoxazol, 3-amino-5-(4-fluorophenyl)isoxazol, 5-amino-3-(4-chlorophenyl)isoxazol, 5-amino-4-(4-bromophenyl)isoxazol, 3-amino-5-(4-bromophenyl)isoxazol, 5-acetyl-3-(4-fluorophenyl)isoxazol, 5-acetyl-3(3-fluorophenyl)isoxazol, 3-methyl-5-[(2S)-1-methyl-2-pyrrolidinyl]isoxazolic acid, 7-methoxy-5-methyl-4,5-dihydronaphtho[2,1-d]isoxazolic acid, 5-methyl-3-phenylisoxazolic acid-4-carboxylic acid methylamide, 5-methyl-3-phenyl-isoxazolic acid-4-carbothioic acid methylamide, 5-methyl-3-phenyl-4-(1h-pyrazole-5-yl)isoxazolic acid, 5-benzyl-3-furan-2-yl-2-phenyl-tetrahydro-pyrrolo(3,4-d)isoxazol,4,6-dione,5-benzyl-3-[4-(dimethylamino)phenyl]-2-phenyldihydro-2h-pyrrolo[3,4]-d]isoxazol-4,6(3h,5h)-dione,5-benzyl-3-(5-br-2-hophenyl)-2-ph-tetrahydro-pyrrolo(3,4 d) Isoxazol-4,6-dione, 5-benzyl-3-(4-nitro-ph)-2-ph-dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 5-benzyl-3-(4)-methoxyphenyl)-2-ph-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione, 5-benzyl-3-(4-fluorophenyl)-2-2-methylphenyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-benzyl-3-(3-nitro-ph)-2-phenyl-tetrahydropyrrolo(3,4-d)isoxazol-4, 6-Dione, 5-benzyl-2-ph-3-(2-pyridinyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-benzyl-2-ph-3-(2-ph-vinyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 5-benzyl-2-(4-chlorophenyl)-3-(2-thienyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-benzyl-2,3-diphenyldihydro2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 5-benzyl-2(4(cl-ph)3-(2)-furyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-benzyl-2(4-cl-ph)-3-(4-f-ph)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-(p-tolyl)isoxazol, 5-(4-methylphenyl)-3-(4-nitrophenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4-methoxyphenyl)-2-phenyl-3-(4-pyridinyl)dihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)-dione,5-(4-methoxyphenyl)-2-phenyl-3-(3-pyridinyl)dihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)-dione,5-(4-methoxyph)-2,3-diphenyldihydro-2h-pyrrolo(3,4- d)Isoxazol-4,6(3h,5h)-dione,5-(4-fluorophenyl)-3-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)dione,5-(4-fluorophenyl)-2-(2-methylphenyl)-3-(4-pyridinyl)dihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)dione,5-(4-ethoxyphenyl)-3-(4-nitrophenyl)-2phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)dione,5-(4-ethoxyphenyl)-3-(4-fluorophenyl)-2-phenyldihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)dione5-(4-ethoxyphenyl)-2-methyl-3-(4-nitrophenyl)dihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,5-(4ethoxy)-ph)-2-ph-3-thiophen-2-yl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione,5-(4-cl-ph)-3-(3-nitro)-ph)-2-phenyltetrahydropyrrolo(3,4-d)isoxazol-4,6-dione,5-(4-bromophenyl)-3-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4]-d]Isoxazol-4,6(3h,5h)-dione,5-(4-bromophenyl)-3-(2-furyl)-2-(2-methylphenyl)dihydro-2hpyrrolo[3,4-d]isoxazol-4,6(3h, 5h)-dione, 5-(4-bromophenyl)-2-phenyl-3-(2-pyridinyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4-br-ph)2-ph-3-(2-ph vinyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-(2-cl-ph)-3-(4-dimethylamino-ph)2-0-tolyl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione, 5-(2-chlorophenyl)-3-[4-(dimethylamino)phenyl]-2-phenyldihydro-2h-pyrrolo[3,4- [d]Isoxazol-4,6(3h,5h)-dione,5-(2-chlorophenyl)-3-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,4-((3-(2-cl-ph)-5-methylisoxazol-4-carbonyl)-amino)-ethyl benzoate,4,5,6,6a-tetrahydro-3ah-cyclopenta[d]isooxa Sazol-3-carboxylic acid, 3-phenyl-3a,6a-dihydrothieno[2,3-d]isoxazol4,4-dioxide, 3-methyl-5-(3-phenylpropyl)isoxazol, 3-methyl-4-nitro-5-[(e)-2-phenylethenyl]isoxazol, 3-methyl-4,5,8,9tetrahydrocycloocta(d)isoxazol, 3-methyl-4,5,5a,6a,7,8hexahydrooxyreno(2',3:5,6)cycloocta(1,2-d) Isooxazol, 3-methyl-3 a, 4, 5, 8, 9, 9 a-Hexahydrocycloocta(d)Isooxazol, 3-Fran-2-yl-2-phenyl-5-p-Tolyl-tetrahydropyrrolo(3,4-d)Isooxazol-4,6-dione, 3-Chloro-4,5-dihydro(1)-benzothiepino(5,4-c)Isooxazol, 3-[4-(dimethylamino)phenyl]-5-(4-methoxyphenyl)-2-(2-methylphenyl)dihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)-dione,3-(5-br-2-ho-phenyl)-2,5diphenyl-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione,3-(5-br-2-ho-)ph)-5-(2-cl-ph)-2-ph-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione,3-(4-meo-phenyl)-5-phenyl-2-0-tolyltetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione,3-(4-fluorophenyl)-5-(4-nitrophenyl)-2-phenyldihydro-2h-pyrrolo[3,4 d]]Isoxazol-4,6(3h,5h)-dione,3-(4-fluorophenyl)-2-(4-methylphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,3-(4-dimethylamino-ph)-5-ph 2-o-tolyl-4h-pyrrolo(3,4-d)Isoxazol-4,6-dione,3-(4-fluorophenyl)-5-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,3-(4-br-ph)-2-ph 5-( 2-trifluoromethyl-ph)-4h-pyrrolo(3,4-d)isoxazol-4,6dione, 3-(3-nitrophenyl)-2,5-diphenyl-tetrahydropyrrolo(3,4-d)isoxazol-4,6dione, 3-(3-br-phenyl)-2,5diphenyldihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 3-(3-br-ph)-5-(2-meo-ph)-2-o-tolyl-teto Lahydropyrrolo(3,4-d)isoxazol-4,6-dione, 3-(2-furyl)-5-[4-(4-morpholinyl))phenyl]-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 3-(2-furyl)-2-(2-me-ph)-5-phdihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 3-(2-furyl)-2,5-diphenyldihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 3-(2-cl-phenyl)-5-methylisoxazol-4carboxylic acid (2,5-dichlorophenyl)amide, 3-(2- Cl)-PH)-5-Isoxazol-4-carboxylic acid (4,5-dihydrothiazolol-2-yl)amide, 3-(2-chlorophenyl)-5-methylisoxazol-4-carboxylic acid cyanomethylamide, 3-(2,4-dichlorophenyl)-5-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 3-(2,4-di-cl-PH)2,5-diphenyldihydro-2h-pyrrolo(3,4-d)isoxazol-4,6 (3h,5h)-dione, 3-(2,2-dichloro-vinyl)-5-phenyl-isoxazol, 3,5-diphenyl-isoxazol, 3,5-dimethyl-4-(1-pyrrolidinylsulfonyl)isoxazol, 3(4-dimethylamino-ph)-5-(4-eth-ph)2-o-tolyl-4h-pyrrolo(3,4-d)isoxazol-4,6-2-(4-cl-ph)5-ph-3-(2-thienyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 2-(4-) cl-ph)-5-(3-meo-ph)-3-(3-nitro-ph)-4h-pyrrolo(3,4-d)isoxazol-4,6-dione,2-(4-cl-)ph)-3-(4-meo-ph)-5-p-tolyl-tetrahydropyrrolo(3,4-d)isoxazol-4,6-dione,2-(4-chlorophenyl)-5-(4-methylphenyl)-3-(2-thienyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5)h)-dione,2-(4. Chlorophenyl)-3-[4-(dimethylamino)phenyl]-5phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)dione, 2-(4-chlorophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 2-(4-chlorophenyl)-3-(2-thienyl))-5-[3(trifluoromethyl)phenyl]dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 2-(4-chlorophenyl)-3-(2,4) Dichlorophenyl)-5-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 2,3-di-ph-5-(3-(tri-f-me))ph)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, danazol, and n-cyclopropyl-5-(thiophen-2-yl)isoxazol-3carboxamide. In further embodiments, the isoxazol compound is danazol or n-cyclopropyl-5-(thiophen-2-yl)isoxazol-3carboxamide, and is also a compound of an isoxazol compound having general formula (1): TIFF0007837302000005.tif7395 where A is a heterocyclic group or phenyl which may be substituted with G, G is a halogen, a C1-C6 alkyl, and an optionally substituted phenyl; B is a halogen-substituted phenyl or heterocyclic group or a C1-C6 alkyl; R is hydrogen or a C1-C6 alkyl.

[0189] Divinostat (GIV) is an orally administered, bioavailable hydroxamic acid histone deacetylase (HDAC) inhibitor with potential anti-inflammatory, anti-angiogenic, and antitumor activity. Divinostat inhibits class I and class II HDACs, leading to the accumulation of highly acetylated histones, induction of chromatin remodeling, and alteration of gene expression patterns. At low concentrations that do not induce apoptosis, the agent inhibits the production of inflammatory cytokines such as tumor necrosis factor (TNF-), interleukin-1 (IL-1), IL-6, and interferon-gamma. Divinostat has also been shown to activate the endogenous apoptosis pathway, inducing apoptosis in hepatoma and leukemia cells. Furthermore, the agent exhibits anti-angiogenic activity, suppressing the production of angiogenic factors such as IL-6 and vascular endothelial growth factor (VEGF) by bone marrow stromal cells.

[0190] Rho-related protein kinase (ROCK) inhibitors belong to the serine-threonine kinase family of AGC (PKA / PKG / PKC) and are primarily involved in regulating cell shape and movement by acting on the cytoskeleton. Non-exclusive examples of such inhibitors include thiazovibin or Y27632 (available from Stemcell Technologies), SR3677 (available from tocris.com), and GSK429286 (available from tocris.com).

[0191] TGF-β-1 receptor inhibitors (meaning activin A receptor type II-like kinases) are inhibitors of membrane-bound receptor proteins for the TGFβ superfamily of signaling ligands. TGFBR1 is its human gene. Such non-exclusive examples include SB431542 and A8301 (available from tocris.com and www.esibio.com), LY2157299 (available from www.selleckchem.com), and LY2109761 (available from www.selleckchem.com).

[0192] DNA methyltransferase inhibitors are small molecule compounds or other drugs that have the ability to suppress hypermethylation and can also restore the expression of suppressor genes and exert antitumor effects in in vitro and in vivo laboratory models. Goffin and Eisenhauer (2002) Ann. Oncol. Nov. 13(11):1699 16716. One such non-limiting example is N-phthaloyl-L-tryptophan (C19H14N204, RG108, marketed under the trade name Sigma-Aldrich). Other examples include 5'-azacitidine, an antisense oligonucleotide against 5'-azacitidine methyltransferase 1, such as MG98 (Amato (2007) Clin. Gentrin Cancer, December, 5(7):422-426) and 1-(β-ribofuranosyl)-2(1H)-pyrimidinone (a nucleoside analog of cytidine, marketed under the trade name Zebralin (Abcam®)).

[0193] DNA hypomethylation refers to a lower-than-normal level of DNA methylation.

[0194] Methods for determining the level of DNA methylation are known in the art, some of which are described herein.

[0195] Sulisoxazoles are sulfonamide antimicrobial agents having oxazole substituents and exhibit antibiotic activity against a broad range of Gram-negative and Gram-positive bacteria. Compounds in this class are available from Sigma-Aldrich, and methods for synthesizing them are known in the art, for example, as described in USPat. No. 2, 721, 200. Non-limiting examples of sulisoxazoles include FDA-approved azogantricin, erythromycin ethyl succinate and sulfaxazole acetyl, erysol, gantricin (with SULFISOXAZOLE active ingredient), gantricin (with SULFISOXAZOLE ACETYL active ingredient), pediatric gantricin, iroson sulfa, LIPO gantricin, pediazol, sozol, soxazole, soxazole, sulfisoxazole, sulfisoxazole diolamine, and sulsoxin. (Drugs @ FDA: FDA-approved drugs on the accessdata.fda.org website)

[0196] Danazol (also known as 17a-ethinyl-17β-hydroxyandrost-4-ene-[2,3-d]isoxazol) is a synthetic steroid primarily used to treat endometriosis. This compound is commercially available and manufactured by various companies.

[0197] As used herein, the term “isolated” refers to a molecular or biological or cellular material that is substantially free of other material, for example, more than 70%, 80%, 85%, 90%, 95%, or 98%. In one aspect, the term “isolated” refers to nucleic acids, DNA, RNA, miRNA, exosomes and microvesicles, proteins or polypeptides, cells or organelles of cells, or tissues or organs that have been isolated from other DNA, RNA, miRNA, exosomes and microvesicles, proteins or polypeptides, cells or organelles of cells, or tissues or organs, respectively. They are present in their natural source and enable the manipulation of the material to achieve results that would not be achievable if they existed in their natural or natural state, for example, recombinant replication or manipulation by mutation. The term “isolated” refers to nucleic acids or peptides that are substantially free of their cellular material, viral material, or culture medium when produced by recombinant DNA technology, chemical precursors, or chemical synthesis with other chemicals. Furthermore, “isolated nucleic acids” means that they include nucleic acid fragments that do not exist naturally as fragments and would not be found in their natural state. The term “isolated” is also used herein to refer to polypeptides isolated from other cellular proteins, and therefore means to include both purified and recombinant polypeptides. For example, purity greater than 70%, 80%, 85%, 90%, 95%, or 98%. The term “isolated” also means to refer to other cells, exosomes or microvesicles, cells isolated from miRNA, exosomes or microvesicles, miRNA and tissues, and means to include both cultured and manipulated cells or tissues and products manufactured or isolated from them.

[0198] The term "phenotype" refers to both a measurable trait or characteristic of an individual and a description of an individual trait or characteristic that is expressed only in a subset of individuals within a population. On the one hand, an individual's phenotype includes the phenotype of a single cell, a substantially homogeneous population of cells, a differentiated population of cells, or a tissue consisting of a population of cells.

[0199] The term pharmaceutically acceptable carrier (or medium) is used interchangeably with the term biocompatible carrier or culture medium and refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with cells and other therapeutically administered drugs, but are also suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other complications commensurate with a reasonable benefit / risk, within the bounds of sound medical judgment. pharmaceutically acceptable carriers suitable for use in the present invention include liquids, semi-solids (e.g., gels), and solid materials (e.g., cell scaffolds and matrices, tube sheets, and other such materials known in the art and described in more detail herein). These semi-solid and solid materials are designed to withstand degradation in the body (non-biodegradable) or to degrade in the body (biodegradable, bioerosive). Biodegradable materials may further be bioabsorbable or bioresorbable. For example, it may dissolve and be absorbed. They degrade and are eventually eliminated from the body through either bodily fluids (water-soluble implants being one example), conversion into other substances, or decomposition and elimination through natural pathways.

[0200] It is one of several cell populations, exosomes, or microvesicles, or a collection of miRNAs, that are phenotypic and / or genotype-identical (cloned) or non-identical. The population may be purified, highly purified, substantially homogeneous or heterogeneous, as described herein.

[0201] The term “proliferation” means increasing or altering the phenotype of a cell or cell population. The term “growth” refers to the proliferation of cells in the presence of a supporting medium, nutrients, growth factors, supporting cells, or any chemical or biological compounds necessary to obtain a desired number of cells or cell type. In one embodiment, cell proliferation results in tissue regeneration. In yet another embodiment, the tissue consists of cardiac progenitor cells or cardiac cells.

[0202] The term "effective dose" refers to the concentration or amount of a reagent or composition, cell population or other pathogen, such as the compositions described herein. This concentration or amount is effective in achieving the intended result, such as cell proliferation or differentiation, in vitro or in vivo, and may be for the treatment of the conditions described herein. Naturally, the number of cells administered will vary depending on the details of the disorder being treated (including, but not limited to, the size or total volume / surface area treated), other factors well known to medicinal biologists, and the proximity of the administration site to the area being treated.

[0203] The terms "effective period (or time)" and "effective conditions" refer to the period or other controllable conditions (e.g., temperature, humidity in the case of an in vitro method) that are necessary or preferred for a drug or composition to achieve its intended result. For example: differentiation or dedifferentiation of cells into a given cell type.

[0204] The terms “subject,” “individual,” or “patient” are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, rodents, rats, monkeys, cattle, dogs, cats, humans, livestock, sports animals, and pets.

[0205] "Substantially homogeneous" refers to a population of cells in which approximately 50%, or more than 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells have the same or similar phenotype. The phenotype may be determined by pre-selected cell surface markers or other markers.

[0206] As used herein, the terms “to treat,” “to cure,” etc., are used herein to mean obtaining a desired pharmacological and / or physiological effect. An effect can be preventive in that it completely or partially prevents a disease or its signs or symptoms, and / or therapeutic in that it partially or completely cures a disorder and / or adverse effect resulting from the disorder. Examples of “treatment” include, for example: preventing the occurrence of a disorder in a subject who may be predisposed to the disorder but has not yet been diagnosed with it; suppressing a disorder, e.g., inhibiting its development; and / or alleviating or improving the symptoms of a disorder (e.g., cardiac arrhythmia). As will be understood by those skilled in the art, “treatment” may include systemic improvement of symptoms related to a pathology and / or delaying the onset of symptoms such as chest pain. The clinical and subclinical evidence of “treatment” varies by pathology, individual and treatment.

[0207] The “administration” or “delivery” of cells, exosomes or microvesicles, mirNAs, therapeutic agents or other drugs, and compositions containing them, can be achieved by continuous, intermittent, or single doses throughout the course of treatment. Methods for determining the most effective means of administration and dosage are known to those skilled in the art and will vary depending on the composition used in treatment, the purpose of treatment, the target cells to be treated, and the subject being treated. Single or multiple doses can be performed at dose levels and patterns selected by the treating physician, or in the case of animals, by the treating veterinarian. Appropriate dosage forms and methods for administering the agent are known in the art. The route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art. Furthermore, these methods will vary depending on the composition used in treatment, the purpose of treatment, the health status and stage of the disease of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, intraperitoneal administration, infusion administration, nasal administration, inhalation, injection, and topical administration.

[0208] [Descriptive mechanism] induced pluripotent stem cells This disclosure provides induced pluripotent stem cells (iPSCs) characterized by DNA hypomethylation. On the one hand, the cells are further characterized by the overexpression of one or more cardiac genes or markers selected from the group of Gai, mir-133, mir-762, CCL7, CXCR2, CXC5, intrinsic membrane protein 2A, and ephrin A3, Nkx 2, 5, ISL1, GATA4, AMHC, Sarcomeric actin, Gai, mir-133, mir-762, CCL7, CXCR2, CXC5, and ephrin A3. On the other hand, the cells below express one or more pluripotency genes or markers. Such non-limiting examples include one or more mir-290-295 clusters, let-7 family, Max, and one or more DNA methyltransferase genes Dnmt1, Dnmt3b. In one embodiment, one or more cardiac genes or markers are Gai. In one embodiment, one or more cardiac genes or markers are overexpressed at least 1.5-fold, or 2-fold, or 3-fold, compared to control cells. Alternatively, the cells are characterized by low expression of one or more other cardiac genes or markers selected from the group of miR-290 cluster, miR-574-5P, let-7 family, Dnmtl, Dnmt3b, and Max. In one embodiment, one or more cardiac genes or markers are not expressed or are underexpressed at least 1.5-fold, 2-fold, or 3-fold, compared to control cells. Methods for identifying and quantifying genes and markers are known in the art and are described herein to supplement these well-known methods.

[0209] The cells may originate from any animal species, such as mammals. For example: horses, mice, cows, dogs, cats, or human patients.

[0210] iPS cells are derived from any suitable parent cells. Such non-limiting examples include, but are not limited to, cells selected from the group consisting of bone marrow cells, myoblasts, cutaneous fibroblasts, umbilical cord blood cells, adult peripheral blood, cardiac progenitor cells, young juvenile stem cells (SJSTs), bone marrow stromal cells, mesenchymal stem cells, hematopoietic stem cells, and mononuclear cells.

[0211] Methods for inducing or preparing iPSCs from these parental cell types are known in the art. On the one hand, iPSCs were prepared by a method that involves contacting parental cells with an effective amount of a DNA methyltransferase inhibitor to upregulate Oct4. Limited examples of non-DNA methyltransferase inhibitors include 5'-azacitidine, 5-aza-2'-deoxycytidine, MG98, zebralin, and RG108. On the other hand, iPS cells were prepared by a method that eliminates the insertion of exogenous genes into parental cells.

[0212] In further embodiments, the parental cells are progenitor cells (e.g., cardiac progenitor cells) prepared by pre-treating cells with electrical stimulation. Alternatively, the parental cells are stem cells expressing Sca1 and pluripotency and cardiac genes, as described herein. The cells are then brought into contact with an isoxazole or isoxazole analog compound as described herein.

[0213] On the one hand, the parental cells are small immature or young stem cells that have not been modified or programmed to become iPSCs. In this case, SJSTs are treated with isoxazole or an isoxazole analogue and used for diagnostic, research, or therapeutic purposes. On the other hand, immature or progenitor cells are directed toward a cardiac precursor phenotype by contacting them with an effective amount of the isoxazole or isoxazole analogue described herein. For example, SJST cells, circulating blood, or heart-derived stem cells. Also, the parental cells are immature or young stem cells that can be identified by low expression of miR-195 compared to cells that do not express the characteristics of young or immature cells as described herein. Furthermore, the parental cells are further characterized by low expression of one or more markers selected from miR-29b, miR-205, miR-378, and miR-542-3p compared to cells that do not express the characteristics of young or immature cells as described herein. Alternatively, the parental cells were pretreated with an effective amount of electrical stimulation. On the one hand, isoxazole or isoxazole analogues are therapeutically injected into the ischemic heart prior to treatment for maximum drug contact to induce regeneration, where BMSCs, skeletal myoblasts, peripheral blood-derived endothelial progenitor cells (EPCs), resident cardiac stem cells / progenitor cells, and fibroblasts are mobilized prior to treatment (Konopliannikov MI, Haider KH, Lai VK, Ahmed RP, Jiang S, Ashraf M, Activation of diverse signaling pathways by extracellular delivery of multiple cytokines for myocardial repair, Stem Cells Dev. 2013 Jan. 15; 22 (2): 204-15).

