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THETWO TORT DIT USU 20180273906A1NANI MINUTA OMULUTNI ( 19) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2018/ 0273906 A1 Ashraf (43 ) Pub . Date : Sep . 27 , 2018 (54 ) MICROVESICLE AND STEM CELL Publication Classification COMPOSITIONS FOR THERAPEUTIC (51 ) Int. Cl. (54 ) COMPOSATIONSAPPLICATIONS C12N 57074 (2006 .01 ) C12N 5 /077 ( 2006 . 01) (71 ) Applicant: Muhammad Ashraf, Cincinnati , OH C12N 5 /071 (2006 . 01) (US ) A61K 35 / 12 ( 2006 .01 ) ( 72 ) Inventor: Muhammad Ashraf , Cincinnati, OH (52 ) U . S . CI. CPC ...... C12N 5 / 0696 ( 2013 .01 ) ; C12N 5 /0657 (US ) (2013 .01 ); C12N 2506 / 45 ( 2013 .01 ); C12N (21 ) Appl. No .: 15 / 881, 693 570661 (2013 . 01 ) ; A61K 35/ 12 ( 2013. 01 ) ; C12N 5 / 069 ( 2013 . 01 ) ( 22 ) Filed : Jan . 26 , 2018 Related U .S . Application Data (57 ) ABSTRACT (63 ) Continuation - in -part of application No . 14 / 255, 789, Provided herein are stem cell and exosome compositions filed on Apr. 17 , 2014 , Continuation - in - part of appli having therapeutic utility to treat a variety of diseases and (63 ) Condcation on No Apr. .14 17 / 951, 20152 , 354 , filed onon Nov;. 5201 24 , 2015, 292 , Con file disorders , e . g . , cardiovascular disease , Duchenne muscular tinuation - in -part of application No . 15 / 201, 292 , filed dystrophy , and fibrotic disease . on Jul. 1 , 2016 . Specification includes a Sequence Listing . Patent Application Publication Sep . 27, 2018 Sheet 1 of 56 US 2018 /0273906 A1

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FIGS. 30A -3B US 2018 /0273906 A1 Sep . 27 , 2018

MICROVESICLE AND STEM CELL to known methods and technology . This disclosure satisfies COMPOSITIONS FOR THERAPEUTIC this need and provides related advantages as well . APPLICATIONS SUMMARY OF THE DISCLOSURE CROSS -REFERENCE TO RELATED [0006 ] This disclosure provides in part methods that use APPLICATIONS just one class of small molecules applied to induced pluri 0001 ] This application is a continuation - in - part under 35 potent stem cells ( IPSCs ) to express and precondition the U . S . C . § 120 of U . S . application Ser. No. 14 /255 ,789 , filed cells propogated from the method ( e. g ., cardiac progenitor Apr. 17 , 2014 ; U . S . application Ser. No . 14 /951 ,354 , filed cells , endothelial cells , smooth muscle cells , myocytes, Nov. 24 , 2015 ; and U . S . Ser. No . 15 / 201, 292 , filed Jul. 1 , myogenic progenitors , cardiomyocytes and blood vessels 2016 , the content of each of which is incorporated herein by and the exosomes or microvesicles produced by the resultant reference in its entirety . cells ). The cells and exosome or microvesicle compositions isolated from the cells activate resident stem cells , car STATEMENT OF GOVERNMENT SUPPORT idomyocytes , myocytes and paracrine factors. In addition , given the existing non efficient process to produce these cells [ 0002 ] This invention was supported in part by National and exosomes or microvesicles, this one step commerciable , Institutes of Health grants , RO1 HL126516 , HL134354 and scaleable and safe process may be the most economical in RO1 AR070029 . Thus , the US government has rights in this producing an unlimited number of cells and exosomes or invention . microvesicles for the treatment of diseases such as CVS , Duchenne muscular dystrophy , muscle and other diseases . REFERENCE TO SEQUENCE LISTING [0007 ] Thus , in one aspect , provided herein is an isolated SUBMITTED VIA EFS - WEB population or a composition comprising , or alternatively [ 0003] This application is being filed electronically via consisting essentially of, or yet further consisting of an EFS - Web and includes an electronically submitted sequence isolated population of exosome or microvesicle , that in one listing in .txt format. The . txt file contains a sequence listing aspect are isolated from a cell selected from the group of: an entitled “ 2018 - 06 - 12 ISPH _ 004 _ ST25 . txt” created on Jun . iPS cell , an embryonic stem cell , or a stem cell , each that had 12 , 2018 and is 3 ,537 bytes in size . The sequence listing been previously contacted with an effective amount of an contained in this . txt file is part of the specification and is isoxazole compound , a derivative or an equivalent of the hereby incorporated by reference herein in its entirety . The isoxazole compound , wherein the exosome or