[0214] The chemically modified iPS cells described herein are prepared by contacting cells with an effective amount of isoxazole or an isoxazole analog. For example, amounts selected from the group of about 0.3 to about 30 μM; about 0.5 to about 25 μM; about 12 to about 25 μM; and about 0.5 μM to about 20 μM. See also Figure 14C.

[0215] While methods for preparing iPS cells are known in the art, the applicant has determined that iPS cells produced by a method involving contacting parental cells with an effective amount of a DNA methyltransferase inhibitor, such as 5'-azacitidine or RG108 (Sigma-Aldrich), are particularly useful for the upregulation of Oct4. On the other hand, iPS cells can be further prepared by a method that eliminates the insertion of exogenous genes into parental cells, thereby increasing the safety of the cells for clinical use. Furthermore, cells can be modified into iPS cells by the insertion of genes and other factors known in the art.

[0216] This disclosure also provides populations of cultured cells described herein by culturing chemically modified cells to grow the cells described herein. On the one hand, the population is substantially homogeneous; for example, at least 70%, 75%, 80%, 85%, 90% identical in phenotype, or at least 95%, 98%, identical in phenotype, or a clone population of cells. On the one hand, the population is a clone population. On the other hand, the cells are cultured under conditions that promote differentiation into a specific cell type, such as cardiac cells. These culture conditions are known in the art.

[0217] Cells and cell populations can be further modified for therapeutic or research applications, for example, by further incorporating detectable labels or exogenous polynucleotides or polypeptides. Methods and suitable polynucleotides for therapeutic use are described in Durrani et al. (2010) Regen. Med. 5 (6): 919-932.

[0218] [Method for preparing iPS cells] This disclosure also provides a method for preparing cardiac lineage cells from stem cells, comprising contacting stem cells with an effective amount of isoxazole or an isoxazole analog. Examples of stem cells useful in this method include, but are not limited to, one or more iPS cells such as bone marrow cells, bone marrow stromal cells, mesenchymal stem cells, hematopoietic stem cells, myoblasts, dermal fibroblasts, umbilical cord blood cells, adult peripheral blood-derived iPS cells, blood, SJSCs, cardiac progenitor cells, or mononuclear cells. Methods for inducing or preparing iPSCs from these parental cell types are known in the art. Cells prepared by this method are characterized by overexpression of one or more cardiac genes or markers, such as: Nkx-2.5, ISL1, Tbx-5, GATA4, AMHC, Sarcomeric actin, Gai, miR-133, miR-762, CCL7, CXCR2, CXC5, endogenous membrane protein 2A, or ephrin A3. Furthermore, these cells underexpress one or more cardiac genes or markers compared to control cells, such as cells that have not been in contact with or exposed to isoxazole or isoxazole analogues. For example: miR-290-295 cluster, let-7 family, Dnmt1, Dnmt3b, and Max. On the other hand, overexpression or underexpression is at least 1.5 times more or less than that of control cells.

[0219] On the one hand, the cells are small, immature stem cells (SJSCs) that have not been modified or programmed to become iPSCs. In this case, the SJSCs are treated with isoxazole or isoxazole analogs and used diagnostically, in research, or therapeutically. The disclosure also provides a method for directing immature or progenitor cells (such as SJST cells, including a population of stem cells derived from circulating blood or the heart) to a pre-cardiac progenitor phenotype by contacting the cells with an effective amount of isoxazole or isoxazole analog as described herein. An effective amount of electrical stimulation can be applied to the cells or tissues requiring treatment, either alone or in combination with isoxazole or isoxazole analogs. The parental cells are selected for chemical modification, characterized by low miR-195 expression and whether or not they have been pre-conditioned by the application of an effective amount of electrical stimulation.

[0220] In certain embodiments, this method involves contacting cells with an effective amount of isoxazole or an isoxazole analog for at least 3, 4, 5, 6, or 7 days to supplement DMEM with approximately 10% to approximately 30%, or 20%, of a knockout serum substitute (KSR; Invitrogen, USA). The mixture contains: F12; approximately 0.05 mM to approximately 0.2 mM or approximately 0.1 mM MEM non-essential amino acid solution (Invitrogen, California, USA); approximately 0.1 mM to approximately 0.3 mM or approximately 0.2 mM L-glutamine (Invitrogen, USA); approximately 0.05 mM to approximately 0.2 mM or approximately 0.1 mM β-mercaptoethanol (Invitrogen, CA, USA); approximately 750 U / ml to approximately 1250 U / ml or approximately 1000 U / ml LIF (Millipore); and effective amounts of 0.5% penicillin and streptomycin. Cells are then retained in the medium for 5 days without the drug. Otherwise, the medium is changed daily for cell number increase, the pH of the medium changes, and this affects cell survival and gene expression. An effective amount of isoxazol or isoxazol analogue contains about 0.3 to about 30 μM, about 0.5 to about 25 μM, about 12 to about 25 μM, or about 0.5 μM to about 20 μM. When used herein, isoxazol or isoxazol analogue refers to the class of compounds described herein.

[0221] In another specific aspect, the method comprises contacting cells with an effective amount of isoxazole or isoxazole analogue in insulin-free RPMI F12 for at least 3 days, or 4, 5, 6, or 7 days. The effective amount of isoxazole or isoxazole analogue includes about 0.3 to about 30 μM, about 0.5 to about 25 μM, about 12 to about 25 μM, and about 0.5 μM to about 20 μM. In yet another specific aspect, isoxazole is ISX-9 as described above herein. Where used herein, isoxazole or isoxazole analogue is intended to refer to the class of compounds described above.

[0222] Next, the cells are kept in RPMI F12 with insulin for approximately 7-10 days to induce cardiomyocyte differentiation. Then, the cells are kept in EGM-2 medium (Lonza, Lonza Walkersville Inc., Walkersville Md. 21793 0127) for approximately 10 days to induce endothelial cell differentiation. Finally, the cells are kept in TGFB (2 ng / ml) and PDGFBB (long / ml, R & D Systems, Inc., Minneapolis, Minn. 55413) for approximately 10 days to induce smooth muscle cell differentiation.

[0223] Methods for identifying and quantifying genes and markers are known in the art and are described herein to supplement these well-known methods.

[0224] The effectiveness of the method disclosed herein can be determined by molecular, clinical, and other techniques. On the one hand, Nkx2.5, GATA4, Tbx5, ISL-1, and Mef2c upregulation were initiated 3 days after contact, and the expression of these markers can be determined by applied histochemistry and / or PCR, if necessary. Additional lineage identification markers are shown in Figure 14C. Cells can be further evaluated for 7 days of treatment by qPCR. Immunofluorescence staining also showed that transcription factors (Nkx2.5, GATA4, and ISL-1) were highly expressed in hiPSCs after 7 days of treatment with small molecule compounds. The purity of Nkx2.5-positive cells in these small molecule compound-treated cells by FACS was 96.5 ± 2.5% cells. Establishing its Nkx2, the 5+ cells were cardiovascular progenitor cells, and the applicant found these to be Nkx2. The 5+ cells were pluripotent and differentiated directly into all three cardiovascular lineages. These include CM, EC, and SMC under basal differentiation conditions without specific induced signaling molecules, in proportions of 95.2% + 2.1% CMS (TnT+), 90.3% + 2.5% EC (CD31+), and 92.3% + 1.8% SMC (Q-SMA+). Furthermore, the applicant determined that differentiated ECs exhibited similar phenotypes and functions to primary endothelial cells (ECS). (Figures 14A-14H)

[0225] The cells may originate from any animal species, such as mammals, e.g., horses, mice, cattle, dogs, cats, or human patients. The cells may be autologous or allogeneic to the animal or patient being treated.

[0226] Contact can be made in vitro (for example, in a tissue culture dish or plate as described herein) or by administering an effective amount of isoxazole or an isoxazole analog to a cell culture in vivo, or by administering an effective amount of the compound, or the compound and iPS cells, in vivo to a patient or subject requiring such treatment. Methods of systemic or topical administration are described below. The compound and / or cells can be combined with a pharmaceutically acceptable carrier for ease of use. In one embodiment, this treatment can be combined with the administration of an effective amount of electrical stimulation to the tissue requiring such treatment. The electrical stimulation can be administered before, simultaneously with, or after the administration of the isoxazole or isoxazole analog.

[0227] On the one hand, stem cells that come into contact with a compound are iPS cells. Methods for producing iPS cells from terminally differentiated cells are known in the art. For example, one method involves contacting parental cells with an effective amount of a DNA methyltransferase inhibitor (such as RG108 (Sigma-Aldrich)) to upregulate Oct4. Alternatively, iPS cells can be produced by methods that eliminate the insertion of exogenous genes into parental cells.

[0228] This disclosure also provides isolated cells prepared by the method herein by further comprising isolating the cells. The method further comprises culturing the cells to prepare a cell population. On the one hand, the population is cultured under conditions for preparing something substantially homogeneous, for example, at least 70%, 75%, 80%, 85%, 90%, or 95%, 98% identical in phenotype. On the other hand, the method further comprises culturing the cells under conditions favorable for the cloning of the cells into a cloning population. The cells are cultured under conditions that promote differentiation into a specific cell type, for example, cardiac cells. These culture conditions are known in the art.

[0229] This method can be further modified by inserting detectable labels or exogenous polynucleotides or polypeptides into cells and cell populations. Methods and suitable polynucleotides for therapeutic use are described by Durrani et al. (2010) Regen. Med. 5 (6): 919 932.

[0230] [Collection of exosomes or microvesicles and composition of exosomes or microvesicles] This disclosure provides a composition comprising an isolated population of exosomes or microvesicles that overexpress exosomes or microvesicles selected from the group consisting of mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-21, mir-30c, mir-214, or mir-548q, or from one or more proteins selected from the group consisting of Tsg101, CD9, Hsp70, Flotilline 1, or GAPDH. The population also comprises two or more, or three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve miRNAs, and / or at least one, two, three, four, or all five proteins. Furthermore, in individuals with exosomes or microvesicles, at least 80%, 85%, 90%, 95%, 98%, or 100% of the exosomes or microvesicles overexpress miRNAs and / or proteins. Population exosomes or microvesicles are isolated from cells selected from cardiac progenitor cells ("CPCs"), cardiomyocytes, endothelial cells, myocytes, or smooth muscle cells.

[0231] Also provided are substantially or substantially comprising cells, cell populations, or compositions comprising one or more isolated populations of the following: cardiomyocyte progenitor cells (CPCs), cardiomyocytes, myocytes, endothelial cells, smooth muscle cells, and skeletal muscle cells generated from cells selected from the following groups: induced pluripotent stem cells (iPSCs), embryonic stem cells, or stem cells in contact with isoxazole compounds or their derivatives or equivalents. The cells or cell populations are identified by the overexpression of microRNAs (miRNAs) selected from the following groups: mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-21, mir-30c, mir-214, mir-548q; and / or the overexpression of muscle genes selected from the following groups: paZ3, PAX7, MYF5, MYOD, MYOG, or dystrophin. In yet another aspect, provided herein are cardiomyocytes or populations of cardiomyocytes expressing one or more selected from cTnT, cTnI, MLC2V, and / or CX43. Further provided are populations of cells or endothelial cells expressing CD31 and VE-cadherin. And further provided are smooth muscle cells or populations of these cells expressing smooth muscle actin (SMA) and calponin.

[0232] Furthermore, the populations described herein include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or a total of eleven miRNAs, or include at least one, two, three, four, five, or a total of six muscle genes. Alternatively, for individuals of exosomes or microvesicles, at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the exosomes or microvesicles overexpress miRNAs and / or genes and proteins.

[0233] Furthermore, this specification also provides isolated cell populations expressing one or more proteins selected from the group cTnl, MLC2v, cTnT, VE-cadherin, CD31, A-SMA (actin), calponin, and Cx43, where, in some cases, at least two, three, four, five, six, seven, or eight of all expressed proteins. On the other hand, the cell population is selected from the group consisting of smooth muscle cells, endothelial cells, vascular cells, or cardiomyocytes.

[0234] In another embodiment, at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the exosomes or microvesicles in a cell population express proteins. Methods for identifying protein expression are known in the art and include hybridization techniques that monitor mRNA expression and utilize protein-specific antibodies. Populations can be prepared by methods that include culturing a population of iPSCs in the presence of an effective amount of isoxazole, its derivatives, or equivalents. Furthermore, populations can be homogeneous, heterogeneous, purified, highly purified, homogeneous, or substantially homogeneous, derived from fibroblasts or skeletal myoblasts. Furthermore, exosomes or microvesicles can be labeled for detection and / or combined with carriers, preservatives, or cryoprotectants. Populations and compositions can be lyophilized. Methods for isolating populations of exosomes or microvesicles and producing isolated populations of cells are known in the art and some of them are described herein. In a further embodiment, populations are derived from fibroblasts or skeletal myoblasts.

[0235] The cell population consists of smooth muscle cells, endothelial cells, and cardiomyocytes. As used herein, "smooth muscle cells" refers to cells expressing markers selected from the following groups: SMTN (smooth muscle cells), Cnn1 (calponin 1), and telokine. As used herein, "endothelial cells" refers to cells expressing markers: VE-cadherin (CD144), CD31, and von Willebrand factor (vWF).

[0236] The composition may further include proteins that promote tissue regeneration and / or improved function, and / or nucleic acids that code for those proteins, and / or agents that inhibit the expression of inflammatory proteins, and optionally cytokines. The proteins are selected from the group consisting of transforming growth factor beta (TGF-beta), WNT proteins, cytokines, or histone deacetylases.

[0237] In one aspect, cells are cultured in serum-free medium. In another aspect, the effective amount of the isoxazole compound, its derivatives, or equivalents is the amount that results in overexpression of mir. In one aspect, the effective amount is about 5 μM to about 25 μM, and in other aspects, the effective amounts are 8 to 20, or about 10 to about 15, or about 5 to about 20 μM, and about 10 μM to about 25 μM.

[0238] These compositions are useful in one or more of the following ways: to regenerate damaged tissue such as muscle and / or cardiac tissue; to improve the survival rate of damaged tissue such as muscle and / or cardiac tissue; to promote the formation of new tissue such as cardiac tissue, muscle tissue, skeletal muscle tissue, blood vessels, capillaries, and muscle cells; to promote cardiac regeneration; to promote cardiac regeneration in subjects suffering from acute cardiac events; to promote cardiac regeneration in subjects suffering from myocardial infarction; and in relation to Duchenne muscular dystrophy (DMD) or Duchenne muscular dystrophy-"DMD" Promote cardiac regeneration in subjects with cardiomyopathy; for example, promote cardiac regeneration in subjects with age-related diseases such as chronic obstructive pulmonary disease ("COPD"), arthritis, osteoporosis, osteoarthritis, diabetes, vascular dementia, or macular degeneration; promote tissue regeneration in tissue damaged by one or more of the following: stroke, arthritis, Alzheimer's disease, memory impairment, cystic fibrosis, inflammatory disorders, and cancer; reduce the thickness of the cardiac wall in tissue damaged by myocardial infarction; protein kinase C, iL-6, mmp, PDGF The method involves altering the expression of one or more genes; reducing or inhibiting the expression of inflammatory proteins, which may be chemokines, macrophages, or cytokines; directly or indirectly stimulating angiogenesis; promoting cardiac regeneration in subjects suffering from diseases selected from the following groups: coronary artery disease, myocardial infarction, heart failure, hypoplastic left heart syndrome, peripheral artery disease (PAD), cardiac hypertrophy, valvular heart disease (aortic stenosis), myocardial hypertrophy, hypertrophic fibrosis; and / or directly or indirectly inhibiting cell replication. The method is achieved by administering an effective amount of exosomes or microvesicles, cells, populations, or compositions containing them to a subject in need. The method may further include administering an effective amount of non-embryonic stem cells or progenitor cells to a subject, which may be the same or a different type of tissue in need of repair. On the one hand, the non-embryonic stem cells or progenitor cells are autologous to the subject. On the other hand, the non-embryonic stem cells or progenitor cells are allogeneic to the subject. Exosomes or microvesicles, cells, or populations can be delivered to the subject locally or systemically, for example, intramyocardial or intracoronary administration.Subjects that can be appropriately treated by these methods include animals, mammals, and human patients. Methods for determining the effectiveness of treatment are known in the art, and some of them are described herein.

[0239] This composition is also useful in methods for one or more of the following therapeutic treatments: providing antioxidant therapy; promoting the activation of local or resident cardiomyocytes; promoting angiogenesis and / or the release of paracrine factors; promoting the activation of WNT, BMP, and / or cytoskeletal remodeling; promoting EMT signaling and cardiac differentiation that facilitates TGF-B; increasing the expression of WNT5 and WNT11, or BMP family proteins, and BMP4 if necessary; increasing the expression of cardiac transcription factors selected from the group consisting of NKX2.5, MEF2C, Gata4, and ISL-1; promoting the development of PIP3 signaling in cardiomyocytes, and gene expression in muscle contraction and hypertrophy signaling pathways; reducing fibrosis and apoptosis; promoting myogenesis and muscle differentiation; and promoting the expression of angiopoietin-2, IL-6NMP, PGFBB, and TIMP. 1. To promote the release of cytokines selected from the group consisting of genes identified in the figures disclosed herein; to promote the upregulation of genes selected from the group consisting of wnt3a, wnt5a, and wnt11; and to promote cytoskeletal remodeling by administering an effective amount of exosomes or microvesicles, cells, populations, or compositions to subjects in need thereof.

[0240] Furthermore, the provided offering is an isolated population of cells expressing one or more proteins selected from the group consisting of cTn1, MLC2v, cTnT, VE-cadherin, CD31, α-SMA (actin), calponin, or Cx43.

[0241] On the one hand, the composition further comprises a protein or nucleic acid that codes for such a protein that promotes tissue regeneration and / or improved function. Non-limiting examples of such proteins include, for example, Bmp7 for the kidney, Fgf-21 for the liver, Op-1 for bone regeneration, and the like. Furthermore, the composition further comprises agents that inhibit the expression of inflammatory proteins. Non-limiting examples of these agents include, for example, phospholipase A2 inhibitors, p38 mitogen-activated protein (MAP) kinase inhibitors, and the like. In a further embodiment, the protein is a cytokine, and non-limiting examples of such cytokines include interleukins and interferons.

[0242] On the one hand, the group described herein further comprises a carrier, which in one embodiment is a carrier not found in nature. On the other hand, the group further comprises a preservative or antifreeze agent, such as polyethylene glycol.

[0243] In a further embodiment, the population described herein is freeze-dried.

[0244] Furthermore, the compositions described herein are isolated from a population of stem cells or progenitor cells cultured in the presence of an effective amount of an isoxazole compound or its derivative. The isoxazole compound is selected from isoxazole-1 (ISX-1) or isoxazole-9 (ISX-9). The isoxazole derivative has the following formula: In formula TIFF0007837302000006.tif91143, both R1 and R2 are hydrogen, or R1 is hydrogen and R2 is selected from the group consisting of substituted or unsubstituted C, or R1 and R2 together may form a ring selected from azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl; R2', Rz and R4 are independently selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C6 cycloalkyl, substituted or unsubstituted aromatic or heteroaromatic ring, cyano, nitro, and acyl. X is 0, NH or S; and Y is 0, NH or S.

[0245] Furthermore, isoxazole compounds have the following formula: In the formula TIFF0007837302000007.tif77129, R1 and R2 are selected from C1-C4 alkyl, phenyl, benzyl, trifluoromethyl, or halogen, R3 is selected from hydrogen, hydroxy, C1-C4 alkyl, or alkoxy, and R4 at position 3 or 5 is selected from hydrogen, trifluoromethyl, C1-C4 alkoxy, C1-C4 alkyl, or C1-C4 hydroxyalkyl. R5 is selected from hydrogen or C4-C4 alkyl, or R4 and R5 together form a tetramethylene group. Z at position 3 or 5 on the heterocycle is selected from -N(R6)-CO-, -CON(RF)-, NORG)-CO-NRG)-, CH(RF)-NHCO, or -NH-COCH(R6), where R6 is selected from hydrogen or C1-C4 alkyl.

[0246] In a further embodiment, isoxazol compounds or derivatives thereof are selected from the following group: 5-(trifluoromethyl)-3-(4-methoxyphenyl)isoxazol-4-carboxylic acid, 5-(trifluoromethyl)-3-(4-fluorophenyl)isoxazol-4-carboxylic acid, 5-(thiophen-2-yl)isoxazol-3-carboxyaldehyde, 5,6,7,8-tetrahydro-4h-cyclohepta[d]isoxazol-3-carboxylic acid, 4,5,6 ,7-tetrahydro-benzo[d]isoxazole-3-carboxylic acid, 3-amino-5-methylisoxazole, 4-amino-n-(5-methyl-3-isoxazolyl)benzenesulfonamide, 3-phenylisoxazole-5-boronic acid pinacol ester, 5-phenylisoxazole, 1-phenyl-1-cyclopentanecarboxylic acid, 3-phenyl-benzo[c]isoxazole-5-carboxylic acid, 5-methyl-3-phenylisoxazole-4-carboxylic acid, 3 a, 4,5,6,7,8,9,9 a-Octahydrocycloocta[d]isoxazol-3-carboxylic acid, 5-(3-nitrophenyl)isoxazol, 3-(4-nitrophenyl)isoxazol, 3-hydroxy-5-aminomethylisoxazol, 5-(morpholinomethyl)isoxazol-3-carboxyhydrochloride, 5-morpholinomethyl)isoxazol-3-carbaldehyde, 3-methyl-5-(trifluoromethyl)isoxazol-4-carboxylic acid, 5-(thiophen-2-yl)isoxazol-3-carboxylate methyl, 3-(methylsulfonyl))-5-(2-thienyl)isoxazol-4-carbonitride, 5-methyl-3-(2-pyrrolidinyl)isoxazol, 3-methyl-5-(2-pyrrolidinyl)isoxazol, 3-(1-methyl-1h-)pyrazole-4-yl)-isoxazol-5-carboxylic acid, 3-(1-methyl-1h-pyrazole-4-yl)-4,5-dihydroisoxazol-5-carboxylic acid, 5-(4-methylphenyl)isoxazol-3- Carboxylic acid, 5-methyl-3-phenylisoxazo-l-4 carboxylic acid, 5-(4-methylphenyl)isoxazo-l-3 carboxaldehyde, 5-methyl-3-(4-phenoxyphenyl)isoxazo-l-4 carboxylic acid, 3-methyl-5-(4-methyl-1,2,3-thiadiazo-l-5-yl)isoxazo-l-4 carboxylic acid, 3-methyl-5-(5-methylisoxazo-l-3-yl)isoxazo-l-4 carboxylic acid, 5-(4-methoxyphenyl)isoxazol-4-carboxylate methyl, 5-(4-methoxyphenyl)isoxazol-3-carboxylate methyl, 5-methylisoxazol, 5-(4-fluorophenyl)isoxazol-4-carboxylate methyl, 5-(4)methyl-fluorophenyl)isoxazol-3-carboxylate, methyl 5-(4-chlorophenyl)isoxazol-4-carboxylate, methyl 5-(4-bromophenyl)isoxazol-4-carboxylate, 5-(4-methoxyphenyl)isoxazol-3-carboxylate. 5-(3-methoxyphenyl)isoxazol-3-carboxylic acid, 3-(2-methoxyphenyl)isoxazol-5-carboxylic acid, 5-(4-methoxyphenyl)isoxazol-3-carboxaldehyde, 3-(4-methoxyphenyl)isoxazol-5-carbaldehyde, 3-(2-methoxyphenyl)isoxazol-5-carbaldehyde, 5-(4-methoxyphenyl)isoxazol, 3-(4-methoxyphenyl)isoxazol,3-(2-methoxyphenyl)-4,5-dihydro-isoxazol-5-carboxylic acid, 3-methoxy-isoxazol-5-carboxylic acid, isoxazol-5-carboxylic acid, isoxazol-4-carboxylic acid, isoxazol-5-carbothioamide isoxazol-5-carbonyl chloride, isoxazol-3-carbonitrile, isoxazol-3-carbaldehyde, isoxazol-4-boronic acid, isoxazol, 5-cyclopropyl-4-[2-(methylsulfonyl)-4-(trifluoromethyl [Isooxazol] 6,-(5-(thiophen-2-yl)isoxazol-3-carboxamide)hexyl 5-((3as,4s,bar)-2-oxohexahydro-1h-thieno[3,4-d]imidazol-4-yl)pentanoethisocarboxazide 5-methyl-3-isoxazol-carboxylic acid 2-benzylhydrazide, 5-isobutylisoxazol-3-carboxylic acid, 4-iodo-5-methylisoxazol, 3,3'-iminobis(n,n-dimethylpropylamine),3. -(3-hydroxyphenyl)-isoxazol-5-carboxylic acid methyl ester, 5-(4-hydroxyphenyl)-isoxazol-3-carboxylic acid, 5-(3-hydroxyphenyl)-isoxazol-3-carboxylic acid, 5-(hydroxymethyl)-3-methylisoxazol, 3-hydroxy-5-methylisoxazol, 5-(1-hydroxyethyl)-3-(4-trifluoromethylphenyl)isoxazol, 3 a, 4, 5, 6, 7, 7α-Hexahydro-benzo[d]isoxazol-3-carboxylic acid, 5-(2-furyl)isoxazol-3-carbaldehyde, 5-furan-2-ylisoxazol-3-carboxylic acid, 6-fluoro-3-(4-piperidinyl)benzisoxazol, 5-(4-fluorophenyl)isoxazol-3-methol, 3-(2-fluorophenyl)isoxazol-5-carboxylic acid, 5-(4-fluorophenyl)isoxazol-3-carboxyaldehyde, 3-(4-fluorophenyl (Phenyl)isoxazol-5-carbaldehyde, 3-(3-fluorophenyl)isoxazol-5-carbaldehyde, 3-(2-fluorophenyl)isoxazol-5-carbaldehyde, 5-(4-fluorophenyl)isoxazol, 3-(4-fluorophenyl)isoxazol, 5-(3-fluoro-4-methoxyphenyl)isoxazol-3-carboxylic acid, ethyl 5-(trifluoromethyl)-3-(4-methoxyphenyl)isoxazol-4-carboxylate, ethyl-5 (Tributylstanyl)isoxazol-3-carboxylate, ethyl 5-(thiophen-2-yl)isoxazol-3-carboxylate, 5-ethyl-isoxazol-4 carboxylic acid, 5-ethyl-isoxazol-3-carboxylic acid, ethyl 5-(4)-fluorophenyl)isoxazol-4-carboxylate, ethyl 5-(4-fluorophenyl)isoxazol-3-carboxylate, ethyl 5-(2,3-dihydrobenzo[b][1,4]dioxin-7-yl)isoxazol -L-3-carboxylate, ethyl 3-(4-chlorophenyl)-5-(trifluoromethyl)isoxazo-L-4-carboxylate, ethyl 5-(4-chlorophenyl)isoxazo-L-3-carboxylate, ethyl 3-(4-bromophenyl)-5-(trifluoromethyl)isoxazo-L-4 carboxylate, ethyl 5-(4-bromophenyl)isoxazo-L-3-carboxylate, ethyl 5-amino-4-(4-chlorophenyl)isoxazo-L-3-carboxylate, eth 5-amino-4-(4-bromophenyl)isoxazo-L-3-carboxylate ethyl, 6b-acetyl-2-(acetyloxy)-4a, 6a-dimethyl-2,3,4,4a,4b,5,6,6a,6b,9a,10,102,10b,11-tetradecahydro-1h-naphtho[2',1':4,5]indeno[2,1-d]isoxazol-9-carboxylate,3,5-dimethyl-4- (Tributylstanyl)isoxazol, 5-(1,5-dimethyl-1h-pyrazole-4-yl)-isoxazol-3-carboxylic acid, 5-(1,3-dimethyl-1h-pyrazole-4-yl)-isoxazol-3-carboxylic acid, 5-(1,5-dimethyl-1h-pyrazole-4-yl)-isoxazol, 3,5-dimethylisoxazol-4-boronic acid pinacol ester, 3,5-dimethylisoxazol, 3-(dimethylamino)-1-(2)-pyridyl)-2propen-1-1, 5-(3,5-difluorophenyl)isoxazol, [2,6-dichloro-4-(trifluoromethyl)phenyl]hydrazine, 5-(2,5-dichlorophenyl Nyl)isoxazol-3-carboxylic acid, danazol, 3-(4-chlorophenyl)-5-(trifluoromethyl)isoxazol-4-carboxylic acid, 5-(4-chlorophenyl)isoxazol-3-propionic acid, 5-(4-chlorophenyl)isoxazol-4-carboxylic acid, 5-(4-chlorophenyl)isoxazol-3-carboxylic acid, 3-(4-chlorophenyl)isoxazol-5-carboxylic acid, 3-(3-chlorophenyl)isoxazol-5-carboxylic acid, 5-(4-chlorophenyl)isoxazol-3-carboxyaldehyde, 3-(4-chlorophenyl)isoxazol-5-carbaldehyde, 3-(3-chlorophenyl) )Isoxazol-5-carbaldehyde, 3-(2-chlorophenyl)isoxazol-5-carbaldehyde, 5-(4-chlorophenyl)isoxazol, 3-(4-chlorophenyl)isoxazol, 5-(chloromethyl)isoxazol-4-carboxylic acid, 3-(chloromethyl)-5-(2-furyl)isoxazol, 4-chloromethyl-3,5-dimethylisoxazol, 5-(chloromethyl)-3-(4-chlorophenyl)isoxazol, 5-(3-chloro-4-methoxyphenyl))-Isoxazol-3-carboxylic acid, 3-chloro-4-fluorobenzaldehyde, 3-(5-chloro-2,4-dimethoxyphenyl)4,5-dihydro-isoxazol-5-carboxylic acid, 5-tert-butyl-4,5,6,7-tetrahydro-benzo[d]isoxazol-3-carboxylic acid, 3-(4-bromophenyl)-5-(trifluoromethyl)isoxazol-4-carboxylic acid, 5-(4-bromophenyl)isoxazol-3-propio nic acid, 5-(4-bromophenyl)isoxazol-3-carboxylic acid hydrazide, 5-(4-bromophenyl)isoxazol-4-carboxylic acid, 5-(4-bromophenyl)isoxazol-3-carboxylic acid, 3-(4-bromophenyl)isoxazol-5-carboxylic acid, 3-(4-bromophenyl)isoxazol, 5-carboxaldehyde, 5-(4-bromophenyl)isoxazol, 5-(3-bromophenyl)isoxazol, 3- (4-bromophenyl)isoxazol, 5-(bromomethyl)-3-(4-methoxyphenyl)isoxazol, 4-(bromomethyl)isoxazol, 5-bromomethyl)-3-(4-fluorophenyl)isoxazol, 5-(bromomethyl)-3-(4-chlorophenyl)isoxazol, 5-(bromomethyl)-3-(4-bromophenyl)isoxazol, 6-bromo-3-methylbenzo[d]isoxazol, 5-bromo-3-methylbenzo[d]isoxazol, 4-bromo-5-(4-methoxyphenyl)isoxazol, 3-bromoisoxazol, 3-bromo-5-(2-hydroxyethyl)isoxazol, 4-bromo-5-(4-fluorophenyl)isoxazol, 3-bromo-5-(4-Fluorophenyl)Isoxazol, 4-Bromo-5-(4-chlorophenyl)Isoxazol, 4-Bromo-5-(4-bromophenyl)Isoxazol, 6-Bromo-benzo[d]Isoxazol3-carboxylic acid, Benzo[d]Isoxazol-3-carboxylic acid, 3-Amino-5-methylIsoxazol, 5-Amino-3-(4)-methoxyphenyl)Isoxazol, 3-Aminoisoxazol, 3-Amino-5-(4-Fluorophenyl)Isoxazol, 5-Amino-3-(4-chlorophenyl)Isoxazol, 5-Amino-4-(4-bromophenyl)Isoxazol, 3-Amino-5-(4-bromophenyl)Isoxazol, 5-Acetyl-3-(4-Fluorophenyl)Isoxazol, 5-Acetyl-3(3-Fluorophenyl)Isoxazol, 3-Methyl-5-[(2 s)-1-methyl-2-pyrrolidinyl]isoxazol hydrochloride, 7-methoxy-5-methyl-4,5-dihydronaphtho[2,1-d]isoxazol, 5-methyl-3-phenylisoxazol-4-carboxylic acid methylamide, 5-methyl-3-phenylisoxazol Zol-4-carbothioic acid methylamide, 5-methyl-3-phenyl-4-(1h-pyrazole-5-yl)isoxazol, 5-benzyl-3-furan-2-yl-2-phenyl-tetrahydropyrrolo(3,4)-d)isoxasole4,6-dione, 5-benzyl-3-[4-(dimethylamino)phenyl]-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-benzyl-3-(5-br-2-hophenyl)-2-ph-tetrahydropyrrolo(3,4 d) Isoxazol-4,6-dione, 5-benzyl-3-(4-nitro-ph)-2-ph-dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 5-benzyl-3-(4-methoxyphenyl)-2-ph-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione, 5-benzyl-3-(4-fluorophenyl)-2-(2-methylphenyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6( 3h,5h)-dione, 5-benzyl-3-(3-nitro-ph)-2-phenyl-tetrahydropyrrolo(3,4-d)isoxazol-4,6-dione, 5-benzyl-2-ph-3-(2-pyridinyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-benzyl-2-ph-3-(2-ph-vinyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 5-benzyl-2-(4-chlorophenyl)-3-(2-thienyl)dihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)-dione, 5-benzyl-2,3-diphenyldihydro2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 5-benzyl-2(4cl-ph)3-(2-furyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-benzyl-2(4cl-ph)-3-(4-f-ph)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-(p-tolyl)isoxazol, 5-(4-methylphenyl)-3-(4-nitrophenyl)-2phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)dione, 5-(4-methoxyphenyl)-2-phenyl-3-(4-pyridinyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4-methoxyphenyl)-2-phenyl-3-(3-pyridinyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4-(methoxy)-2,3-diphenyldihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 5-(4-fluorophenyl)-3-(4-methoxyphenyl)-2phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4, 6(3h,5h)dione, 5-(4-fluorophenyl)-2-(2-methylphenyl)-3-(4-pyridinyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4, 6(3h,5h)dione, 5-(4-ethoxyphenyl)-3-(4-nitrophenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4, 6(3h,5h)dione, 5-(4-)ethoxyphenyl)-3-(4-fluorophenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4, 6(3h,5h)dione, 5-(4-ethoxyphenyl)-2-methyl-3-(4-nitrophenyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4ethoxy-ph)-2-ph-3-thiophen-2-yl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione, 5-(4-cl-ph)-3-(3-nitro-ph)-2-phenyltetrahydropyrrolo(3,4-d)isoxazol-4,6-dione, 5-(4-bromophenyl)-3-(4-methoxyphenyl)-2-phenyldihydro-2hpyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4-bromophenyl)-3-(2-furyl)-2-(2-methylphenyl)dihydro-2h-pyrrolo[3, 4-d]Isoxazol-4,6(3h,5h)-dione,5-(4-bromophenyl)-2-phenyl-3-(2-pyridinyl)dihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,5-(4-br-ph)2-ph-3-(2-ph vinyl)dihydro-2h-pyrrolo(3,4-d)Isoxazol-4,6(3h) ,5h)dione, 5-(2-cl-ph)-3-(4-dimethylamino-ph)2-o-tolyl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione, 5-(2-chlorophenyl)-3-[4-(dimethylamino)phenyl]-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(2-chlorophenyl)-3-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 4-(3-(2-cl-p h)-5-methylisoxazol-4-carbonyl)-amino)-benzoate ethyl ester, 4,5,6,6a-tetrahydro-3ah-cyclopenta[d]isoxazol-3-carboxylic acid, 3-phenyl-3a,6a-dihydrothieno[2,3-d]isoxazol 4,4-dioxide, 3-methyl-5-(3-phenylpropyl)isoxazol, 3-methyl-4-nitro-5-[(e)-2-phenylethenyl]isoxazol, 3-methyl-4,5,8,9tetrahydrocycloocta(d)isoxazol, 3-methyl-4,5,5a,ba 1,7,8hexahydrooxyreno(2',3':5,6)cycloocta(1,2-d)isoxazol, 3-methyl-3a,45,8,9,9 a-Hexahydrocycloocta(d)isoxazol,3-Fran-2-yl-2-phenyl-5-p-tolyl-tetrahydropyrrolo(3,4-d)isoxazol-4,6-dione,3-chloro-4,5-dihydro(1)-benzoteepino(5,4-c)isoxazol,3-[4-(dimethylamino)phenyl]-5-(4-methoxyphenyl)-2-(2-methylphenyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4 ,6(3h,5h)-dione,3-(5-br-2-ho-phenyl)-2,5-diphenyl-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione,3-(5-br-2-ho-ph)-5-(2-cl-ph)-2-ph-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione,3-(4-meo-phenyl)-5-phenyl-2-0-tolyltetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione,3-(4-fluorophenyl)-5-(4-nitrophenyl)-2-phenyldihydro-2h-pyrrolo[3,4d]isoxazol-4 ,6(3h,5h)-dione,3-(4-fluorophenyl)-5-(4-methylphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione,3-(4-dimethylamino-ph)-5-ph 2-o-tolyl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione,3-(4-fluorophenyl)-5-(4)-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione,3-(4-br-ph)-2-ph 5-(2-trifluoromethyl-)ph)-4h-pyrrolo(3,4-d)isoxazol-4,6 dion e,3-(3-nitrophenyl)-2,5-diphenyl-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione,3-(3-br-phenyl)-2,5-diphenyldihydro-2h-pyrrolo(3,4-d)Isoxazol-4,6(3h,5h)-dione,3-(3-br-ph)-5-(2-meo-ph)-2-0-tolyl-tetrahydropyrrolo(3,4-d)Isoxazol-4,6-dione,3-(2-furyl)-5-[4-(4-morpholinyl)phenyl]-2-phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,3-(2-furyl)-2-(2-me-ph)-5-phdihydro-2h-pyrrolo(3,4-d)Isoxazol-4,6(3h,5h)-dione,3-(2-furyl)-2,5-diphenyldihydro-2h-pyrrolo(3,4- d) Isoxazol-4,6(3h,5h)-dione, 3-(2--cl-phenyl)-5-methylisoxazol-4-carboxylic acid (2,5-dichlorophenyl)amide, 3-(2-cl-ph)-5-m-isoxazol-4-carboxylic acid (4,5-dihydrothiazoll(2-yl)amide, 3-(2-chlorophenyl)-5-methylisoxazol-4-carboxylic acid cyanomethylamide, 3-(2,4-dichlorophenyl)-5-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3,5h) )-dione, 3-(2,4-di-cl-ph)2,5-diphenyldihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 3-(2) , 2-dichloro-vinyl)-5-phenyl-isoxazol, 3,5-diphenyl-isoxazol, 3,5-dimethyl-4-(1-pyrrolidinylsulfonyl)isoxazol, 3(4-dimethylamino-ph)-5-(4-eto-ph)2-o-tolyl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione, 2-(4-cl-ph)5-ph-3-(2-thienyl)-dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 2-(4-cl-ph)-5-(3-meo-ph)-3-(3-nitro-ph)-4h-pyrrolo(3,4-d)Isoxazol-4,6-dione,2-(4-cl-ph)-3-(4-meo-ph)-5-p-tolyl-tetrahydro-pyrrolo(3,4-d)Isoxazol-4,6-dione,2-(4-chlorophenyl)-5-(4-methylphenyl)-3-(2-thienyl)dihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,2-(4-chlorophenyl)-3-[4-(dimethylamino)phenyl)-5phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)dione,2-(4-chlorophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)dihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)-dione,2-(4-chlorophenyl)-3-(2-thienyl)-5-[3(trifluoromethyl)phenyl]dihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,2-(4-chlorophenyl)-3-(2,4-dichlorophenyl)-5-phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,2,3-di-ph-5-(3-(tri-f-me)ph)dihydro-2h-pyrrolo(3) , 4-d) isoxazol-4,6(3h,5h)-dione, danazol, and n-cyclopropyl-5-(thiophen-2-yl)isoxazol-3-carboxamide.

[0247] On the other hand, an effective dose includes amounts that result in the overexpression of miRNA, such as approximately 5 μM to 25 μM.

[0248] Furthermore, the cells are cultured in serum-free medium.

[0249] On the other hand, stem cells or progenitor cells are selected from the group of iPSC-derived cardiac progenitor cells or iPSC-derived embryoid bodies. Methods for inducing iPSC-cardiac progenitor cells from stem cells are known in the art and are described herein. Methods for inducing stem cells from iPSC-derived embryoid bodies are known in the art as described in the following specifications: Michael W. Nestor et al., "Differentiation of human induced pluripotent stem cells from serum-free embryoid bodies into networks," Stem Cell Research, Vol. 10, No. 3, 2013, Pages 454-463, ISSN 1873-5061, https: / / doi.org / 10.1016 / j.scr.2013.02.001; Steven D. Sheridan et al., "Analysis of human induced pluripotent stem cell-derived embryoid bodies as a means of evaluating pluripotency," Stem Cell International, Vol. 2012, Specification ID 738910, 9:2012; Heming Wei et al., "One-step induction of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells," Stem Cell Research, Vol. 9, No. 2, 2012, P 87-100; and Di Pasquale E. et al. J.Vis Exp. 2013 Jun.28; (76) Generation of human cardiomyocytes: Differentiation protocol from feeder-free human induced pluripotent stem cells.

[0250] On the other hand, isoxazole compounds are selected from isoxazole-1 (isx-1) or isoxazole-9 (isx-9). Furthermore, isoxazole derivatives are compounds having the following formula: TIFF0007837302000008.tif88153 R1 and R2 are both hydrogen, or R2 is hydrogen. Also, R2 is selected from the group consisting of substituted or unsubstituted C1-C4 alkyl, C3-C4 cycloalkyl, C2-C4 alkenyl, C2-C6, alkynyl, and benzyl. Also, R1 and R2 together may form a ring selected from azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl. R2', R2 and R4 are independently selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C8 cycloalkyl substituted or unsubstituted aromatic or heteroaromatic ring, cyano, nitro, and acyl. X is 0, NH or S; Y is 0 or NH.

[0251] Furthermore, isoxazole compounds have the following formula: TIFF0007837302000009.tif95166 Here, R1 and R2 are selected from C1-C4 alkyl, phenyl, benzyl, trifluoromethyl or halogen, respectively, and R3 is selected from hydrogen, hydroxy, C1-C4 alkyl or alkoxy. R4 at position 3 or 5 is selected from hydrogen, trifluoromethyl, C1-C4 alkoxy, C1-C4 alkyl, or C1-C4 hydroxyalkyl, and R5 is selected from hydrogen or C4-C4 alkyl, or R4 and R5 together form a tetramethylene group. Z at position 3 or 5 on the heterocycle is selected from -N(R6)-CO-, -CON(R6)-, _N(R6)CO_NRG)-, CH(RF)-NHCO…, or -NHCOCH(RO). Here, R 6 is selected from hydrogen or a C1-C4 alkyl group.