the microve content of the sequence listing information recorded in sicle overexpresses a microRNA miRNA or mir ) selected computer readable form is identical to the written sequence from the group of mir - 373 , mir - 210 , mir - 377 , mir - 367 , mir - 520 , mir -548ah , mir - 335 , mir -30c , mir -214 , or mir listing and includes no new matter 5487 , and / or one or more of a protein selected from Tsg101, CD9, Hsp70 , Flotillin - 1 , or GAPDH . In a further aspect the BACKGROUND exosome or the microvesicle can further overexpress one or 10004 ] Throughout this disclosure , various technical and both of mir - 30c and / or mir- 21 . In one aspect, at least 70 % , patent publications are referenced to more fully describe the or 80 % , or 85 % , or 90 % or 95 % , or 98 % of the cells express state of the art to which this invention pertains , the full the stated mir and/ or protein . bibliographic citations for some of the publications are 0008 ] Also provided herein is a cell or an isolated popu found at the end of the specification , immediately preceding lation of one or more of: a cardiac progenitor cell (CPC ) , a the claims. All publications noted in the present specification cardiomyocyte, a myocyte , an endothelial cell , a smooth are incorporated by reference, in their entirety , into this muscle cell , a skeletal muscle cell , each generated from a application . cell selected from an iPSC , an embryonic stem cell , a a stem [ 0005 ] Induced pluripotent stem ( iPS ) cells are important cell, each that had previously contacted with contacted with source for progenitors, such as cardiac progenitors , for drug an isoxazole compound , derivative , or an equivalent of each discovery and the treatment of disease , e . g ., infarcted myo thereof, wherein the cells of the population overexpress one cardium . Due to inherent properties of iPS cells to form or more of mir - 373 , mir- 210 mir - 377 , mir - 367, mir- 520 , teratomas , it becomes very important to generate iPS cells mir - 548ah , mir - 335 , mir - 30c , mir - 214 , or mir - 548q ; and /or without producing tumors for use in clinics . Given the a muscle gene selected from the group of: paZ3 , PAX7, importance of these concerns , efforts have been made to MYF5 , MYOD , MYOG , or dystrophin . Also provided is a improve the reprogramming efficiency and several methods cardiomyocyte that expresses one or more of cTnT, cTnl, c have been devised for the non - viral generation of iPS cells . MLC2V , and / or VE - cadherin . In a further aspect , provided Various growth factors and chemical compounds, such as herein is a cell or a population of cells of a -smooth muscle DNA methyltransferase inhibitor ( 5 ' - azacytidine and actin ( SMA ) and calponin . In one aspect, at least 70 % , or RG108 ) , histone deacetylase inhibitors ( e . g . , valproic acid ) , 80 % , or 85 % , or 90 % or 95 % , or 98 % of the cells overex histone methyltransferase inhibitor (BIX -01294 ), Wnt3A , press the stated mir and / or muscle gene and / or protein . The and ALK5 inhibitor , have been found to improve the induc exosomes or microvesicles can be isolated for any one of the tion efficiency . ( 1 - 6 ) With different experimental manipula following: a cardiac progenitor cell , a cardiomyocyte , a tions , iPS cells can be induced to cardiac lineage prior to skeletal muscle , an endothelial cell , a myocyte , or a smooth their transplantation ; however , these procedures are labor muscle cell . intense , expensive and involve multiple growth factors [0009 ] In one aspect , the population or cells further com and / or smallmolecules that limit the consistency and repro prising , or alternatively consisting essentially of, or yet ducibility required for drug development and to enter the further consisting of, a pharmaceutically acceptable carrier clinic . Therefore there is a need in the art for improvements that in one aspect , is a non -naturally occurring carrier. In US 2018 /0273906 A1 Sep . 