[0252] In a further embodiment, isoxazol compounds or derivatives thereof are selected from the following group: 5-(trifluoromethyl)-3-(4-methoxyphenyl)isoxazol-4-carboxylic acid, 5-(trifluoromethyl)-3-(4-fluorophenyl)isoxazol-4-carboxylic acid, 5-(thiophen-2-yl)isoxazol-3-carboxyaldehyde, 5,6,7,8-tetrahydro-4h-cyclohepta[d]isoxazol-3-carboxylic acid, 4,5,6,7-tetrahydro-benzo[d]isoxazol-3-carboxylic acid, 3-amino-5-methylisoxazol, 4-amino-n-(5-methyl-3-isoxazolyl)benzenesulfonamide, 3-phenylisoxazol-5-boronic acid pinacol ester, 5-phenylisoxazol, 1-phenyl-1-cyclopentanecarboxylic acid, 3-phenyl-benzo[c] Isoxazo-5-carboxylic acid, 5-methyl-3-phenylisoxazo-4-carboxylic acid, 3 a, 4, 5, 6, 7, 8, 9, 9 a-octahydrocycloocta[d]isoxazo-3-carboxylic acid, 5-(3-nitrophenyl)isoxazolic acid, 3-(4-nitrophenyl)isoxazolic acid, 3-hydroxy-5-aminomethylisoxazolic acid, 5-(morpholinomethyl)isoxazo-3-carboxylic acid salt, 5-(morpholinomethyl)isoxazolic acid-3-carbaldehyde, 3-methyl-5-(trifluoromethyl)isoxazolic acid-4-carboxylic acid, methyl-5-(thiophen-2-yl)isoxazolic acid-3-carboxylic acid, 3-(methylsulfonyl) )-5-(2-thienyl)isoxazol-4-carbonitride, 5-methyl-3-(2-pyrrolidinyl)isoxazol, 3-methyl-5-(2-pyrrolidinyl)isoxazol, 3-(1-methyl-1h-pyrazole-4-yl)-isoxazol-5-carboxylic acid, 3-(1-methyl-1h-pyrazole-4-yl)-4,5-dihydroisoxazol-5-carboxylic acid, 5-(4-methylphenyl)isoxazol-3-carboxylic acid, 5-methyl-3-phenylisoxazol-4-carboxylic acid, 5-(4-methylphenyl)isoxazol-3 Carboxaldehyde, 5-methyl-3-(4-phenoxyphenyl)isoxazo-l4-carboxylic acid, 3-methyl-5-(4-methyl-1,2,3-thiadiazo-l5-yl)isoxazo-l4-carboxylic acid, 3-methyl-5-(5-methylisoxazo-l3-yl)isoxazo-l4-carboxylic acid, methyl-5-(4-methoxyphenyl)isoxazo-l4-carboxylic acid, 5-(4-methoxyphenyl)isoxazo-l3-carboxylic acid methyl, 5- Methyl isoxazole, 5-(4-fluorophenyl)isoxazole-4-carboxylate methyl, 5-(4-fluorophenyl)isoxazole-3-carboxylate methyl, 5-(4)methyl-chlorophenyl)isoxazole-4-carboxylate, 5-(4-bromophenyl)isoxazole-4-carboxylate, 5-(4-methoxyphenyl)isoxazole-3-carboxylic acid, 5-(3-methoxyphenyl)isoxazole-3-carboxylic acid,3-(2-methoxyphenyl)isoxazol-5-carboxylic acid, 5-(4-methoxyphenyl)isoxazol-3-carboxyaldehyde, 3-(4-methoxyphenyl)isoxazol-5-carbaldehyde, 3-(2-methoxyphenyl)isoxazol-5-carbaldehyde, 5-(4-methoxyphenyl)isoxazol, 3-(4-methoxyphenyl)isoxazol, 3-(2-methoxyphenyl)-4,5-dihydroisoxazol-5-carboxylic acid, 3-methoxyisoxazol-5-carboxylic acid, isoxazol-5-carboxylic acid, isoxazol-4-carboxylic acid, isoxazol-5-carbothioamide, isoxazol-5-carbonyl chloride, isoxazol-3-carbonitrile, isoxazol- 3-Carbaldehyde, isoxazole-4-boronic acid, isoxazole, 5-cyclopropyl-4-[2-(methylsulfonyl)-4-(trifluoromethyl)benzoyl]isoxazole, 6-(5)-(thiophen-2-yl)isoxazole-3-carboxamide)hexyl 5-((3as,4s,bar)-2-oxohexahydro-1h-thieno[3,4-diimidazole-4-yl)pentanoate, isocarboxazide 5-methyl-3isoxazole-carboxylic acid 2-benzylhydrazide, 5-isobutylisoxazole-3-carboxylic acid, 4-iodo-5-methylisoxazole, 3,3'-iminobis(n,n-dimethylpropylamine), 3-(3-hydroxyphenyl)-isoxazole-5-carboxylic acid methyl ester , 5-(4-hydroxyphenyl)-isoxazo-3-carboxylic acid, 5-(3-hydroxyphenyl)-isoxazo-3-carboxylic acid, 5-hydroxymethyl)-3-methylisoxazole, 3-hydroxy-5-methylisoxazole, 5-(1-hydroxyethyl)-3-(4-trifluoromethylphenyl)isoxazole, 3 a, 4,5,6,7,7 a-hexahydro-benzo[diisoxazole-3-carboxylic acid, 5-(2-furyl)isoxazole-3-carbaldehyde], 5-furan-2-ylisoxazole-3-carboxylic acid, 6-fluoro-3-(4-piperidinyl)benzisoxazole,5-(4-fluorophenyl)isoxazol-3-methyl, 3-(2-fluorophenyl)isoxazol-5-carboxylic acid, 5-(4-fluorophenyl)isoxazol-3-carboxyaldehyde, 3-(4-fluorophenyl)isoxazol-5-carbaldehyde, 3-(3-fluorophenyl)isoxazol-5-carbaldehyde, 3-(2-fluorophenyl)isoxazol-5carbaldehyde, 5-(4-fluorophenyl)isoxazol, 3-(4)fluorophenyl)isoxazol, 5-(3-fluoro-4-methoxyphenyl)isoxazol-3-carboxylic acid, ethyl 5-(trifluoromethyl)-3-(4-methoxyphenyl)isoxazol-4-carboxylate, ethyl-5( Tributylstanyl) isoxazol-3-carboxylic acid ester, ethyl 5-(thiophene 2-yl) isoxazol-3-carboxylic acid ester, 5-ethyl isoxazol-4 carboxylic acid, 5-ethyl isoxazol-3-carboxylic acid, ethyl 5-(4-fluorophenyl) isoxazol-4-carboxylate, ethyl 5-(4-fluorophenyl) isoxazol-3-carboxylate, ethyl 5-(2,3-dihydrobenzo[b][1,4]dioxin-7-yl) isoxazol-3-carboxylate, ethyl 3-(4-chlorophenyl)-5-(trifluoromethyl) isoxazol-4-carboxylate, ethyl 5-(4-chlorophenyl) isoxazol-3-carboxylate, ethyl 3-(4-bro 5-(4-bromophenyl)isoxazol-3-carboxylate ethyl, 5-amino-4-(4-chlorophenyl)isoxazol-3-carboxylate ethyl, 5-amino-4-(4-bromophenyl)isoxazol-3-carboxylate, ethyl 6b-acetyl-2-(acetyloxy)-4a,6a-dimethyl 2,3,4,4a,4b,5,6,6a,6b,9a,10,10a,106,11-tetradecahydro-1h-naphtho[2',1:4,5]indeno[2,1-d]isoxazol-9-carboxylate, 3,5-dimethyl-4-(tributylstanyl)isoxazol, 5-(1,5-dimethyl1h-pyrazole-4-yl)-isoxazol-3-carboxylate,5-(1,3-dimethyl-1h-pyrazole-4-yl)-isoxazole-3-carboxylic acid, 5-(1,5-dimethyl-1h-pyrazole-4-yl)-isoxazole, 3,5-dimethylisoxazol-4-boronic acid pinacol ester, 3,5-dimethylisoxazol, 3-(dimethylamino)-1-(2-pyridyl)-2-propen-1-one, 5-(3,5-difluorophenyl)isoxazol, [2,6-dichloro-4-(trifluoromethyl)phenyl]hydrazine, 5-(2,5-dichlorophenyl)isoxazol-3-carboxylic acid, danazole, 3-(4-chlorophenyl)-5-(trifluoromethyl)isoxazol-4-carboxylic acid, 5-(4-chlorophenyl)isoxazol-3-propionic acid, 5-(4-chlorophenyl)isoxazol-4-carboxylic acid, 5-(4)-chlorophenyl)isoxazol-3-carboxylic acid, 3-(4-chlorophenyl)isoxazol-5-carboxylic acid, 3-(3-chlorophenyl)isoxazol-5-carboxylic acid, 5-(4-chlorophenyl)isoxazol-3-carboxyaldehyde, 3-(4-chlorophenyl)isoxazol-5-carbaldehyde, 3-(3-chlorophenyl)isoxazol-5-carbaldehyde, 3-(2-chlorophenyl)isoxazol-5-carbaldehyde, 5-(4-chlorophenyl)isoxazol, 3-(4-chlorophenyl (L)isoxazol, 5-(chloromethyl)isoxazol-4-carboxylic acid, 3-(chloromethyl)-5-(2-furyl)isoxazol, 4-chloromethyl-3,5-dimethylisoxazol, 5-(chloromethyl)-3-(4-chlorophenyl)isoxazol, 5-(3-)chloro4-methoxyphenyl)-isoxazol-3-carboxylic acid, 3-chloro4-fluorobenzaldehyde, 3-5-chloro-2,4-dimethoxyphenyl)4,5-dihydro-isoxazol-5-carboxylic acid, 5-tert-butyl-4,5,6,7-tetrahydro-benzo[d]isoxazol-3-carboxylic acid, 3-(4)bromophenyl)-5-(trifluoromethyl)isoxazol-4-carboxylic acid, 5-(4-bromophenyl)isoxazol-3-propionic acid,5-(4-bromophenyl)isoxazol-3-carboxylic acid hydrazide, 5-(4-bromophenyl)isoxazol-4-carboxylic acid, 5-(4-bromophenyl)isoxazol-3-carboxylic acid, 3-(4-bromophenyl)isoxazol-5-carboxylic acid, 3-(4-bromophenyl)isoxazol-5-carboxyaldehyde, 5-(4)-bromophenyl)isoxazol, 5-(3-bromophenyl)isoxazol, 3-(4-bromophenyl)isoxazol, 5-(bromomethyl)-3-(4-methoxyphenyl)isoxazol, 4-(bromomethyl)isoxazol, 5-bromomethyl)-3-(4-fluorophenyl)isoxazol, 5- (bromomethyl)-3-(4-chlorophenyl)isoxazol, 5-(bromomethyl)-3-(4-bromophenyl)isoxazol, 6-bromo-3methylbenzo[d]isoxazol, 5-bromo-3-methylbenzo[d]isoxazol, 4-bromo-5-(4-methoxyphenyl)isoxazol, 3-bromoisoxazol, 3-bromo-5-(2-hydroxyethyl)isoxazol, 4-bromo-5-(4)-fluorophenyl)isoxazol, 3-bromo-5-(4-fluorophenyl)isoxazol, 4-bromo-5-(4-chlorophenyl)isoxazol, 4-bromo-5-(4-bromophenyl)isoxazol, 6-bromobenzo[d] ]Isoxazol 3-carboxylic acid, benzo[d]isoxazol-3-carboxylic acid, 3-amino-5-methylisoxazol, 5-amino-3-(4-methoxyphenyl)isoxazol, 3-aminoisoxazol, 3-amino-5-(4-fluorophenyl)isoxazol, 5-amino-3-(4-chlorophenyl)isoxazol, 5-amino4-(4-bromophenyl)isoxazol, 3-amino-5-(4-bromophenyl)isoxazol, 5-acetyl-3-(4-fluorophenyl)isoxazol, 5-acetyl-3(3-fluorophenyl)isoxazol, 3-methyl-5-[(25)-1-methyl-2-pyrrolidinyl]isoxazol hydrochloride, 7-methoxy-5-methyl-4, 5-dihydronaphtho[2,1-d]isoxazol,Methyl 5-methyl-3-phenylisoxazole-4-carboxamide, methyl 5-methyl-3-phenylisoxazole-4-carbothioamide, 5-methyl-3-phenyl-4-(1H-pyrazol-5-yl)isoxazole, 5-benzyl 3-furan-2-yl-2-phenyl-tetrahydro-pyrrolo(3,4-d)isoxazole-4,6-dione, 5-benzyl-3-[4-(dimethylamino)phenyl]-2-phenyldihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione, 5-benzyl-3-(5-br-2-ho-phenyl)-2-ph-tetrahydro-pyrrolo(3,4d)isoxazole-4,6-dione, 5-benzyl-3-(4-nitro-ph)-2 - ph-dihydro-2H-pyrrolo(3,4 - d) isoxazole-4,6(3h), 5h)-dione, 5-benzyl-3-(4-methoxy-phenyl)-2-ph-tetrahydro-pyrrolo(3,4-d) isoxazole-4,6-dione, 5-benzyl-3-(4)-fluorophenyl)-2-(2-methylphenyl)dihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione, 5-benzyl-3-(3-nitro-ph)-2-, Phenyl-tetrahydropyrrolo(3,4-d)isoxazol-4,6-dione, 5-benzyl-2-ph-3-(2-pyridinyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-benzyl-2-ph-3-(2-(ph-vinyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 5-benzyl-2-(4-chlorophenyl)-3-(2-thienyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(34,5h)-dione, 5-benzyl-2,3-diphenyldihydro2h-pyrrolo(3,4-d)isoxazol-4,6(3h) ,5h)-dione,5-benzyl-2(4cl-ph)3-(2-furyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione,5-benzyl-2(4-cl-ph)-3-(4-f-ph)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione,5-(p-tolyl)isoxazol,5-(4-methylphenyl)-3-(4-nitrophenyl)-2phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)dione,5-(4-methoxyphenyl)-2-phenyl-3-(4-pyridinyl)dihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)-dione,5-(4-methoxyphenyl)-2-phenyl-3-(3-pyridinyl)dihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,5-(4-methoxyphenyl)-2,3-diphenyldihydro-2h-pyrrolo(3,4-d)Isoxazol-4,6(3h,5h)-dione,5-(4-fluorophenyl)-3-(4-methoxyphenyl)-2phenyldihydro-2h-pyrrolo[3,4]-d]Isoxazol-4,6(3h,5h)dione,5-(4-fluorophenyl)-2-(2-methylphenyl)-3-(4-pyridinyl)dihydro-2h-pyrrolo[3,4 - d]Isoxazol-4,6(3h,5h)5-(4-ethoxyphenyl)-3-(4-nitrophenyl)-2-phenyldihydro-2h-pyrrolo[3,4 - d]Isoxazol-4,6(3h,5h)dione, 5-(4-ethoxyphenyl)-3-(4-fluorophenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)dione, 5-(4-ethoxyphenyl)-2-methyl-3-( 4-(nitrophenyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4ethoxy-ph)-2-ph-3-thiophen-2-yl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione, 5-(4-cl-ph)-3-(3-nitro-ph)-2-phenyltetrahydropyrrolo(3,4-d)isoxazol-4,6-dione, 5-(4-bromophenyl)-3-(4-methoxyphenyl)-2-phenyldihydro-2hpyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4-bromophenyl)-3-(2-furyl)-2-( 2-methylphenyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4-bromophenyl)-2-phenyl-3-(2-pyridinyl)dihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(4-br-ph)2-ph-3-(2-ph vinyl)dihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 5-(2-cl-ph)-3-(4-dimethylamino-ph)2-0-tolyl-4h-pyrrolo(3,4-d) )Isoxazol-4,6-dione, 5-(2-chlorophenyl)-3-[4-(dimethylamino)phenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 5-(2-chlorophenyl)-3-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 4-(3-(2-cl-ph)-5-methylisoxazol-4-carbonyl)-amino)-ethyl benzoate, 4,5,6,6a-tetrahydro-3ah-cyclopenta[d]isoxazol-3-carboxylic acid, 3-phenyl-3a,ba-dihydrothieno[2,3-d]isoxazol-4,4-dioxide, 3-methyl-5-(3-phenylpropyl)isoxazol, 3-methyl-4-nitro-5-[(e)-2-phenylethenyl]isoxazol, 3-methyl-4,5,8,9-tetrahydrocycloocta(d)isoxazol, 3-methyl-4,5,5a,ba,7,8-hexahydrooxyleno(2,3':5,6)cycloocta(1,2-d)isoxazol, 3-methyl-3a,4,5,8,9,9a-hexahydrocycloocta(d)isoxazol, 3-furan-2-yl-2-phenyl-5-p-tolyl-tetrahydropyrrolo(3,4-d)isoxazol-4,6-dione, 3-chloro-4,5-dihydro(3). 1)-Benzothiopino(5,4-c)isoxazol, 3-[4-(dimethylamino)phenyl]-5-(4-methoxyphenyl)-2-(2-methylphenyl)dihydro-2h-pyrrolo[3,4-diisoxazol-]4,6(3h,5h)-dione, 3-(5-br-2-ho-phenyl)-2,5diphenyl-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione, 3-(5-br-2-ho-ph)-5-(2-cl-ph)-2-ph-tetrahydro-pyrrolo(3,4-d)isoxazol-4,6-dione, 3-(4-meo-phenyl)-5-phenyl-2-0-tolyltetrahydropyrrolo(3,4-d)isoxazol-4,6-dione, 3-(4-fluorophenyl)-5-(4-nitrophenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 3-(4-fluorophenyl)-5-(4-methylphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 3-(4-dimethylamino-ph)-5-ph 2-0-tolyl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione, 3-(4-fluorophenyl)-5-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4- d] Isooxazol-4,6(3h,5h)-dione, 3-(4-br-ph)-2-ph 5-(2-trifluoromethyl-ph)-4h-pyrrolo(3,4-d)isoxazol-4,6dione, 3-(3-nitro-phenyl)-2,5-diphenyl-tetrahydropyrrolo(3,4-d)isoxazol-4,6dione, 3-(3-br-phenyl)-2,5-diphenyldihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)dione, 3-(3-br-ph)-5-(2-meo-ph)-2-0-tolyl-tetrahydropyrrolo(3,4-d)isoxazol-4, 6-dione, 3-(2-furyl)-5-[4-(4-morpholinyl)phenyl]-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 3-(2-furyl)-2-(2-me-ph)-5-phdihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 3-(2-furyl)-2,5-diphenyldihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 3-(2-cl-phenyl)-5-methylisoxazol-4carboxylic acid (2,5-dichlorophenyl)amide, 3-(2-cl-ph)-5-oxazol-4carboxylic acid (4,5-Dihydrothiazo-l-2-yl)amide, 3-(2-chlorophenyl)-5-methylisoxazol-4 carboxylic acid cyanomethylamide, 3-(2,4-dichlorophenyl)-5-(4-methoxyphenyl)-2-phenyldihydro-2h-pyrrolo[3,4-d]isoxazol-4,6(3h,5h)-dione, 3-(2,4-di-cl-ph)2,5-diphenyldihydro-2h-pyrrolo(3,4-d)isoxazol-4,6(3h,5h)-dione, 3-(2,2-dichloro-)vinyl)-5-phenyl- Soxazol, 3,5-diphenyl-isoxazol, 3,5-dimethyl-4-(1-pyrrolidinylsulfonyl)isoxazol, 3(4-dimethylamino-ph)-5-(4-eth-ph)2-o-tolyl-4h-pyrrolo(3,4-d)isoxazol-4,6-dione, 2-(4-cl-ph)5-ph-3-(2-thienyl)dihydro-2h-pyrrolo(3,4)-d)isoxazol-4,6(3h,5h)dione, 2-(4-cl-ph)-5-(3-meo-ph)-3-(3-nitro-ph)-4h-pyrrolo(3) ,4-d)Isoxazol-4,6-dione,2-(4-cl-ph)-3-(4-meo-ph)-5-p-tolyl-tetrahydro-pyrrolo(3,4-d)Isoxazol-4,6-dione,2-(4-chlorophenyl)-5-(4-methylphenyl)-3-(2-thienyl)dihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,2-(4-chlorophenyl)-3-[4-(dimethylamino)phenyl]-5phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)dione,2-(4-chlorophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)dihydro-2h-pyrrolo[3,4- d]Isoxazol-4,6(3h,5h)-dione,2-(4-chlorophenyl)-3-(2-thienyl)-5-[3(trifluoromethyl)phenyl]dihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,2-(4-chlorophenyl)-3-(2,4-dichlorophenyl)-5-phenyldihydro-2h-pyrrolo[3,4-d]Isoxazol-4,6(3h,5h)-dione,2,3-Di-ph - 5-(3-(tri-f - me)ph)dihydro-2H-pyrrolo(3,4 - d)isoxazole-4,6(3H,5H)-dione, danazol, and N-cyclopropyl-5-(thiophen-2-yl)isoxazole-3-carboxamide.,

[0253] Also provided are cell populations prepared by the above method, as well as exosomes or microvesicles isolated and / or purified from these cell populations. The populations can be allogeneic, xenogeneic, purified, highly purified, syngeneic or substantially syngeneic. In one embodiment, at least 80%, 85%, 90%, 95%, 97% or 99%, 100% of the cells are expressing protein.

[0254] Further provided are a cell population and exosomes or microvesicles isolated from said cell population, wherein said cell population is a cardiac or skeletal myogenic progenitor cell prepared by culturing a population of human induced pluripotent stem cells (hiPSC) with an effective amount of givinostat (GIV: Givinostat) for an effective amount of time. In one aspect, said effective amount of givinostat (GIV) is about 100 nM to about 30 nM per 1×10 6 cells, or about 100 nM to about 50 nM per 1×10 6 cells, about 90 nM to about 30 nM per 1×10 6 cells, about 30 nM to about 75 nM per 1×10[[ID=**15**]] 6 cells, or about 70 nM to about 50 nM per 1×10 6 cells. In yet another aspect, the population of said cells, exosomes or microvesicles can be prepared by a method further comprising culturing said cells in the presence of a cell-free medium and a Rho-associated kinase (ROCK) inhibitor. Non-limiting examples of Rho-associated kinase (ROCK) inhibitors include thiazovivin or Y27632, commercially available from R&D Systems, Sigma Aldrich, and Stemgent. Methods for isolating the compositions are known in the art and some of them are described herein.

[0255] The aforementioned cells, exosomes, or microvesicles are useful for regenerating skeletal muscle or treating DMD by administering the cells and / or exosomes or microvesicles to subjects in need. Methods for determining the efficacy of the treatment are known in the art, some of which are described herein.

[0256] In another aspect, the population can be prepared by culturing the cells in serum-free medium and in the presence of a TGF-β1 receptor inhibitor. Non-limiting examples of such TGF-β1 receptor inhibitors include SB431542 and A8301, commercially available from Cayman Chemical and Sigma Aldrich; LY2157299, commercially available from AdooQ Bioscience (catalog no. A11017); and LY2109761, commercially available from AdooQ Bioscience and Selleckchem (catalog no. S2704). The cell population may be heterogeneous, homogeneous, substantially homogeneous, or highly purified. In one aspect, the population may contain at least 80%, 85%, 90%, 95%, 97%, or 99%, 100% of the cells expressing the exosome or microvesicles. Methods for isolating the population are known in the art, some of which are described herein.

[0257] These cells and exosomes or microvesicles are useful for regenerating myocardium by administering an effective amount of the population or exosome or microvesicles to subjects in need. They are also useful for treating cardiac dysfunction associated with Duchenne muscular dystrophy (DMD) by administering an effective amount of the cell population or exosome or microvesicles to subjects in need. Methods for determining the efficacy of treatment are known in the art, some of which are described herein.

[0258] In one aspect, the cell population overexpresses at least one skeletal muscle-forming gene selected from the group Meox1, Meox2, Tcf15, Pax3, Pax7, MyoDi, dystrophin, Myf5, or DESMIN. In another aspect, the population overexpresses at least one, or two, three, four, five, six, seven, eight, or nine skeletal muscle-forming genes. The cells are prepared, in some cases, by contacting iPSCs with an effective amount of divinostat (GIV) or by culturing them in the presence of an effective amount of divinostat (GIV) in serum-free medium. The effective amount of GIV is approximately 100 nM to approximately 30 nM per 1 × 10 cells. In another aspect, the cells are also contacted with an effective amount of ROCK inhibitor or cultured in the presence of an effective amount of ROCK inhibitor. Non-limiting examples of ROCK inhibitors include Thiazovivin, Y27632, SR3677, or GSK429286. In one aspect, the cells are cultured (or in contact) in the presence of a TGF-β1 inhibitor. Non-limiting examples of TGF-β1 inhibitors include SB431542, A8301, LY2157299, or LY2109761. The cell population may be heterogeneous, homogeneous, substantially homogeneous, or highly purified. In one aspect, at least 80%, 85%, 90%, 95%, 97%, or 99%, 100% of the cells in the population express the gene.

[0259] The cells and / or exosomes or microvesicles isolated from the cells are advantageously used in methods for regenerating skeletal muscle or treating Duchenne muscular dystrophy (DMD) by administering an effective amount of the cell population and / or exosomes or microvesicles isolated from the cells to a subject requiring them. Methods for determining the efficacy of the treatment are known in the art, some of which are described herein.

[0260] In one aspect, a population of cells overexpressing xESI myogenic genes selected from the following groups, and exosomes or microvesicles isolated from said cells are also provided: Pitx2, ISL1, Nkx2.5, Hand1, GATA4, Tbx5, TnnT2, My17, MLC2v, Myf2c, Cdh4, or Lhx2. In one aspect, the population overexpresses at least one, or two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of the xESI myogenic genes. The population of said cells or exosomes and microvesicles may be heterogeneous, homogeneous, substantially homogeneous, or highly purified. In one aspect, at least 80%, 85%, 90%, 95%, 97%, or 99%, or 100% of the cells in said population overexpress said genes. Cells that overexpress one or more of the aforementioned genes, and methods for isolating exosomes or microvesicles isolated from such populations, are known in the art and are described herein.

[0261] The aforementioned population and exosome or microvesicles are useful for myocardial regeneration and / or cardiac dysfunction associated with Duchenne muscular dystrophy (DMD) by administering an effective amount of the aforementioned cell population and / or exosome or microvesicles isolated from the aforementioned cells to subjects in need. Methods for determining the efficacy of the treatment are known in the art, some of which are described herein.

[0262] Compositions are also provided for the repair or regeneration of damaged or affected cardiac tissue, or for the treatment of one or more of them: Hoyeraal-Hreidarsson syndrome, congenital keratosis, pulmonary fibrosis, congenital keratosis, bone marrow failure, lung disease, endocrine disorders, polycystic ovary syndrome (PCOS), Cushing's syndrome, and acromegaly, cerebrovascular disease (stroke), hypertension, Parkinson's disease, dementia, Alzheimer's disease, age-related hearing loss, celiac disease (COP), bipolar disorder, hydroxyurea, sickle cell anemia, hypertension, atherosclerosis, arthritis, osteoporosis, osteoarthritis, vascular dementia or macular degeneration, cancer, type 2 diabetes, or diseases with telomere dysfunction, shortened telomere length, a composition comprising synthetic MicroRNA 146a and a pharmaceutically acceptable carrier, and administration of an effective amount of danazol. These compositions are useful for regenerating tissue in subjects requiring it by administering one or more microRNA fragments or derivatives thereof to an individual. Following administration of one or more microRNA fragments, they alter gene expression in the damaged tissue, improve the viability of the damaged tissue, and promote the formation of new tissue in the subject. Methods for determining the efficacy of the treatment are known in the art, some of which are described herein.

[0263] For the purposes of this disclosure, the exosome or microvesicle has an average diameter of about 20 nm to about 90 nm.

[0264] [composition] The present invention also provides compositions comprising cells, exosomes or microvesicles, mirNA, and populations comprising them in combination with carriers such as biocompatible scaffolds or pharmaceutically acceptable carriers. Furthermore, the compositions also comprise preservatives and / or cryoprotectants. The compositions can be freeze-dried, dried, or lyophilized, and can be provided in one or more dosage forms. In one embodiment, the composition is intended for therapeutic use, and therefore effective amounts of modified cells, exosomes or microvesicles, mirNA, and populations are provided in the composition, either alone or in combination with isoxazole or isoxazole analogs. Methods for preparing specific cell populations, exosomes or microvesicles and mirNA populations, and their use.

[0265] Methods for preparing isolated populations of exosomes or microvesicles overexpressing microRNAs (mirNAs) selected from the following groups are also provided herein: mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-30c, mir-214, or mir-548q, or one or more proteins selected from Tsg101, CD9, Hsp70, Flotillin-1, or GAPDH. On the one hand, at least 70%, 80%, or 85%, 90%, 95%, or 98% of cells express the described mir and / or proteins.

[0266] On the one hand, the following method is provided. Briefly, the applicant evaluated the applicant's exosome data for more than 50 mice with MI and cardiac function assessments at three time points during a one-month observation period. miRNAs secreted by exosomes derived from specific cardiac progenitor cells exerted potent therapeutic effects against myocardial infarction. The paracrine effects of precursor-derived cardiac exosomes on cell survival, migration, and differentiation play a crucial role in cardiac regeneration. miRNA signatures are unique in exosomes among different parental cells. Therefore, the applicant isolated and characterized exosomes from iPSCs and isoxasols, induced cardiac progenitor cells (Exo-CPCSOs), and determined their effects on cardiac cells in vitro and in mouse hearts after myocardial infarction (MI). The applicant characterized the exosomes isolated from iPSCs and CPCs. Transmission electron microscopy (TEM) of exosomes isolated from iPSCs and CPC SX-9 showed typical morphology. Western blotting further confirmed that exosomes from iPSCs and CPC SX-9 were rich in the exosome-specific marker Tsg101. Other common exosome proteins, including CD9, Hsp70, and flochylin-1, were not present in the exosomes. Calnexin was also absent from the exosomes. No significant difference was observed in the mean size of exosomes from iPSCs and CPC / SX-9.

[0267] The applicant also investigated the mirNA cargo content in exosomes from CPCs. Significant overexpression of miR-373, miR-367, miR-520, and miR-548 was confirmed in Exo-CPCs. Therefore, these miRNA microarray data were confirmed by real-time PCR compared to exosomes from iPSCs, commercially available CPCs, and embryoid bodies (EBs). Go enrichment analysis based on miRNA-targeted genes showed that the biological processes involved organelles, molecular function, stress response, and the mitotic cell cycle. The applicant further investigated the effects of Exo-CPCSO on cultured fibroblasts. PKH26-labeled exosomes from CPCISA-9 were observed inside fibroblasts (calcein AM) that were mostly located in the perinuclear region. Interestingly, the expression of fibrosis genes (CTCF, FN1, TIMP1, TIMP2, MMP2, and MMP9) in TGF-B-stimulated cultured fibroblasts was significantly downregulated after treatment with Exo-CPCiso. Loss-of-function analysis using a miR-373 inhibitor confirmed that enrichment of miR-373 was the primary contributor to the recovery of TGF-B stimulation in fibroblasts. The applicant also tested the therapeutic effect in a mouse MI model. The applicant found that intramyocardial injection of Exo-CPCiso exerted favorable cardioprotective effects on cardiomyocyte proliferation, angiogenesis, and preservation of cardiac function in mice one month after MI, compared to PBS and Exo-iPSC-treated mice. Quantitative analysis of ki67-positive CM (cTnT-positive) and proliferating CMS in Exo-CPCSX-9 treated mice 30 days after MI showed that Exo-CPCSX-9 significantly increased CM proliferation in the periinfarct area 30 days after MI (data not shown). Quantitative analysis of arteriole density showed that Exo-CPCSX-9 significantly increased arteriole density after MI. Furthermore, functional measurements demonstrated that Exo-CPC / SX-9 treatment significantly improved EF and FS (data not shown). Quantitative analysis showed smaller fibrotic areas from Exo CPC SA-9 treated mice after MI (data not shown).Therefore, the Micro-RNA 373 mimetic will overexpress Micro-RNA 373, which will reduce scar tissue in fibrous diseases.

[0268] Populations of exosomes or microvesicles may be heterogeneous, homogeneous, substantially homogeneous, or highly purified. On the one hand, populations may contain at least 80%, or 85%, 90%, 95%, 97%, or 99%, 100% of exosomes or microvesicles, and express mirNA.

[0269] Also provided is a method for preparing and isolating one or more populations of cardiac progenitor cells, cardiomyocytes, muscle cells, endothelial cells, smooth muscle cells, and skeletal muscle cells, wherein these cells are generated from cells selected from the following groups: iPSCs, embryonic stem cells, or stem cells in contact with isoxazole compounds, derivatives thereof, or equivalents thereof. Herein, the cells or population overexpress one or more of mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-30c, mir-214, or mir 548q and / or one or more muscle genes selected from the group paZ3, PAX7, MYF5, MYOD, MYOG, or dystrophin. In one aspect, at least 70%, or 80%, 85%, 90%, 95%, or 98% of the cells express the above-mentioned mir and / or muscle genes. Exosomes or microvesicles can be isolated from any of the following cells: cardiomyocyte progenitor cells, cardiomyocytes, skeletal muscle, endothelial cells, myocytes, or smooth muscle cells.

[0270] Also provided herein is a method for preparing cells that overexpress one or more of mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-30c, mir-214, or mir-548q, and / or one or more muscle genes selected from the group paX3, PAX7, MYF5, MYOD, MYOG, or dystrophin. In one aspect, at least 70%, 80%, 85%, 90%, 95%, or 98% of the cells overexpress the aforementioned mir and / or muscle genes. The expression level is determined using methods described herein and known in the art, such as high-throughput assays, ELISA, and hybridization techniques. One or more of the aforementioned mir and / or genes may be overexpressed.

[0271] Methods for preparing compositions comprising exosomes or microvesicles and / or cells are also provided. The compositions may further include proteins that promote tissue regeneration and improved function, nucleic acids that code for these proteins, and agents that inhibit the expression of inflammatory proteins, which may be cytokines. Examples are described herein.

[0272] Populations are prepared by culturing a population of stem cells or progenitor cells cultured for an effective period in the presence of an effective amount of isoxazole compounds, their derivatives, or their equivalents. Exemplary isoxazoles and their derivatives are provided herein. On the one hand, the effective amount includes amounts that result in overexpression of miRNA, e.g., about 5 μM to about 25 μM, or intermediate ranges as described above. Non-limiting and exemplary stem cells or progenitor cells are selected from the group of iPSC-derived CPCs, cardiomyocytes, skeletal muscle cells, endothelial cells, myocytes, smooth muscle cells, or iPSC-derived embryoid bodies.

[0273] After culturing the cells under appropriate conditions, exosomes or microvesicles are isolated from the cells or cell culture supernatant using the methods described herein and methods known in the art. Depending on the purification method, the composition can be heterogeneous, purified, highly purified, homogeneous, or substantially homogeneous at the desired level of purity, as described herein. Exosomes or microvesicles and cells can be detectably labeled by adding a detectable label to the cells and / or exosomes or microvesicles by binding the label to them using the methods described herein or methods known in the art.

[0274] Depending on the purification method, the cell composition can be purified, highly purified, homogeneous, or substantially homogeneous and have the desired purity. Cells can be detectably labeled by adding a detectable label to the cells by binding the label to them using the methods described herein or methods known in the art.

[0275] Cells can be mixed or combined with a carrier, which in some cases is a carrier that does not occur naturally.

[0276] Preservatives or cryoprotectants can be combined or mixed with the cells or compositions containing them. These compositions can be lyophilized using methods known in the art and / or formulated into a suitable dosage form to facilitate use.

[0277] Cells and / or exosomes or microvesicles can be mixed or combined with a carrier, where the carrier is in some cases a carrier that does not occur naturally. On the one hand, the cell population is cultured in serum-free medium.

[0278] Preservatives or cryoprotectants can be combined or mixed with the cells, exosomes or microvesicles or compositions containing them. These compositions can be lyophilized using methods known in the art and / or formulated into a suitable dosage form to facilitate use.

[0279] MicroRNAs (miRNAs) prepared by these methods and selected from the following group: mir-373, mir-210, mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-21, mir-30c, mir-214, or mir-548q; and / or cells and compositions containing one or more proteins selected from the following group: Tsg101, CD9, Hsp70, flotirin-1, or GAPDH; or, mir-373, mir-210, etc. Cells or cell populations overexpressing one or more of the following muscle genes: mir-377, mir-367, mir-520, mir-548ah, mir-335, mir-30c, mir-214, or mir-548q; and / or one or more muscle genes selected from the group of paX3, PAX7, MYF5, MYOD, MYOG, or dystrophin may be advantageously used for one or more of the following subjects that require them: To regenerate damaged tissue such as muscle and / or cardiac tissue; to improve the survival rate of damaged tissue such as muscle and / or cardiac tissue; to promote the formation of new tissue such as cardiac tissue, muscle tissue, skeletal muscle tissue, blood vessels, capillaries, or muscle cells; to promote cardiac regeneration; to promote cardiac regeneration in subjects suffering from acute cardiac events; to promote cardiac regeneration in subjects suffering from myocardial infarction; to promote cardiac regeneration in subjects suffering from Duchenne muscular dystrophy (DMD) or Duchenne muscular dystrophy ("DMD")-associated cardiomyopathy; to promote cardiac regeneration in subjects suffering from age-related diseases, such as chronic obstructive pulmonary disease ("COPD"), arthritis, osteoporosis, osteoarthritis, diabetes, vascular dementia, or macular degeneration; stroke, arthritis, alopecia To promote the regeneration of tissue damaged by one or more of Zheimer's disease, amnesia, cystic fibrosis, inflammatory diseases, and cancer; to reduce cardiac wall thickness in tissue damaged by myocardial infarction; to alter the gene expression of one or more of the following: protein kinase C, IL-6, mmp, and PDGF; to reduce or inhibit the expression of inflammatory proteins, which may be chemokines, macrophages, or cytokines; to directly or indirectly stimulate angiogenesis and promote cardiac regeneration in subjects suffering from diseases selected from the following group: coronary artery disease, myocardial infarction, heart failure, hypoplastic left heart syndrome, peripheral artery disease (PAD), cardiac hypertrophy, valvular heart disease (aortic stenosis), myocardial hypertrophy, hypertrophic fibrosis; or to directly and / or indirectly inhibit cell replication. The method is achieved by administering an effective amount of exosome or microvesicles, cells, populations, or compositions containing them to a subject in need. The method may further include administering an effective amount of non-embryonic stem cells or progenitor cells to a subject, which may be the same or a different type of tissue requiring repair. On the one hand, the non-embryonic stem cells or progenitor cells are autologous to the subject. On the other hand, the non-embryonic stem cells or progenitor cells are allogeneic to the subject. Exosomes or microvesicles, cells, populations can be delivered to the subject locally or systemically. Subjects appropriately treated by these methods include animals, mammals, and human patients.Methods for determining the effectiveness of a treatment are known in the art, some of which are described herein, and are carried out by administering an effective amount of cells, exosomes or microvesicles or compositions containing them to a subject in need. As described above, methods for determining the effectiveness of a treatment are known in the art, some of which are described herein.

[0280] The method may further include administering an effective amount of non-embryonic stem cells or progenitor cells to a subject, which may be of the same type as the tissue requiring repair, but of a different type than the type of tissue being repaired. The cells may be non-embryonic stem cells, or the progenitor cells may be autologous or allogeneic to the subject.

[0281] Any appropriate method of administration may be used, for example, local, infusion, or intravenous administration as determined by the treating veterinarian or physician. The appropriate route of administration and dosage also depend on the age, health status, and sex of the subject being treated, as well as the formulation. The effective dose may be determined empirically by the treating veterinarian or physician. On the one hand, cells, exosomes, or microvesicles or compositions are delivered locally or systemically to the target tissue, or by intramyocardial or intracoronary routes.

[0282] A method is also provided for preparing a population of cardiac or skeletal myogenic progenitor cells from a population of human induced pluripotent stem cells (hiPSCs) by contacting the hiPSCs with an effective amount of givinostat (GIV), or by culturing the hiPSCs for an effective period in the presence of an effective amount of givinostat (GIV). In one aspect, the effective amount of GIV is 1 × 10⁻⁶ 6The concentration per cell is approximately 100 nM to 30 nM and may have a range as described herein. In one aspect, the cells may be cultured for an effective period in serum-free medium and in the presence of an effective amount of a Rho-related quinase (ROCK) inhibitor. Non-limiting examples of ROCK inhibitors are described above and incorporated herein by reference. In another aspect, an effective amount of a TGF-β1 receptor inhibitor is added to the cell culture medium. Non-limiting examples of TGF-β1 receptor inhibitors are described above and incorporated herein by reference.

[0283] After culturing the cells under appropriate conditions, the cells are isolated from the cell culture supernatant using the method described herein and methods known in the art.

[0284] Markers may be used to isolate and / or purify the composition. Depending on the purification method, the cell composition may be purified, highly purified, homogeneous, or substantially homogeneous, and / or have the desired purity percentage as described above. These cells may be labeled detectably by attaching a detectable label to the cells and by adding a detectable label to the cells using the methods described herein or methods known in the art. In one aspect, exosomes or microvesicles may be isolated from the cells using methods described herein and methods known in the art.

[0285] Cells and / or exosomes or microvesicles can be mixed or combined with a carrier, where the carrier may be one that does not exist in nature.

[0286] Preservatives or cryoprotectants may be combined with or mixed with cells, exosomes, or microvesicles or compositions containing them. These compositions may be freeze-dried using methods known in the art and / or formulated into appropriate dosage forms for ease of use.

[0287] Cells, exosomes, or microvesicles and compositions containing them are useful in methods of treating DMD by administering an effective dose of a population of cells to a subject that needs to regenerate skeletal muscle or cardiac muscle and / or so. Any suitable method of administration may be used, for example, local, infusion, or intravenous administration as determined by the treating physician. It also depends on the age, health status, and sex of the subject being treated, as well as the formulation. The effective dose may be determined empirically by the treating physician. On the one hand, cells, exosomes, or microvesicles or compositions are delivered to the tissue of the subject locally or systemically, or by intramyocardial or intracoronary pathways. Methods for determining the efficacy of treatment are known in the art, some of which are described herein.

[0288] Compositions for the repair or regeneration of damaged or affected cardiac tissue, comprising synthetic microRNA-146a (miRNA-146a) or fragments thereof, are also provided, optionally pharmaceutically acceptable carriers, and optionally the composition is purified, highly purified, or substantially homogeneous. Furthermore, the composition may be heterogeneous. Synthetic mRNA-146a is commercially available from manufacturers such as Sigma Aldrich or can be manufactured using conventional techniques. On the one hand, microRNA fragments or derivatives thereof are synthetically produced using techniques such as artificial microRNA (amiRNA) technology that utilizes the microRNA (miRNA) biosynthesis pathway to produce artificially designed small RNAs using the miRNA gene backbone in the following steps: Step 1: Predict the target miRNA from the gene code sequence; Step 2: Predict the artificial miR* sequence from the miR; Step 3: Predict the primer from the secondary structure of the premicroRNA. Furthermore, microRNA fragments, or their derivatives, are synthesized using sequences that mimic one or more endogenous microRNA molecules, mimicking endogenous miRNAs and enabling miRNA functional analysis through upregulation of miRNA activity. This is done using techniques such as miRNA mimics, which are small, chemically modified double-stranded RNA molecules. miRNA inhibitors are small, chemically modified single-stranded RNA molecules designed to specifically bind to and inhibit endogenous miRNA molecules, enabling miRNA functional analysis through downregulation of miRNA activity. Here, microRNA fragments or their derivatives are also modified to enhance their stability using techniques such as anti-miRNA oligonucleotides, which inhibit miRNA activity to fine-tune specific signaling pathways or block disease-induced miRNA function. These anti-miRNA oligonucleotides can be modified to enhance stability, target affinity, and promote cellular uptake.AntagomiR is an anti-miRNA modified in one or more of the following ways: (1) 2'-O-methylation of ribose sugar to enhance nuclease resistance and improve binding affinity to target RNA; (2) phosphorothioate bonding between nucleotides to enhance and provide stability of the oligonucleotide against nucleases by balancing phosphodiester and phosphorothioate; and (3) cholesterol conjugation at 3'UTR to promote cellular uptake. Locked nucleic acid (LNA-)-modified oligonucleotides are anti-miRNAs with 2' sugar modifications in which ribose is fixed to the C3-terminal conformation by a 2-O,4-C methylene bridge, strongly increasing affinity to complementary RNA, and the increased miRNA inhibition at double-strand melting temperature is achieved by anti-miRNAs that are single-strand oligonucleotides complementary to the target miRNA. AntagomiR and LNA oligonucleotides are anti-miRNAs modified to improve uptake and stability. Examples of Micro-ma 195 inhibitors are known and marketed by Sigma-Aldrich (HSTUD0320 SIGMA MISSION® Synthetic MicroRNA Inhibitor, Human hsa-miR-195-5p) and abmgood.com (Inhibitory hsa-miR-195-5p miRNA / MicroRNA Lentictor).

[0289] An example of a micro-MA 373 mime is known and marketed by Sigma-Aldrich as MISSION® microRNA mime hsa-miR-373* (sigmaaldrich.com / catalog / product / sigma / hmi0531lang=en®ion=US). Additional micro-MA 373 mimes, mir-373 inhibitors, mir-373 oglios, mir-373 expression vectors, and mir-373 precursors are known and marketed by: Switchgear genomics (switchgeargenomics.com / products / lightswitch-mirna-mimics-inhibitors / inhibitors / mir-300-399), mir373 inhibitor (switchgeargenomics.com / products / lightswitch-mirna-mimics-inhibitors / mir-300-399). miR373 mimics (switchgeargenomics.com / products / synthetic-Zutr-goclone-reporters / mir-300-399 (miR 3' UTR reporter)); Thermo Fisher (thermofisher.com / us / en / home / life-science / epigenetics-noncoding-rna-research / mirna-analysis / mirna-mimics-inhibitors / mirvana-mimics-inhibitors.html and thermofisher.com / us / en / home / life-science / epigenetics-noncoding-rna-research / mirna-analysis / mirna-mimics-inhibitors / ambion-pre-mir-precursors.html).