27 , 2018 another aspect, the composition also contains a preservative ened telomere length ) ; promoting tissue regeneration in and / or a cryopreservative that in one aspect facilitates tissue damaged from one or more of stroke , arthritis , lyophilization and /or preservation of the composition . In a Alzheimer ' s , memory loss disorders, cystic fibrosis , inflam yet further aspect , the composition further comprising, or matory disorders or cancer; decreasing cardiac wall thick consisting essentially of, or yet further aspect, consists of, a ness in a tissue damaged from a cardiac infarction ; altering protein that facilitates regeneration and /or improved func gene expression of one or more of protein kinase C , iL -6 , tion of a tissue or a nucleic acid that encodes the protein . mmp , PDGF; reducing or inhibiting the expression of an Non - limiting examples of such include TGF - B , WNT pro inflammatory protein ; that is optionally a cytokine , a tein , a cytokine, or a histone deacetylase. chemokine , or a macrophage ; directly or indirectly stimu [ 0010 ] Yet further provided is a composition for the repair lating angiogenesis ; directly or indirectly inhibiting cellular or regeneration of damaged or diseased cardiac tissue , the replication ; promoting cardiac regeneration in a subject composition comprising synthetic microRNA - 146a . In one suffering from a disease selected from the group of: coronary aspect, the composition further comprises , or alternatively artery disease ,myocardialinfaction , heart failure , hypoplasic consisting essentially of, or yet further consisting of, a left heart syndrome, peripheral artery disease ( PAD ) , cardiac pharmaceutically acceptable carrier that in one aspect, is a hypertrophy , valvular heart disease ( aortic stenosis ) , myo non - naturally occurring carrier. In another aspect, the com cardial hypertrophy mi, hypertrophy fibrosis by administer position also contains a preservative and /or a cryopreserva ing to a subject in need of the method , an effective amount tive that in one aspect facilitates lyophilization and / or pres of a population of exosome or microvesicle and / or isolated ervation of the composition . cells as described herein , as well compositions containing [0011 ] Yet further provided is a composition comprising the same. In a further aspect, the methods further comprise , one or more of a synthetic microRNA -373 , a micro -ma 373 or consist essentially of, or yet further consist of, adminis mimic or micro -ma 373 exosome. In one aspect, the com tering an effective amount of a non - embryonic stem cell or position further comprises, or alternatively consisting essen a progenitor cell to the subject, that is optionally of the same tially of, or yet further consisting of, a pharmaceutically type as the tissue in need of repair of an type different from acceptable carrier that in one aspect , is a non -naturally the type of tissue of repair . The stem cells can be non occurring carrier . In another aspect, the composition also embryonic stem or progenitor cell is autologous to the contains a preservative and /or a cryopreservative that in one subject . They also can be delivered systemically or locally to aspect facilitates lyophilization and / or preservation of the the tissue of the subject. composition . These compositions are useful to treat fibrotic [0014 ] Also provided herein are methods for preparing a diseases by administering an effective amount to a subject in population of cells as described herein from a population of need thereof. A non - limiting example of the fibrotic disease human induced pluripotent stem cells (hiPSCs ) , comprising is myocardial fibrosis . contacting the hiPSCs with an effective amount of Givinos [0012 ] The exosome or microvesicle population or the tat (GIV ). cells is isolated from a population of stem cells or progenitor [0015 ]. Further provided are methods for one or more of: cells cultured in the presence of an effective amount of an providing anti - oxidative therapy; promoting activation of isoxazole compound or a derivative thereof. In one aspect , local or resident cardiomyocytes ; promoting the release of the isoxazole compound is selected from isoxazole - 1 ( isx - 1 ) , angiogenesis and / or paracrine factors ; promoting activation isoxazole - 9 ( isx - 9 ) , or Danazol. of one or more of: wnt, a BMP, and / or cytoskeleton remod [ 0013 ] Also provided herein are methods for one or more eling ; promoting TGF- B induced emt signaling and cardiac of providing in a subject in need thereof: regenerating differentiation ; increasing expression of wnt5 and wnt11 or damaged tissue; improving the viability of damaged tissue; a BMP family protein , optionally BMP4 ; increasing expres facilitating the formation of new tissue, optionally a cardiac sion of a cardiac transcription factor selected from the group tissue, a muscle tissue, a skeletal muscle , a blood vessel, a consisting of nkx2 . 