[0290] IDT oligonucleotides can be used and are commercially available (see idtdna.com / pages / decoded / decoded-articles / product-spotlight / decoded / 2012 / 04 / 11 / inhibiting-mirnasusing-antisense-oligonucleotides). Others can be purchased from Qiagen (qiagen.com / us / shop / genes-and-pathways / mirna-details.aspx ? mirnaid=7344).

[0291] miRNA compositions can be labeled detectably by attaching a detectable label to the miRNA by conjugating the label to it using the methods described herein or methods known in the art. miRNAs may be mixed or combined with carriers, which may optionally not be naturally occurring. Preservatives or cryoprotectants may be combined or mixed with miRNAs or compositions containing them. These compositions may be freeze-dried using methods known in the art, or formulated into appropriate dosage forms for ease of use.

[0292] Furthermore, methods for preparing these compositions are encapsulated or conjugated in synthetic liposomes using techniques known in the art. A "liposome" is a microscopic vesicle consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions ranging from several hundred angstroms to a fraction of a millimeter. Vesicle-forming lipids are selected to achieve a certain degree of fluidity or rigidity of the final complex, providing the lipid composition of the outer layer. These are neutral (cholesterol) or bipolar and include, but are not limited to, phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), and other types of bipolar lipids, including dioleoylphosphatidylethanolamine (DOPE), with hydrocarbon chain lengths ranging from 14 to 22 and saturated or having one or more double C=C bonds. Lipids that can produce stable liposomes, either alone or in combination with other lipid components, are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, stearoylphosphatidylethanolol-olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and palmitoyl phosphate phosphatidylcholine. (POPC) is palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimide-triethyl)cyclohexane-1-carboxylate (DOPE-mal).Further phosphorus-free lipids that may be incorporated into liposomes include, for example, stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-allyl sulfate, acetyl palmitate, glycerol lysinolate, hexadecyl sterol, amphoteric acrylic polymers, polyethyl oxy-fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoyl phosphatidylglycerol (DPPG), diotailyl phosphatidylglycerol, and (DOPG), and dicetyl phosphates that can form vesicles. Typically, liposomes can be classified into three categories based on their overall size and the nature of their lamellar structure. The three classifications, developed by the New York Academy Scientific Conference, “Liposomals and Their Use in Biology and Medicine” (December 1977), are multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar vesicles (LUVs). Biologically active agents described herein can be encapsulated in such forms for administration according to the methods described in, for example, U.S. Patents 5,512,295, 7,401,707, and 9,554,999. These compositions are further provided herein.

[0293] Synthetic miRNA compositions are useful for tissue regeneration in subjects requiring them. By administering one or more microRNA fragments or derivatives thereof to a subject, the microRNA fragments alter gene expression in the damaged tissue after administration, thereby improving the viability of the damaged tissue and promoting the formation of new tissue in the subject.

[0294] Any appropriate method of administration may be used, for example, local, infusion, or intravenous administration as determined by the treating veterinarian or physician. It also depends on the age, health status, and sex of the subject being treated, as well as the formulation. The effective dose may be determined empirically by the treating veterinarian or physician. Methods for determining whether a method is effective are known in the art, and some of them are described herein.

[0295] A method is also provided for isolating a population of exosomes or microvesicles, which involves culturing a population of non-embryonic human regenerative cells in the presence of a hydrolase enzyme to induce cells to secrete exosomes or microvesicles, thereby generating exosomes or microvesicles. Furthermore, the secreted exosomes or microvesicles are isolated or purified from the culture medium. Such non-limiting examples include sphingomyelinases selected from the group of lysosome acidic sphingomyelinase, secreted zinc-dependent acidic sphingomyelinase, neutral sphingomyelinase, and alkaline sphingomyelinase. Also, neutral sphingomyelinase includes one or more of magnesium-dependent neutral sphingomyelinase and magnesium-independent neutral sphingomyelinase. Such non-limiting examples include neutral sphingomyelinase type I, neutral sphingomyelinase type 2, and neutral sphingomyelinase type 3.

[0296] Methods and Uses of Cell Populations Another embodiment described herein is a method for restoring cardiac function in a tissue or host where it is needed. This use and other therapeutic, diagnostic, and research uses are described herein.

[0297] In one embodiment, the present invention provides one or more methods of: regenerating myocardial tissue, which is scar tissue of a damaged or affected heart; improving cardiac function, or treating a heart disease or condition in a patient in need thereof; by contacting the tissue to be regenerated with an effective amount of isoxazole or an isoxazole analog, or by administering an effective amount of the above-mentioned chemically modified cells or population of chemically modified cells to a subject in need thereof and / or a population of such cells. Furthermore, isolated cells, such as iPS cells or other progenitor cells or stem cells, are administered topically in an effective amount of isoxazole or an isoxazole analog. On the one hand, the treated iPS cells differentiate into muscle cells that form muscle fibers in the scar tissue of the heart. These cells may be autologous or allogeneic to the host or patient. The subject and cells may be any species as described herein.

[0298] A further embodiment of the present invention is a method for regenerating myocardial tissue in a suitable host by administering to the host an effective amount of chemically modified cells or a population of chemically modified cells as described above. The cells may be autologous or allogeneic to the host or patient. The subject and cells may be of any species as described above.

[0299] The method also includes administering an effective amount of iPS cells and an effective amount of isoxazole or an isoxazole analog to a patient in need. The administration may be local to the site of injury and may include direct injection of the cells and isoxazole or isoxazole analog into the patient's heart. The method also includes combination therapies as described herein. For example, the method can be combined with an effective amount of electrical stimulation before, after, or concurrently with this treatment. The method can also be combined with an effective amount of other cardiac induction small molecule compounds:

[0300] 1) Wnt / β-catenin inhibitors, such as IWR-1 and IWP 1. It is combined with the FDA-approved drug Pyrvinium (common trade name: Vanquin). Other novel small molecule compounds include ICG-001 (chemical name: (65,9 aS)-hexahydro6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxamide), which targets the Wnt / β-catenin pathway.

[0301] 2) TGF-B inhibitors such as the low molecular weight compound ITD-1, chemical name: (4-[1,1'-biphenyl]-4-yl 1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinoline ethyl carboxylate)

[0302] 3) Prostaglandins and COX-2. The production of prostaglandin E2 (PGE2) induced by activation of cyclooxygenase 2 (COX-2) and subsequent MI is an FDA-approved treatment for inducing labor under the brand name Dinoprostone. Another example is the low molecular weight compound agonist of PGI2, which is a synonym for 7,8-dihydro-5-[](E)-[[c-(3-pyridyl)benzylidene-aminooxyethyl]-1-naphthyloxy]acetic acid, supplied by Sigma-Aldrich, containing the low molecular weight compound ONO-1301.

[0303] 4) DPP-IV inhibitors combined with granulocyte colony-stimulating factor or G-CSF. (This approach combines two molecules: a small molecule inhibitor of dipeptidyl peptidase IV (DPP-IV), enzyme-1a which degrades SDF, and granulocyte colony-stimulating factor (G-CSF), a biomolecule that promotes the release of stem cells from the bone marrow via matrix metalloproteinase 2.)

[0304] 5) Angiotensin (1-7) and Mas receptor (formula: C41H62N 2011, supplied by Tochris).

[0305] Combination therapy may be sequential or simultaneous, as determined by the condition being treated, the patient's health status, and the choice of specific combination therapy.

[0306] On the one hand, stem cells or iPS cells are administered in the form of stem cell-derived cardiac patches (e.g., adult or bone marrow-derived progenitor cells, iPS cells, iPS cell-derived cardiac lineage cells, small immature stem cells, and / or artificial tissue cardiac patch transplantation for cardiac repair in heart disease). Thus, in one embodiment, stem cells include very small immature stem cells present in the bone marrow (contained within bone marrow stem cells), or peripheral blood cells (contained within circulating blood and cardiac-derived stem cells). These cells have remarkably high proliferative and differentiation potential and can also be readily reprogrammed and / or converted into cardiac precursors using isoxasol or isoxasol analogs or Wnt inhibitors. Thus, this method does not require the induction of iPS cells for safe cell transplantation. This disclosure also provides a method for promoting stem cell survival and differentiation by applying a cardiac patch with iPS cell derivatives treated with isoxazole or isoxazole analogues and a Wnt inhibitor administered across a scar area, thereby enabling better penetration, migration, and integration of patch cells (muscle cells, endothelial cells, smooth muscle cells (Figures 14A-14H)) with host cells.

[0307] This disclosure also provides a method for promoting the survival and differentiation of stem cells in vitro, comprising applying an effective amount of electrical stimulation to the stem cells. On the one hand, the effective amount includes electrical stimulation for about 1 to about 5 hours, and about 1.0 V / 1.5 cm to about 2.0 V / 2.0 cm.

[0308] This disclosure also provides a method for generating iPSC-derived muscle progenitor cells (MPCs) using isoxazoles and isoxazole-like compounds, and a method for pretreatment thereof or treatment of skeletal muscle, which induces functional CXCR4 expression in MPCs to promote the recruitment and engraftment of progenitor cells in dystrophic skeletal muscle. See Figures 15A-15B. For MPCs to be a viable therapeutic option for DMD, dystrophin gene expression must be sufficient to restore muscle contractility. MPCs obtained by the methods disclosed herein provide an abundant cell source for transplantation and offer an effective and safe treatment for dystrophic muscle regeneration. Transplantation of iPSC-derived progenitor cells combined with methods to optimize the host muscle microenvironment, such as treatment with effective doses of steroids, would more effectively improve dystrophy and improve the quality of life for DMD patients.

[0309] Patients who are appropriately treated by this method include those with diseases or disorders associated with cardiac dysfunction. This cardiac dysfunction includes, but is not limited to, congestive heart failure, isolated diastolic heart failure, myocardial infarction, and cardiac arrhythmias. Several forms of cardiac arrhythmias that can be treated include, but are not limited to, arrhythmia syndromes, bradyarrhythmic function, abnormal sinoatrial node function, atrioventricular block, and atrial and ventricular tachyarrhythmias.

[0310] Topical or systemic administration of cells or compositions by using catheters or cardiac patches with iPS cells treated with isoxasol or similar compounds can be performed in a single dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means of administration and dosage are known to those skilled in the art and will vary depending on the composition and / or cells used in the treatment, the purpose of the treatment, and the subject being treated. Single or multiple doses can be performed at dose levels and patterns selected by the treating physician. Appropriate dosage forms and methods for administering cells or drugs are known in the art. In a further aspect, the cells and compositions of the present invention can be administered in combination with other treatments.

[0311] Cells and cell populations are administered to a host using methods known in the art, for example, as described in U.S. Patent 6638369. Such administration of the cells or compositions of the present invention can be used to treat diseases as described herein and to create animal models of desired diseases, disorders, or conditions for experiments and screening assays. Screening assay

[0312] This invention provides methods for screening various agents that modulate gene expression, cell differentiation, exosome, or microvesicular expression, as well as therapeutic functions described herein. For the purposes of this invention, “agents” are intended to include, but are not limited to, biological or chemical compounds. For example, simple or complex organic or inorganic molecules, peptides, proteins (e.g., antibodies), polynucleotides (e.g., antisense), or ribozymes. A vast number of compounds, such as polymers including polypeptides and polynucleotides, as well as synthetic organic compounds based on various core structures, can be synthesized and are also included in the term “agents.” Furthermore, various natural sources, such as plant or animal extracts, can provide compounds for screening. It should be understood that it is not always explicitly stated that agents used alone or in combination with other agents will have the same or different biological activity as the agents identified by screening in this invention.

[0313] To carry out the screening method in vitro, a suitable cell culture or tissue culture containing modified cells is first provided. If the active substance is a composition other than DNA or RNA, such as a low molecular weight compound as described above, the active substance can be added directly to the cell culture or added to the medium for addition. As will be obvious to those skilled in the art, an "effective" mount, which can be determined empirically, must be added. If the active substance is a polynucleotide, it can be added directly by the use of a gene gun or electroporation. Alternatively, it can be inserted into cells using a gene delivery vehicle or other methods as described above. Positive and negative controls can be assayed to confirm the intended activity of the drug or other agent.

[0314] Methods for selecting a cell or cell population for initialization are also provided herein, including determining the expression level of miR-195 in a sample. Here, low expression of miR-195 selects the cell or population, and lack of low expression of miR-195 does not select the cell or population. Further, the method further includes determining the expression level of one or more markers selected from miR-29b, miR-205, miR-378, and miR-542-3p. Here, low expression of one or more markers selects the cell or population, and lack of low expression of miR-195 does not select the cell or population.

[0315] The following methods are useful for practicing the invention described herein. Experiment 1

[0316] In vitro generation of muscle progenitor cells (MPCs) from hiPSCs

[0317] Human induced pluripotent stem cells (iPS cells) (ATCC® ACS-1021TM) derived from human cardiac fibroblasts were cultured on a 6-well plate coated with vitronectin XF (STEMCELL Technologies Inc.) using mTeSR TM 1 (STEMCELL Technologies Inc.). The iPS cells were passaged every 4 - 6 days with ReLeSR TM (STEMCELL Technologies Inc.).

[0318] For the differentiation of iPS cells into MPCs, the iPS cells were dissociated into single cells using ACCUTASE TM (STEMCELL Technologies Inc.) and seeded at 1x10 cells / cm TM using mTeSR 2 supplemented with 5 μM of a RHO / ROCK pathway inhibitor (Y-27632, STEMCELL Technologies Inc.). After 24 hours, the medium was changed to fresh mTeSR TM 1. mTeSR TMThe culture medium was changed daily for the first three days. After three days, the medium was supplemented with 20 μM ISX-9 (MedChem Express) in mTeSR. TM The medium was changed to 1. The medium was changed every other day. After 6 days, the medium was changed to RPMI 1640 medium (Thermo Fisher Scientific) supplemented with N2 supplement (Thermo Fisher Scientific) and 20 μM ISX-9, and the cells were cultured for a further 3 to 6 days, changing the medium every other day. Methods for the generation of exosomes or microvesicles and Cpcs include cell culture and maintenance of hiPSCs.

[0319] Human iPS cells (ACS-1021, ATCC, USA) were maintained in mTeSR1 medium (STEMCELL Technologies Inc.) on a 6-well plate coated with vitronectin, with daily medium changes. Following the manufacturer's protocol (STEMCELL Technologies Inc.), cells were reconditioned every 4–7 days. TM The cells were subcultured using reagents. CPC Generation

[0320] In short, hiPSCs maintained on a 6-well vitrod plate in mTeSR1 medium (Stem Cell Technology) were dissociated into single cells using Accutase solution (Invitrogen) at 37°C for 10 minutes. Subsequently, 1 × 10¹³ cells were added to mTeSR1 supplemented with 5 UM ROCK inhibitor (Y-27632, Stem Cell Technology). 6 Cells were seeded at a rate of one cell / well onto a 6-well plate coated with vitronectin for 24 hours. Starting the following day, the cells were cultured in mTesR1 for 3 days, with daily cell changes. Subsequently, the culture medium was switched to insulin-free RPMI / B27 supplemented with ISX-9 (20 μM, dissolved in DMSO, Stem Cell Technology) for 7 days. EB Generation

[0321] The applicant generated EB cells using the hanging-drop method in RPMI / B27 minus insulin medium. Generation and isolation of exosomes or microvesicles from cardiac progenitor cells, EB cells, or hiPSCs.

[0322] Exosomes or microvesicles were generated from human iPS cell line ACS-1021 (ATCC, USA), embryoid bodies (EB), and CPCs induced by ISX-9. CPCs were generated as described in Experiment 10. Acclimatized medium was collected from the hiPSCs and CPCs. The acclimatized medium was centrifuged at 3000 rpm for 30 minutes to remove cells and debris, followed by filtration through a 0.22 μm filter to remove remaining debris. The medium was then further concentrated to 500 μl using an Amicon Ultra-15 100 kDa centrifugal filter unit (Millipore). Isolation of exosomes or microvesicles in the concentrated medium was performed using a qEV size exclusion column (Izon Science). Exosome fractions or microvesicle fractions were collected and concentrated to a final volume of <100 μl using an Amicon Ultra-4 10 kDa centrifugal filter unit. Purified exosomes or microvesicles were stored at -80°C. They were then characterized by nanotracking analysis, protein analysis, and ultrastructural analysis. Particle size and concentration distribution were measured by variable resistance pulse sensing.

[0323] Particle size and concentration distribution for exosome or microvesicle isolation analysis were performed using a qNano instrument (Izon Science) with adjustable resistance pulse sensing. Briefly, particle counts were performed at least 600–1000 events using an NP200 nanopore film stretched between 46.5–47.5 mm and under a pressure of 20 mmbars. Calibration was performed using a CPC200 bead (diameter: 210 nm) of known concentration. Data were processed using Izon Control Suite software. Transmission electron microscopy

[0324] Exosomal or microvesicular pellets were fixed with 4% paraformaldehyde (PFA). After washing a total of eight times with distilled water, the grid was contrasted with uranyl oxalate solution for 5 minutes, and then transferred to methylcellulose-uranyl acetate on ice for 10 minutes, as described in previous studies. The samples were examined using a JEOL JEM-1220 transmission electron microscope (TEM) (JEOL USA, Inc.). Selected exemplary embodiments

[0325] The applicant discovered that iPSc can be chemically induced to induce upregulation of cardiac genes in response to DNA hypomethylation, and that this may enable successful proliferation in affected hearts with little to no chance of tumorigenesis.

[0326] Skeletal myoblasts (SMS) purified from young male Oct3 / 4-GFP+ transgenic mice were treated for 5 days with 500 μMRG-108, a DNA methyltransferase inhibitor, in 0.5% DMSO in knockout DMEM. Two weeks later, GFP-expressing SM-derived iPS cells (SiPS) with GFP+ colonies and morphological features of mouse embryonic stem cells were isolated and grown in vitro. The SiPS were positive for alkaline phosphatase activity, expressed SSEA1, and exhibited a panel of pluripotency markers similar to those of ES cells (Figure 1), and developed teratomas in nude mice. Although the studies described herein were performed in a mouse model, the use of the same markers and agents would likely yield similar, if not identical, results in human cells and tissues. To direct SiPS cells towards cardiac lineage cells, they were treated with a small molecule compound (isoxasol, 20 μM, Sigm α-Aldrich or Isox 9, StemCell Technologies, Inc. Vancouver, BC) for 5 days, after which DNA methyltransferase (DNMT) activity, cell proliferation, and cardiac gene expression were analyzed. DNMT activity was completely abolished in SiPS treated with the small molecule compound, with a 95% reduction in overall DNA methylation (Figure 2). These SiPS cells showed increased proliferation activity (p < 0.01 vs. untreated SiPS) as assessed by cell proliferation assays, and also became more tolerant to apoptosis, a key consideration for preventing cell death in an ischemic environment (Figures 3A-3B). IPS cells treated with the small molecule compound showed a significant reduction in cytoplasmic cytoplasmic cytoplasmic migration compared to untreated IPS cells (Figure 4).

[0327] To direct SiPS cells towards cardiac lineage cells, they were treated with a small molecule (Isoxazole, 20 μM, Sigma-Aldrich or Isox 9, StemCell Technologies, Inc. Vancouver, BC) for 5 days, and DNA methyltransferase (DNMT) activity, cell proliferation, and cardiac gene expression were analyzed. DNMT activity was completely abolished in small molecule-treated SiPS cells, with a 95% reduction in overall DNA methylation (Figure 2). These SiPS cells showed increased proliferation activity as assessed by cell proliferation assays (p<0.01 vs. untreated SiPS cells) and also became more tolerant to apoptosis (Figures 3A-3B), which is an important consideration for preventing cell death in an ischemic environment. Small molecule-treated iPS cells showed a significant reduction in cytoplasmic cytochrome c translocation compared to untreated iPS cells (Figure 4).

[0328] RT-PCR analysis revealed cardiomyocyte-like gene expression profiles with significant upregulation of Nkx-2.5 (p < 0.01 vs. untreated iPS cells; see Figures 5A-5B). Approximately 60% of treated iPS cells were Nkx-2.5 positive. Given the critical importance of post-transcriptional regulation to gene expression and the fact that cell viability molecular phenotyping analysis was performed, Affymetrix array-based gene expression profiling further confirmed 2-3-fold downregulation of Dnmt1, Dnmt3b, and Max gene-related proteins associated with global DNA hypomethylation and myc-dependent cell transformation (see Table 1). Furthermore, there was 2-3-fold concomitant upregulation of CCL7, CXCR2, CXCR5, intrinsic membrane protein 2A, and ephrin A3 (Figure 6). These were associated with DNA synthesis, cell proliferation, cell matrix interactions, and chemoattractions. miR microarray analysis showed upregulation of cardiac-specific miR-133 and 762, and downregulation of pluripotency-related miR-290 cluster, miR-574-5p, and let-7 family (see Figures 7A–7D). Western blot analysis showed significant upregulation of Gai protein levels compared to untreated iPS cells (see Figure 7E). TIFF0007837302000010.tif125167

[0329] Sips treated with a low molecular weight compound were stained with PKH 26 to locate them within the heart and transplanted into the myocardium 6 weeks after 30 minutes of coronary artery ligation. Cardiac function was monitored by echocardiography before heart harvesting to visualize the fate of the transplanted Sips. The treated Sips were differentiated into muscle cells that form muscle fibers in the scar tissue. The scar area of ​​the left ventricle was more myofascial with treatment with isox-treated SiPS (Figures 8B, 8C, 8D) compared to the untreated / control heart (Figure 8A). These are significant findings and have been previously reported in the initial patent application, as only new photographs are shown graphically in the figures (see Figures 8A–8F).

[0330] Cardiac function: Changes in overall cardiac function over time, including left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS), were measured in control and treated infarcted hearts. Pathological remodeling of left ventricular chamber dimensions during systolic (LVD) and diastolic (LVDd) was also significantly reduced in hearts treated with CP (2.97 mm and 3.99 mm) compared to hearts treated with DMEM (4.77 mm and 4.78 mm). See Figure 8E. CP implantation significantly improved LVEF and LVFS (56.8 ± 1.32%; 25.5 ± 0.4%) compared to DMEM-injected infarcted hearts (n=4; 32.42 ± 1.03% and 13.0 ± 0.4%). See Figure 8F.

[0331] The applicant also discovered that isoxazoles induce DNA hypomethylation and myc-dependent cell transformation in iPS cells and are associated with DNA synthesis, cell proliferation, cell matrix interactions, and chemotaxis. Isooxazole compounds upregulated CXC chemokine receptors and intrinsic membrane proteins, the Epherin family, and related receptors particularly involved in the development of erythropoiesis.

[0332] The applicant further discovered that isoxazol upregulated the downregulation of cardiac-specific miR-133, miR-762, and pluripotency-associated miR-290 clusters and the let-7 family in iPS cells compared to untreated iPS cells. (10333) Thus, the above report indicates that small molecule compound-mediated modifications of iPS cells can grow in infarcted hearts and replace scar tissue with active muscle cells coupled together with electrical connections (gap junctions). Small molecule compounds of the sulfonylhydrazone family can induce cardiac genes in myoblast-derived iPS cells. These small molecule compounds are known to induce muscle differentiation in Notch-activated epicardial-derived progenitor cells (Russell JL et al. (2012) ACS Chem Biol. 7(6): 1067-1076). Small molecule compound-induced iPS cells were transplanted into a post-ischemic model and improved overall cardiac function compared to untreated iPS cells. Recovery of cardiac function depended on the survival of iPS cell-derived progenitor cells. Small molecule compounds are innovative in inducing tissue-specific gene expression in iPS cells toward tissue differentiation, as well as in determining the temporal and spatial patterns of development.

[0333] Therefore, the above report indicates that small molecule-mediated modifications of iPS cells can proliferate in infarcted hearts and replace scar tissue with active muscle cells bound together with electrical connections (gap junctions). Small molecules of the sulfonylhydrazone family can induce cardiac genes in myoblast-derived iPS cells. These small molecules are known to induce muscle differentiation in Notch-activated epicardial progenitor cells (Russell J. L. et al. (2012) ACS Chem Biol. 7(6): 1067-1076). Small molecule-induced iPS cells were transplanted into a post-ischemic model and showed improved overall cardiac function compared to untreated iPS cells. Recovery of cardiac function depended on the survival of iPS cell-derived progenitor cells. Small molecules are innovative in inducing tissue-specific gene expression in iPS cells toward tissue differentiation, as well as in determining the temporal and spatial patterns of development.

[0334] The applicant found that when induced pluripotent stem cells are treated with appropriate small molecule compounds, DNA methylation can be pharmacologically inhibited, thus playing a crucial role in regulating cardiac development genes. Without being constrained by theory, the effects of small molecule compounds on the efferin family may manipulate the process of hematopoietic progenitor cell generation and could be beneficial for clinical hematopoietic malignancies.

[0335] The applicant also found that isoxazol upregulated the downregulation of heart-specific miR-133, miR-762, and pluripotency-associated miR-290 clusters and let-7 family in iPS cells compared to untreated iPS cells.

[0336] Eric Oslon previously reported the use of these compounds in neurogenesis, epicardial progenitor cells in U.S. Patent No. 8318951 (951 patent). Regardless of these studies, the findings reported herein are unique and innovative in that they address small molecule compound-induced epigenetic changes that can be manipulated to render iPS cells for proliferation in infarcted hearts. This study highlights iPS cells that can be generated from patient cells (skeletal muscle in this case) and reintroduced into the same patient after pretreatment with small molecule compounds. These findings differ from Oslon's studies because the iPS cells in this disclosure were therapeutically pre-designed (or pre-treated) for proliferation in scar tissue of mouse hearts after coronary artery ligation or heart attack. There is significant regeneration of scar tissue by iPS cells. Furthermore, Oslon injected drugs into living mice to act on cardiac ancestors present in the heart. The applicant believes the drugs acted on various cells of the heart. Here, the composition and method involved pre-designing iPS cells into cardiac progenitor cells in a dish, and then reintroducing them into a damaged heart.

[0337] The applicant also found that pretreatment of iPS cells with small molecule compounds reduces apoptosis, which is another novel finding. For example, less apoptosis was observed in small molecule compound-induced iPS cells compared to uninduced iPS cells. The results reported in the '951 patent were generated solely using a trypanble-exclusion assay for cell viability, and not for cell apoptosis, which is important for cell survival under ischemic conditions.

[0338] The applicant also found that DNA methyltransferase (DNMT) activity was completely abolished in chemically modified iPS cells. In iPS cells modified with DNA hypomethylation and small molecule compounds associated with cell transformation, there was 2-3x downregulation of Dnmt1, Dnmt3b, and Max gene-related proteins. 2-3x upregulation was observed of CCL7, CXCR2, CXCR5, intrinsic membrane protein 2A, and EphrinA3, which are associated with cell recruitment, chemotaxis, cancer, and erythropoiesis. The applicant further observed upregulation of cardiogenicity-specific miR-133 and miR-762, and downregulation of pluripotency markers, as well as pluripotency-related miR-290-295 clusters and the let-7 family. This confirms the induction of cardiac regulatory genes by microRNAs by downregulating pluripotency genes.

[0339] Furthermore, while the 951 patent did not observe Ga protein levels compared to the studies reported here, this report observed an upregulation of Ga protein levels, which were blocked when chemically modified iPS cells were treated with GPCR blockers, negating all in vitro effects in small molecule compound-induced iPS cells. Oslon has previously reported the use of these compounds in neurogenesis and epicardial progenitor cells. Regardless of previous research, the applicant believes that the current findings addressing small molecule compound-induced epigenetic changes that can be manipulated to make iPS cells suitable for proliferation in infarcted hearts are unique and innovative. Another novelty shown here is that iPS cells first stimulated with isoxasol and similar small molecule compounds can be converted not only into muscle cells but also into vascular (endothelial) progenitor cells and smooth muscle cells.

[0340] Based on the applicant's knowledge and results to the best of their knowledge, as reported in Example 2 below, it is understood that isoxazole and other similar small molecules act on other stem cells in the body. For example, bone marrow and heart-derived stem cells are directly reprogrammed with small molecule compounds such as isoxazole into muscle cells, endothelial cells, and smooth muscle cells. Thus, isoxazole and similar small molecule compounds can be administered directly, such as by injection into the patient's heart. For example, in heart attack patients to convert inflammatory cells and stem cells mobilized to the ischemic site to replace scar tissue into cardiac cells. The ultimate effect is to help regenerate the heart damaged after a heart attack. The disclosed method has the advantage of being non-viral based, using previously approved compounds, which simplifies clinical adoption. Furthermore, the applicant believes that the disclosed method will dramatically increase cardiac progenitor cells in a one-step treatment of monolayer iPSCs within weeks after treatment with isoxazole or other similar cardioniodin-like small molecule compounds without converting them into embryoid bodies. CDNG1 / vuc230, CDNG2 / vuc198, and CDNG3 / vuc247.

[0341] Current approaches to using iPS cells are limited by genetic mutations and / or tumor (cancer, etc.) proliferation versus the applicant's methods, approaches, and techniques. This method is beneficial in that it does not require genetic mutations and improves safety for clinical use.

[0342] This is the applicant's belief, and to the best of the applicant's knowledge, currently reported research provides the first evidence that iPS cells can be made therapeutically safe by altering their chromatin arrangement for upregulation of cardiac genes. These so-called cardiac progenitor cells can proliferate and regenerate dying cardiomyocytes with limited cell death. Reprogramming iPS cells into a desired cardiac lineage is a prudent strategy in cardiac therapy. Experimental method Mouse SiPS maintenance

[0343] SiPS cells were maintained on mitomycin C-treated mouse embryonic fibroblast (MEF) dishes in knockout Dulbecco's modified Eagle medium (knockout-DMEM, Invitrogen, California, USA) supplemented with 20% knockout serum substitute (KSR; Invitrogen, USA), 0.1 mM MEM non-essential amino acid solution (Invitrogen, CA, USA), 0.2 mM L-glutamine (Invitrogen, USA), 0.1 mM β-mercaptoethanol (Invitrogen, California, USA), and 1000 U / ml LIF (Millipore) 0.5% penicillin and streptomycin. The colonies thus generated were periodically detached at 3-4 day intervals using 0.2% collagenase-1V (Invitrogen, CA, USA) dissociated into a single-cell suspension containing 0.025% trypsin (Sigm a Aldrich, MO, USA), and then replated onto MEF. Cell proliferation assay

[0344] A cell proliferation assay was performed using the MTS[3-(4,5-dimethylthiazolyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, as recommended by the manufacturer (Promega). Using an automated ELISA plate reader, the amount of formazan product, directly proportional to the number of viable cells in culture, was read from the plate at 490 nm. DNA methyltransferase (DNMT) activity assay

[0345] Nuclear extracts were isolated using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, IL, USA). Total DNMT activity was determined using the EpiQuik DNA methyltransferase activity assay kit (Epigen tek, Brooklyn, NY) according to the manufacturer's protocol. Enzyme activity of samples and controls was measured at 450 nm using a microplate recorder (Hidex Chameleon, Finland), and DNMT activity (OD / h / ml) was calculated according to the formula (sample OF - blank AND) / (sample volume)1000. RT-PCR and quantitative RT-PCR

[0346] Total RNA from low-molecular-weight compound-treated and untreated SiPS cells was isolated using the RNe asy mini-kit (Qiagen, Merriland, USA), and the OmniScript reverse transcription kit (Qiagen, Merriland, USA) was used for cDNA synthesis according to the manufacturer's instructions. For PCR amplification, 1 μg of cDNA from the reverse transcription reaction was added to a PCR mixture containing the recommended amounts of PCR buffer, Q solution, dNTP mixture, reverse and forward primers, Taq DNA polymerase, and distilled water. PCR conditions included initial denaturation at 95°C for 4 minutes, 32 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 1 minute, and final extension at 72°C for 7 minutes. PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, visualized, and photographed on a UV transilluminator (Bio-Rad, USA).

[0347] Myocardial infarction model The animals were anesthetized with ketamine / xylazine (0.05 ml intraperitoneally). A midline cervical skin incision was made for intubation. The animals were mechanically ventilated with oxygenated room air (1.5 L / min) using a rodent ventilator (Model 683, Harvard, Massachusetts, USA). Body temperature was carefully monitored with a probe (Cole-Parmer Instrument, Illinois, USA) and maintained at 37°C throughout the surgical procedure. The heart was exposed by a limited left thoracotomy, and the left anterior descending coronary artery was ligated with Prolen #9-0 sutures. Myocardial ischemia was confirmed by a change in the color of the left ventricular wall. Animals were divided into groups (n = 12 per group) for intramyocardial injection of 10 μL of basal DMEM without cells (Group 1), or containing 3.6 x 10^5 SiPS (Group 2), or containing 3.6 x 10^5 SiPS-CPs (Group 3). Ten minutes after coronary artery ligation, cells were injected under direct visualization into multiple sites (3-4 sites per heart) around the ischemic area of ​​the free wall of the left ventricle. For follow-up of transplanted cells after engraftment and determination of their fate, cells were labeled with PKH26 (Sigm a, product number PKH26-GL) according to the manufacturer's instructions. To reduce pain, Buprinex (0.05 ml) was administered subcutaneously during the first 24 hours after surgery. For assessment of cardiac function, animals were euthanized 4 days and 6-8 weeks after transthoracic echocardiography. The hearts were either frozen or fixed in 10% formalin solution and then paraffin-embedded for immunohistochemical study. Transthoracic echocardiography

[0348] Animals (n = 8 per group) were anesthetized and lightly fixed in a supine position on a warm pad. After shaving the chest, acoustic gel was applied, and transthoracic echocardiography was performed using an HDI-5000 SONOS-CT(HP) ultrasound system with a 7 MHz transducer. The heart was imaged in two-dimensional mode in parasternal long-axis and / or parasternal short-axis views, and then an M-mode cursor was used to position it perpendicular to the interventricular septum and left ventricular posterior wall. Subsequently, modal images were obtained. For each animal, measurements were obtained from 4-5 consecutive cardiac cycles. Interventricular septal thickness (VST), left ventricular dimensions (LVID), and left ventricular posterior wall thickness (LVPW) were measured from two-dimensional oriented M-mode images of the left ventricle in both systolic and diastolic states. An index of left ventricular systolic function was determined using the mean values ​​from all measurements in the animals. For example, the left ventricular shortening ratio (LVFS) and left ventricular ejection fraction (LVEF) are expressed as percentages using the following relationship: LVFS = (LVEDd2LVESd) / LVEDd6100 and LVEF = [(LVEDd32LVESd3) / LVEDd3]6100. immunocytochemistry

[0349] For immunocytochemistry, differentiated SiPS colonies were immunostained with their respective specific primary antibodies (anti-Oct3 / 4, anti-Sox2, anti-N anog antibody, all at 1:100 concentrations; Cell Signaling, Danvers, USA). For immunostaining, low-molecular-weight compound-treated SIPs were seeded on 0.1% gelatin-coated chamber slides. Cells were fixed in PBS containing 4% paraformaldehyde at room temperature for 10 minutes. After washing with PBS, cells were blocked by CAS block (Invitrogen, CA, USA) at room temperature for 45 minutes and immunostained with antigen-specific primary antibodies Nkx-2.5, Gata 4, and Sarcomeric actin (Santa Cruz, Calif., USA). Primary antibody-antigen reactions were detected using fluorescently conjugated specific secondary antibodies. Nuclei were stained with 5 μg / ml 4'6-diamidino-2-phenylindole (DAPI; Invitrogen, CA, USA). Fluorescence signals were observed and photographed using fluorescence microscopy (Olympus; Tokyo, Japan). Gene expression profiling

[0350] Affymetrix array-based gene expression profiling further confirmed 2-3-fold downregulation of Dnmt1, Dnmt3b, and Max gene-related proteins associated with global DNA hypomethylation and myc-dependent cell transformation. In addition, there was 2-3-fold concomitant upregulation of CCL7, CXCR2, CXCR5, intrinsic membrane protein 2A, and ephrin A3. Experiment No. 2

[0351] The applicant identified a population of bone marrow stem cells, named small immature stem cells (SJSCs), derived from aged mice that express pluripotency and cardiac markers. The applicant predicted when these cells should be treated with isoxazole compounds to be more suitable for cardiac differentiation and can be used as an alternative to iPS cells (Igura, K. et al. (2013) Am J Physiol Heart Circ Physiol. 305(9): H1354-H1362). Experiment No. 3

[0352] This experiment discloses an alternative method for generating cardiac progenitor cells by pretreatment with electrical stimulation supplemented with cardiogenic small molecule compounds. The applicant identified another cell population expressing hematopoietic precursor markers (c-kit), pluripotency markers (Oct-4, Sox2, Nanog), stem cell population marker (Bcrpl), early cardiac lineage markers (Nkx 2.5, GATAL, MEF2C), and vascular precursor marker (Fiki) (Kim, SW et al. (2013) Cardiovasc Res. 100 (2): 241-251). Preconditioning (PC) of stem cells by short-term ischemia / anaxia or treatment with alternative mimetic agents improves their post-engraft survival and differentiation characteristics. However, there are no known reports on the role of PC using electrical stimulation (EleS) in stem cell survival, adhesion, and cardiac differentiation. The heart generates a constant electric field, and its effect has not been investigated in pre-transplant stem cells. This study demonstrates that EleS provides a PC effect on the survival of cardiac stem cells (Sca-1+ CSCs) through increased cell adhesion via focal adhesion kinase (FAK) activation, and releases connective tissue growth factor (CTGF) through miR-378 downregulation. Connective tissue growth factor (CTGF) was found to be responsible for the survival and adhesion of EleS-induced CSCs (Eles CSCS). Furthermore, miR-378 was identified as a candidate Ctgf regulator for Ele-CSCs. This is another stem cell type that expresses both pluripotency and cardiac genes, and these cells can be further developed using isoxazole for cardiac progenitor cells. Isolation of Sca-1 + CSC

[0353] C57BL6 mice (H arl an) were used for the isolation of CSCs. 12-week-old C57BL6 mice were anesthetized by intraperitoneal injection of ketamine / xylazine (87-100 mg and 13-15 mg / kg, respectively). The depth of anesthesia was monitored by pinching the toes to create a positive pressure and relaxing the muscles. The heart was removed and washed with ice-cold PBS to remove blood cells. After removing the aorta, pulmonary artery, and pericardium, the entire heart was finely chopped and digested at 37°C for 20 minutes with 0.1% type II collagenase (Invitrogen) and 0.01% DNase I (Worthington Biochemical Corporation). The obtained cells were passed through a 40 μm filter to remove debris, fractionated with 70% Percoll (Fluka), and cultured in maintenance medium containing serum-free DMEM / F12 (Invitrogen) supplemented with B27 (Invitrogen), 20 ng / ml EGF (Sigma), and 40 ng / ml bFGF (basic fibroblast growth factor, Peprotech). After one week, cells were transferred to a new dish containing serum-free maintenance medium at a density of 100 cells / cm² to initiate colony formation, and each colony was mechanically picked up for individual subculturing in 24 welldishes in DMEM / F12 (Invitrogen) supplemented with 2% FBS, B27 supplement, 20 ng / ml EGF, 40 ng / ml bFGF, and 10 ng / ml LIF (leukemia suppressor, Millipore). Colony-derived cells were seeded into new dishes to achieve 90% confluence and maintained in DMEM / F12 containing 2% FB S. CSCS Cheer

[0354] Cells were seeded at a cell density of 3 × 10⁶ cells / 35 mm dish, and 24 hours later, the cells were serum-starved for 15 hours, followed by the use of Eles (ElesCSC) with a cell culture pacer system (IonOptix). Cells were subjected to Eles at 1.5V / 1.8cm for 0, 1, and 3 hours using a biphasic square pulse (5ms) at a frequency of 5Hz. Cells without Eles (Non-ElesCSC) were used as baseline control. The cells were subsequently harvested and used in various molecular and cellular studies. Experiment No. 4

[0355] Isolation of old mesenchymal stem cells (OMSCs) and young mesenchymal stem cells (YMSCs) from bone marrow

[0356] This study followed the Guidelines for the Management and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication #85-23, revised 1985) and protocols approved by the Institutional Animal Management and Use Committee of the University of Cincinnati. BMSCs were isolated from the bone marrow of C57BL / 6 mice (young; 2 months, 2 months; 24 months, respectively) as previously described in Igura K et al. (2013) Am J Physiol. Heart Circ. Physiol 305: H1354 H1362. Briefly, BMSCs were cultured in 100 mm dishes containing low-glucose Dulbecco's modified Eagle medium (DMEM) (HyClone Laboratories, Logan, Utah, http: / / www.hyclone.com) for 6–10 days, supplemented with 10% fetal bovine serum (FBS). Hematopoietic bone marrow cells that were not attached were discarded during routine fresh medium changes. YMSCs were isolated from old bone marrow by sieving through wells with 3 μm pores. (Cell culture insert; BD Bioscience, San Diego, CA http: / / www.bdbiosciences.com) Homogeneity of the YMSC population was confirmed by surface marker expression by fluorescence-activated cell sorting analysis, as previously described in Igura K et al. (2013) Am J Physiol Heart Circ Physiol 305: H1354-H1362. Quantitative fluorescence in situ hybridization for telomere length measurement

[0357] Telomeres were detected by fluorescence in situ hybridization (FISH) using a PNA telomere Cy3-labeled probe (F1002; TelC-Cy3, PNA Bio) according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde for 1 hour. After washing with phosphate buffer (PBS), the cells were incubated with RNase 100 ng / 1L for 20 minutes and then with 0.005% pepsin at 37°C for 5 minutes. After dehydration with 70%, 85%, and 100% cold ethanol, the cells were incubated with a 200 nM PNA telomere probe at 85°C for 10 minutes and then incubated at room temperature for 2 hours. Next, the cells were incubated with 2 × 3 saline sodium citrate (SSC) buffer at 60°C for 10 minutes, and 4',6-diamidino-2-phenylindole (DAPI) and Cy3 signals were simultaneously acquired in separate channels using a confocal microscope (Fluoview FV1000, Olympus, Tokyo, http: / / www.olympus-global.com). For image quantification, the maximum projection from the image stack (10 compartments in 1 mm steps) was generated. Telomere length was analyzed using TFL-TeloV2-2 free software (Vancouver, Canada). The program was used to calculate and present the integrated fluorescence intensity value of each telomere, proportional to the number of hybridized probes. TFL-Telo is an application program used to estimate telomere length from captured images of the midterm phase stained for quantitative FISH (Q-FISH) analysis, as described by Poon et al. (1999) Cytometry 36: 267-278. Reverse transcription-polymerase chain reaction analysis

[0358] Following the manufacturer's instructions, total RNA was isolated from various cell treatment groups using the RNeasy Mini Kit (Qiagen, MD, http: / / wwwl.qiagen.com), and cDNA was prepared using the Omniscript-RT Kit (Qiagen). For polymerase chain reaction (PCR) amplification, 1 μg of cDNA from the reverse transcription reaction was then added to a PCR mixture containing the recommended amounts of Qiagen PCR buffer, Q-Solution, dNTP mixture, reverse and forward primers, Taq DNA polymerase, and distilled water. Each PCR reaction was performed using a specific primer. Isolation and detection of miRNAs

[0359] As previously described in Kim et al. (2012) J.Mol.Med.90:997-1010, miRNAs were extracted using the mirVana miRNA isolation kit, and miR-195 expression was detected using the mirVana qRT-PCR miRNA observation kit (Ambion, Life Technologies, Austin, Tex., http: / / www.ambion.com) and the QuantiTect SYBR green PCR kit (Qiagen). Specific miRNA primers were purchased from Ambion. miRNA microarray

[0360] Total RNA samples obtained from OMSC and YMSC were sent to Exiqon (Denmark, http: / / www.exiqon.com / ) for miRNA microarray profiling. The data was analyzed by Exiqon using an in-house developed computer program. Intensity values ​​were converted to a log 2 scale, and magnification changes were shown on a log 2 scale. A t-test was performed between OMSC and YMSC profiling, and statistically significant differences were considered at p < 0.01. FISH to detect miR-195

[0361] In situ detection of miR-195 was performed in OMSC and YMSC cells plated on chamber slides. For cell culture, samples were fixed in 4% paraformaldehyde at room temperature for 20 minutes, followed by two washes in PBS. The fixed cells were then pre-hybridized in hybridization solution (Biochain, California) at room temperature for 3 hours prior to hybridization. A probe complementary to miR-195 (3 pmol; LNA-modified and fluorescein isothiocyanate (FITC)-labeled oligonucleotide; purchased from Exiqon) was hybridized to the cells at 22°C for 13–16 hours. The Tm was lower than the predicted Tm of the probe. After washing with SSC buffer following hybridization, the in-situ hybridization signal was detected by confocal microscopy (Fluoview FV1000, Olympus). Luciferase activity assay

[0362] The precursor miR-195 expression vector was constructed in a feline immunodeficiency virus-based lentiviral vector system (Geneco-poei a). The luciferase reporter construct, containing the 3' untranslated region (UTR) of mouse Tert, was designed to include the mmu-miR-195 binding site. Cells were seeded in triplets on 24-well plates and co-transfected with the miR-195 vector (or scrambled vector) and reporter construct using lipofectamine 2000 (Invitrogen, Carlsbad, Calif., http: / / www.invitrogen.com). Firefly luciferase activity was measured using a dual luciferase reporter assay system kit (Promega, Madison, Wis., http: / / www.promega.com) according to the manufacturer's instructions. Transfection efficiency was standardized by sea oyster cipherase activity. Lentivirus-mediated suppression of miR-195 OMSC

[0363] Lentivirus-containing miR-195 inhibitor expression vectors were generated using the Lenti-Pac HIV expression packaging system (GeneCopoeia) according to the manufacturer's protocol. A 2.5 μl lentivirus miR-195 inhibitor expression plasmid or scrambled mixture, along with 5.0 mL of EndoFectin Lenti and EndoFectin Lenti reagent, was added to Opti-MEM I to form a DNA-endofectin complex. After incubating the complex at room temperature for 10–25 minutes, the DNA-endofectin complex was added to 293Ta cells in DMEM containing 10% FBS. The cells were then incubated overnight at 37°C in 5% CO2. The culture medium was replaced with fresh DMEM containing 5% FBS and 1 / 500 volume of TiterBoost reagent relative to the medium. The virus-containing pseudovirus medium was collected 48 hours after transfection, filtered, and concentrated. For transduction of OMSC by lentivirus, 10 × 10⁶ cells were used. 6 Cells were plated with OMSCs and 20 μl of viral suspension was added. To enhance lentiviral transduction efficiency, cells were left at 4°C for 2 hours and incubated in 5% CO2 at 37°C for 48 hours. Aging-related B-Ga lactosidase (gal) staining. Aging-related B-Gal was detected by an aging observation kit (BioVision, CA, http: / / www.biovision.com / ) as instructed by the manufacturer. Terminal deoxynucleotidyltransferase dUTP nick end labeling assay.