5 , mef2c , gata4 and isl - 1 ; promoting capillary , or a myocyte ; promoting cardiac regeneration ; expression of genes for development of pip3 signaling in promoting cardiac regeneration in a subject suffering from cardiomyocytes , muscle contraction and nf- at hypertrophy an acute cardiac event; promoting cardiac regeneration in a signaling pathways ; reducing fibrosis and apoptosis ; pro subject suffering from a myocardial infarction ; promoting moting myoangeneis and muscle differentiation ; promoting cardiac regeneration in subject suffering from Duchenne the release of a cytokine selected from the group consisting muscular dystrophy or Duchenne muscular dystrophy - asso of angiopoietin - 2 , il- 6nmp, pgfbb , timp 1 or a gene identified ciated cardiomyopathy ; or promoting cardiac regeneration herein or in the Figures ; promoting upregulation of a gene in a subject suffering from age - related diseases selected selected from the group of wnt3a , wnt5a , wnt11 ; and /or from the group of: such as Hoyeraal -Hreidarsson syndrome, promoting cytoskeletal remodeling, the method comprising, dyskeratosis congenita , pulmonary fibrosis, aplastic anemia , or alternatively consisting essentially of, or yet further liver fibrosis , dyskeratosis congenita , bone marrow failure, consisting of, administering an effective amount of the lung disease , endocrine diseases , polycystic ovary syndrome exosome or microvesicle population or cells , or composition (PCOS ) , Cushing ' s syndrome, and acromegaly , Cerebrovas containing them , to a subject in need thereof . cular Disease (Strokes ) , High Blood Pressure Hyperten [0016 ] Further provided are cells or populations of cells , sion , Parkinson ' s Disease , Vascular Dementia , dementia , as well as cells and populations prepared by method as macular degeneration , Alzheimer ' s Disease, Age -related disclosed herein , wherein at least 80 % , or alternatively 85 % , hearing loss, Celiac disease (CD ) , COPD , bipolar disorder, or alternatively 90 % , or alternatively 95 % , or alternatively hydroxyurea , sickle cell diseases, hypertension , atheroscle 97 % or alternatively 99 % , or alternatively 100 % , of the cells rosis , arthritis , osteoporosis , osteoarthritis , vasculardemen overexpress one or more protein selected from the group of: tia or macular degeneration , cancer , type 2 diabetes, or cTN1, MLC2v , TNT, VE - cadherin , CD31 , a - SMA ( actin ) , diseases with telomerase dysfunction dealing with a short calponin , or Cx43 . Further provided is a a cell or a popu US 2018 /0273906 A1 Sep . 27 , 2018

lation of cells that overexpress skeletal myogenic genes tion of the heart in patients such as elderly patients with seleted from the group of: Meox1, Meox2 , Tcf15 , Pax3 , cardiac diseases such as heart failure , and heart attack Pax7 , MyoD1, MYF5 , dystrophin , or DESMIN . Determin patients . ing the expression of the genes, miRNA and / or proteins can [0022 ] Also provided are methods comprising, or alterna be performed using methods known in the art and briefly tively consisting essentially of, or yet further consisting of described herein . The cells and /or microvesicles or exo administering one or more microRNA fragments, or deriva somes isolated from the cells are useful in method for tives thereof to the subject , wherein after administration of regenerating skeletal muscle cells , treating DMD . the one or more microRNA fragments , the one or more [ 0017 ] Also provided is a population of cells that overex microRNA fragments alter gene expression in the damaged press xESI myogenic genes selected from the group of: tissue , improve the viability of said damaged tissue , and Pitx2 , IS11, Nkx2 . 