[0364] Apoptosis cell death was detected by terminal deoxynucleotidyltransferase DUTP nick-end labeling (TUNEL) according to the manufacturer's instructions (TMR Red; Roche Applied Science, http: / / www.roche-applied-science.com). For quantification, the number of TUNEL-positive cells was counted in at least five randomly selected high-magnification fields (magnification 3200) using three independent samples. Western blot analysis

[0365] Western blotting was performed as previously described in Kim et al. (2009) Cardiovasc Res. 100: 241-251. Cells were lysed once in lysis buffer, pH 7.4 [(in mM) 50 HEPES, 5 EDTA, 50 NaCl], 1% Triton X-100, protease inhibitors [(10 mg / mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 mg / mL leupeptin)] and phosphatase inhibitors [(mM) 50 sodium fluoride, 1 sodium orthovanadate, 10 sodium pyrophosphate]. Protein samples (40 mg) were electrophoresed using SDS-polyacrylamide gel and then electroimmunoblotted. The specific antibody used for the detection of Tert (SC-68720) was from Santa Cruz (Santa Cruz, Calif., http: / / www.scbt.com). p53 (#2524), Sirt-1 (#2028), Phospho-Fox01 (#2599), Bcl-2 (#3498), cleavage caspase-3 (#9661), Akt (#2920), Phospho Akt (#4060), and actin (#4968) were purchased from Cell Signaling (Beverly, Mass., http: / / www.cellsignal.com). All antibodies were used diluted 1:1,000. Measurement of cell proliferation rate

[0366] Cell proliferation rates were determined by cell proliferation assays and colony formation assays, as previously reported in Igura, K. et al. (2013) Heart Circ. Physiol. 305: H1354-1362. Experimental models of acute myocardial infarction and cell transplantation

[0367] Cell transplantation using a mouse acute myocardial infarction (AMI) model in vitro was described by Kim et al., the present applicant, (2013) Cardiovasc.Res.100:241-251. C57BL / 6 mice (male, 24 months old, body weight 30-35 g per animal, n 5-10 animals per group) were prepared for ligation of the left anterior descending coronary artery (LAD). Intraperitoneal anesthesia was administered using 0.1% ketamine and 0.02% xylene per g of body weight for anesthesia. After endotracheal intubation and mechanical ventilation using a rodent ventilator (Harvard Apparatus Model 683), the heart was exposed by a minimal left thoracotomy, and the LAD coronary artery was ligated with 6-0 silk. Immediately after ligation, 20 μl of DMEM (scrOMSC or anti-1950MSC) containing either no cells or 2 × 10 cells was injected into the infarcted and border regions. After injection, the opened chests of the mice were sutured, and all mice were allowed to recover. miR-195 expression was significantly upregulated with OMSC.

[0368] To profile age-induced miRNA expression, the applicant isolated total RNA from OMSCs and YMSCs and performed microarray analysis in both OMSCs and YMSCs. These results showed that the expression of miR-140, miR-146a / b, and miR-195 was significantly upregulated in OMSCs, while the expression of miR-29b, miR-205, miR-378, and miR-542 3p was downregulated in OMSCs. Reverse transcriptase PCR (RT-PCR) and real-time PCR confirmed the microarray results showing significantly upregulated miR-195 expression in OMSCs compared to YMSCs. FISH analysis for miR-195 visualization further confirmed high expression of miR-195 in OMSCs. Based on these results, the applicant hypothesized that miR-195 is induced with age, and that its depletion in OMSCs may restore the regenerative capacity of OMSCs. The applicant transfected OMSCs with anti-miR195 and confirmed that the transfected OMSCs showed reduced miR-195 expression. Scrambling did not affect this, as shown by real-time RT-PCR, indicating that the miR-195 inhibitor used by the applicant was specific to the reduction of miR-195 expression in stem cells. Tert is a direct target of miR-195 in senescent stem cells.

[0369] To investigate the biological relevance of miR-195 induction during stem cell aging and its involvement in OMSC aging, the applicant first performed target prediction analysis using in silico search to identify potential target genes of miR-195 that are involved in aging and the causes of aging.

[0370] Interestingly, computer analysis predicted that mmu-miR-195 directly binds to the 3'-UTR of the mouse Tert gene. To experimentally verify this result, the applicant performed RT-PCR and Western blot analysis to examine whether Tert expression was altered by miR-195 knockdown in OMSCs. Interestingly, the expression of both the mRNA and protein of TeMS was significantly increased by the deactivation of miR-195 in OMSCs (Figures 12B and 12C), indicating that Tert is a putative target of miR-195. Tert as a target gene of miR-195 was confirmed by a luciferase activity assay showing co-transfection with its miR-195 expression vector (PEZX miR-195). When the miR-195 expression vector contained the 3'-UTR of the Tert gene, luciferase activity was significantly reduced compared to co-transfection with the miR-scramble vector (pEZX-miR-Sc) (Figure 12D). These results demonstrate that Tert is a direct target of miR-195 in stem cell aging. Deactivation of miR-195 activates OMSCs through telomere sensitization and reactivation of anti-aging markers.

[0371] To investigate the mechanistic involvement of miR-195 in stem cell senescence, the applicant knocked down miR-195 expression by using a lentiviral miR-195 inhibitor-vector (Lenti-anti-miR-195) that successfully transfected OMSCs to achieve miR-195 inactivation (Figure 4). The mCherry signal (red fluorescence) in this vector system allowed the applicant to recognize OSMCs transfected with either the miR-195 inhibitor or the scrambled vector (Figure 13B, top panel). Interestingly, compared to scrambled-transfected OMSCs, the deactivation of miR-195 significantly reduced senescence-related B-gal expression in OSMCs (Figure 13B, 20.6% vs. 64.9% vs. 54.5% vs. 68.1%, p < 0.01). Furthermore, OMSCs transfected with the miR-195 inhibitor showed a 2.9-fold higher telomere rejoining compared to scrambled transfection (Figure 13C). Inhibition of miR-195 also reduced TUNEL-positive apoptosis cell death in OMSCs (Figure 13D, 19.7% 6 4.5% vs. 60.8% 6 2.7%, p < 0.01). This indicates that under apoptosis conditions such as ischemia, the survival rate of activated OMSCs improved by deactivating miR-195. Furthermore, the expression of anti-aging markers (Tert and Sirt1) and survival-promoting markers (p-Akt and Bcl-2) was significantly increased by transfection with anti-miR-195 in OMSCs, while the expression of aging-related markers (p53) and pro-apotosis markers (cleavage caspase-3) was significantly decreased by miR-195 deactivation (Figure 13E). This supports the applicant's hypothesis that age-related suppression of miR-195 can activate OMSCs by reactivating anti-aging factors and suppressing aging-related markers. It is important to note that, as assessed by cell proliferation assays and colonization assays, knockdown of miR-195 significantly restored damaged cell proliferation capacity in OMSCs (Figures 13F and 13G).These results demonstrate that miR-195 induced during stem cell aging plays a crucial role in their fate and behavior.

[0372] [Experiment No. 5] Generation of cardiac progenitor cells from human iPS cells (hiPSCs) and generation of multiple cell lineages therefrom.

[0373] Human iPS cells from the American Type Culture Collection (ATCC) in Manassas, Virginia, USA. C Stock (A C S-1021 TM The cells were maintained on a 6-well plate coated with vitronectin in mTeSR1 medium. Then, the cells were treated with acutase (Invitrogen). 10 minutes at 37℃ Use One The cells were dissociated. Then, the cells were divided into 5 u M's RO C K inhibitors (Y -27632, StemCell Technologies, Vancouver, British Columbia Canada)( - (Day 3) In mTeSR1 that was replenished, 1 x 1 0 6 Cells were seeded at a rate of one cell / well in a vitronectin-coated 6-well plate for 24 hours. Furthermore, the pluripotency of these iPS cells was assessed using protocols such as OCT4, SOX2, and TRA. -1- 60, TRA -1- The expression of markers 81 and SSEA4 was confirmed by immunohistochemical staining (Figures 14A-14B).

[0374] Next, cells mTe S Culture in R1 medium culture medium The medium was changed daily according to the illustrative schematic diagram shown in Figure 14C. On day 0, the medium was changed to 20 μM ISX for 7 days. - 9 (Stem C The medium was replaced with RPMI / B27 medium without insulin, supplemented with ell Technologies. Day 7 process At the end, Nkx 2.5and GATA4 CPC Marker expression is controlled by RT-P C Observation was performed using R and immunohistochemical staining (Figure 14D).

[0375] For the differentiation of cardiomyocytes, 7 days ISX - 9 processes After The culture medium was then switched to RPMI / B27 medium containing insulin for an additional 7-10 days. At the end of day 20, all cells began to pulsate spontaneously, as shown in Figure 14E.

[0376] For endothelial cell differentiation, 2 × 10 5 / cm 2 ISX - 9-lead CPC is performed using EGM. - 2 Culture Mediums (Lonza, Lonza Walkersville Inc. Walkers v The cells were cultured for 10 days in ille Md. 217930127. process At the end, endothelial cells It is a marker. CD31 and VE-cadherin were expressed (Figure 14F). In vitro On Matrigel in To analyze tube formation, 1.2 × 10⁶ 24-well plates were used. 5 Cells were seeded on a thin layer of Matrigel at a cell / well density. After 16 hours, the cells were treated with calcein AM (Corning, Tewksbury Matrigel). ss. The samples were labeled with 01876 (USA) and examined under a fluorescence microscope to visualize the formation of tubular structures (Figure 14G).

[0377] For smooth muscle cell differentiation, the induced CPC is used in TGF β1 (2 ng / ml) and PDGFBB ( 10 ng / ml, Minneapolis, Minnesota, R&D Systems , DMEM (55413) was supplemented. - The cells were cultured in F12 medium for 10 days. At the end of this period, the expression of α-SMA and calponin was observed by immunostaining (Figure 14H).

[0378] One-step generation of cardiac progenitor cells from monolayer human induced pluripotent stem cells using isoxazol or isoxazol-like compounds.

[0379] Currently, to generate large quantities of cardiac progenitor cells (CPCs) from induced pluripotent stem cells (iPSCs) to repair the heart after a sudden heart attack or chronic heart failure due to congestive heart failure, iPSCs must be converted into embryoid bodies or treated with multiple small molecule compounds. These steps are costly and time-consuming. Despite these developments, progress is slow, and at the same time, the purity of the CPC preparations cannot be guaranteed. The one-step use of isoxazoles or isoxazole compounds, such as ISX-9, is extremely efficient in generating CPCs without the use of other growth factors (activin, BMP4), glycogen synthase-zekinase-3 inhibitors, Wnt inhibitors, and other small molecule compounds. This small molecule compound has been reported to be involved in the differentiation of nerve cells (Schneider et al. (2008) Nat Chern Bioi. 4(7):408-10. doi: 1 0.1 03 8 / nchembio .9 5) and the reprogramming of fibroblasts into neurons in combination with other small molecule compounds (Li et al. (2015) Cell Stem Cells 17(2): 195-203).

[0380] To test this theoretical basis, the applicant also purchased a human iPSC strain (ACS-1021(trademark)) from the American Type Culture Collection, Manassas, Va., 20110 USA (ATCC). The iPSCs, maintained on a 6-well plate of vitronectin-coated medium in mTeSR1, were dissociated into single cells using accutase (Invitrogen) at 37°C for 10 minutes, and then 1 × 10¹⁶ cells were added to mTeSR1 supplemented with 5 f / L of the ROCK inhibitor (Y-27632, StemCell Technologies, Canada, British Columbia, Canada). 6Cells were seeded at a rate of cells / well on a 6-well plate coated with vitronectin (Day 3) (Figure 14A). iPSCs were confirmed for their pluripotency by immunostaining (Figures 14A, B). The cells were then cultured in mTesR1 medium, which was changed daily according to our schematic diagram (Figure 14C). On Day 0, the medium was replaced with insulin-free RPMII B27 medium supplemented with 20 μM ISX-9 (Stem Cell Technologies) for 7 days. At the end of treatment on Day 7, the expression of CPC markers, including Nkx2.5 and GATA4, was observed by RT-PCR and immunostaining (Figure 14D). For cardiomyocyte differentiation, 7 days after ISX-9 treatment, the medium was switched to insulin-containing RPMI / B27 medium for an additional 7-10 days. At the end of Day 20, all cells began spontaneous beating (Figure 14E). For endothelial cell differentiation, 2 × 10⁵ / cm² of ISX-9-induced CPCs were cultured in EGM-2 medium (Lanza, Lanza Walkersville Inc., Walkersville Md. 21793-0127) for 10 days. At the end of treatment, endothelial cell makers, CD31, and VE-cadherin were expressed (Figure 14F). To analyze tube formation on Matrigel in vitro, cells were seeded onto a thin layer of Matrigel at a density of 1.2 × 10⁵ cells / well in a 24-well plate. After 16 hours, cells were labeled with AM calcein (Coming, Tewksbury Mass. 01876, USA) and observed under a fluorescence microscope to visualize the formation of tubular structures (Figure 14G). For smooth muscle cell differentiation, induced CPCs were cultured for 10 days in DMEM-F12 medium supplemented with TGF (2 ng / ml) and PDGFBB (long / ml, R&D Systems, Inc., Minneapolis, Minn. 55413). At the end of this period, α-SMA and calponin expression was observed by immunostaining (Figure 14H).

[0381] In conclusion, Isx-9 was a highly effective small molecule compound for consistently converting iPSCs into progestin-containing hearts, including endothelial / vascular precursors and smooth muscle cells. Experiment No. 7

[0382] Pluripotent cardiac progenitor cells (CPCs) offer a promising source for cardiac repair due to their ability to proliferate and develop into cardiac lineage cells. In this experiment, the applicant explored a novel strategy for generating human CPCs from human induced pluripotent stem cells (hiPSCs) using a cardiogenic small molecule compound, isoxazole (ISX-9), and its proliferative capacity in scar tissue for improving the function of infarcted myocardium. Methods and Results

[0383] In short, CPCs were induced from hiPSCs using ISX-9. CPCs were characterized by immunocytochemistry and RT-PCR. In vivo transplantation of CPCs in immunodeficient mice (NOD / SCID mice) after LAD ligation determined their survival and differentiation in infarcted hearts. Their effects were determined to be fibrosis and functional improvement. The applicant found that ISX-9 simultaneously induced the expression of cardiac transcription factors Nkx2.5, ISL1, GATA4, and Mef2c hiPSCs within 3 days of treatment, and successfully differentiated into three cardiac lineages in vitro. mRNA and miRNA sequencing results indicated that ISX-9 targets multiple cardiac differentiation and proliferation signaling pathways, increasing the expression of myogenesis and cardiac hypertrophy-related miRNAs. CPC transplantation promoted angiogenesis, suppressed fibrosis, and resulted in functional improvement in mice after myocardial infarction.

[0384] This study demonstrates a novel technique for producing pure CPCs from hiPSCs using ISX-9, a single cardiogenic small molecule compound with potent muscle differentiation potential via diverse signaling pathways. These CPCs were pluripotent and differentiated into three cardiac lineages, forming new CMs and blood vessels in the infarcted heart, thereby suppressing fibrosis and improving cardiac function.

[0385] Stem cell-based therapies hold great promise and potential for cardiac regeneration. Human induced pluripotent stem cells (hiPSCs) have the potential to generate an unlimited number of functional human cell types in vitro for home-based and personalized medicine. Previous studies have reported that iPSC-derived cardiomyocyte (CM) transplantation resulted in functional improvement in rodent models of myocardial infarction (MI) (Wang Y. et al., Medical Physics. 2016); (Wang Y. et al., IEEE International Conference on: IEEE; 2011). However, poor engraftment of cardiomyocytes after transplantation into infarcted hearts is a common problem affecting the final outcome. The beneficial effects of CM transplantation are thought to be due to paracrine mechanisms rather than integration of transplanted cells with host cardiomyocytes. These limitations are likely due to poor cell survival and retention under ischemic conditions, the limited proliferative capacity of differentiated CMs, and the lack of angiogenesis in the graft. Furthermore, the risk of proarrhythmic complications after cardiac transplantation due to the immaturity and heterogeneity of iPSC-CM or embryonic stem cell (ESC)-CM remains unresolved before full-scale application (Li H. et al., Bioinformatics. 2009; Robinson MD et al., Bioinformatics. 2010); (Benjamini Y et al., Royal Statistical Society Journal Series B - Methodology. 1995).

[0386] Cardiac progenitor cells (CPCs) offer a promising means for cardiac repair due to their pluripotency and proliferative capacity. Previous studies have demonstrated that CPCs derived from hiPSCs or ESCs can differentiate into multiple cardiac lineages without teratoma formation. Furthermore, transplantation of CPCs derived from hiPSCs and mouse iPSCs improved cardiac function more effectively than iPSC-CMs (Kozomara A. et al. Nucleic Acids Res. 2014); (Xuan W. et al., Cardiovasc Res. 2011). Recently, two studies reported that mouse fibroblast-derived CPCs spontaneously differentiated into CM, EC, and SMC in the hearts of infarcted mice, improving cardiac function after myocardial infarction (Zhang et al., Cell stem cell, 2016; Vol. 18; pgs. 368-381); (Lalit PA et al., Cell stem cell, 2016; Vol. 18; pgs. 354-367). In addition, several clinical trials are underway on cell therapy for heart disease using adult stem cells (Sheridan C. Cardiac, Nature biotechnology, 2013; Vol. 31; pgs. 5-6); (Malliaras et al., Stem Cell Translational Medicine, 2014; Vol. 3; pgs. 2-6). For example, the European Consortium CARE-MI uses human CPCs isolated from the right atrial appendage of donors for the treatment of acute myocardial infarction (Gomes-Alves et al., Translational Research: Laboratory and Clinical Medicine, 2016; Vol. 171; pgs. 96-110 & 111-113). More recently, it has been reported that SSEA-1+ CPCs derived from human ESCs were delivered to the infarcted area of ​​a single patient with severe heart failure, and they resulted in improved cardiac function (Menasche et al., European Heat Journal, 2015; Vol. 36; pgs. 2011-(2017)). This clinical trial demonstrated the potential to generate a clinical-grade population of human ESC-derived CPCs and provided a strong encouragement for future clinical applications of hiPSC or ESC-derived CPCs.Current strategies for human CPC generation include isolation from donor atrial appendages and in vitro expansion (Koninckx et al., Cardiovascular Research, 2013; Vol. 97; pgs. 413-423); induction from hiPSCs or ESCs; conversion to embryoid bodies or treatment with multiple small molecule compounds and growth factors (activin, BMP4) (Yang et al., Nature, 2008; Vol. 453; pgs. 524-528); (Lei et al., Visualization Experiment Journal: JoVE 2015: 52047; Moretti et al., FASEB Journal: Official Publication of the American Association for Experimental Biology, 2010; Vol. 24; pgs. 700-711). These strategies are labor-intensive, time-consuming, and have high production costs that limit clinical application. Furthermore, cardiac tissue-derived CPCs are limited by the availability of limited human tissue sources. Therefore, a novel platform for large-scale human CPC production is needed to ensure safety, high reproducibility, high purity, cost-effectiveness, and ease of manufacturing for further clinical applications.

[0387] In some reports, ISX-9 was referred to as ISX-1. (Russell et al., ACS Chemical Biology, 2012; Vol. 7; pgs. 1067-1076; Burchfield et al., Investigational Drug Journal: Official Publication of the American Coalition for Clinical Research, 2016; Vol. 64; psg. 50-62.) ISX-9 is a small molecule compound belonging to the isoxazol family. ISX-9 has been reported as a cardiac small molecule compound. Previous studies have shown that direct injection of...

Claims

1. A method for preparing a population of skeletal myogenic progenitor cells from a population of human induced pluripotent stem cells (hiPSCs), comprising contacting the hiPSCs with an effective amount of ISX-9.

2. The effective amount of ISX-9 is 1 × 10 ６ The method according to claim 1, wherein each cell contains approximately 100 nM to approximately 30 nM.

3. The method according to claim 1, further comprising culturing the cells in the presence of serum-free medium and a Rho-related quinase (ROCK) inhibitor.

4. The method according to claim 3, wherein the Rho-related quinase (ROCK) inhibitor is selected from thiazovibin, Y27632, SR3677, or GSK429286.

5. The method according to claim 3, further comprising culturing the cells in the presence of a serum-free medium and a TGF-β1 receptor inhibitor.

6. The method according to claim 5, wherein the TGF-β1 receptor inhibitor is selected from the group consisting of SB431542, A8301, LY2157299, or LY2109761.

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