5 , Handi , GATA4 , Tbx5 , TnnT2 , My17 , facilitate the formation of new tissue in the subject . In one MLC2v , Myf2e , Cdh4 or Lhx2. In one aspect, at least 80 % , aspect, the microRNA fragments , or derivatives thereof , are or alternatively 85 % , or alternatively 90 % , or alternatively synthetically generated . In a further aspect, wherein the 95 % , or alternatively 97 % or alternatively 99 % , or alterna microRNA fragments , or derivatives thereof are synthesized tively 100 % , of the cells overexpress the xESI myogenic with a sequence that mimics one or more endogenous genes. Determining the expression of the genes, miRNA microRNA molecules . In another aspect , wherein the micro and / or proteins can be performed using methods known in RNA fragments , or derivatives thereof are modified to the art and briefly described herein . These populations and enhance their stability. The microRNA fragments can be cells are useful in methods of regenerating cardiac muscle administered by any appropriate method as determined by tissue, or yet further treating cardiac dysfunction associated the treating physician or professional. Non - limiting with Duchenne Muscular Dystrophy (DMD ) , the methods examples of such comprise administration of a plurality of comprising, or alternatively consisting essentially of, or yet synthetic liposomes that comprise said one or more micro further consisting of, administering to a subject in need RNA fragments , or derivatives thereof. thereof an effective amount of the population of cells as [ 0023 ] Yet further provided are methods of generating described herein and /or an effective amount of the popula exosomes or microvesicles , the methods comprising , or tion of exosomes or microvesicles as disclosed herein . alternatively consisting essentially of, or yet further consist ing of, culturing a population of non - embryonic human [0018 ] Further provided are populations of exosomes or regenerative cells in the presence of a hydrolase enzyme to microvesicles isolated from these cell populations. induce the cells to secrete exosomes or microvesicles , [0019 ] Yet further provided are methods of regenerating thereby generating exosomes or microvesicles . In a further cardiac muscle comprising , or alternatively consisting aspect, the methods further comprise , or alternatively consist essentially of, or yet further consisting of, administering to essentially of, or yet further consist of , isolating the exo a subject in need thereof an effective amount of the popu somes or microvesicles from the culture media and / or the lation of cells as described herein and / or an effective amount cells . of the population of exosome or microvesicle as described [ 0024 ] In one aspect , the hydrolase comprises a member herein . of the DNAse I superfamily of enzymes, a non - limiting [0020 ] Also provided herein are methods for repairing or example of such includes a sphingomyelinase. In one aspect, regenerating damaged or diseased cardiac tissue in a subject the sphingomyelinase is of a type selected from the group consisting of lysosomal acid sphingomyelinase , secreted in need thereof. zinc -dependent acid sphingomyelinase , neutral sphingomy [0021 ] Further provided are compositions for the repair or elinase , and alkaline sphingomyelinase. In a further aspect , regeneration of damaged or diseased cardiac tissue or for the the neutral sphingomyelinase comprises one or more of treatment of one or more of Hoyeraal- Hreidarsson syn magnesium - dependent neutral sphingomyelinase and mag drome, dyskeratosis congenita , pulmonary fibrosis , aplastic nesium - independent neutral sphingomyelinase . In a further anemia , liver fibrosis , dyskeratosis congenita , bone marrow aspect , the neutral sphingomyelinase comprises one or more failure , lung disease , endocrine diseases , polycystic ovary of neutral sphingomyelinase type I, neutral sphingomyeli syndrome ( PCOS ) , Cushing ' s syndrome, and acromegaly , nase type 2 , and neutral sphingomyelinase type 3 . Cerebrovascular Disease ( Strokes ) , High Blood Pressure Hypertension , Parkinson ' s Disease , Dementia , Alzheimer 's Disease , Age- related hearing loss , Celiac disease ( CD ) , BRIEF DESCRIPTION OF THE FIGURES COPD , bipolar disorder, hydroxyurea , sickle cell diseases , [ 0025 ] The patent or application file contains at least one hypertension , atherosclerosis , arthritis, osteoporosis , drawing executed in color. Copies of this patent or patent osteoarthritis , vasculardementia or macular degeneration , application publication with color drawing ( s ) will be pro cancer , type 2 diabetes, or diseases with telomerase dys vided by the Office upon request and payment of necessary function dealing with a shortened telomere length , the composition comprising a synthetic microRNA - 195 inhibi fee . tor. Non - limiting examples of such include for example [0026 ] FIG . 1 shows characterization of IPS cells for the HSTUD0320 (SIGMA MISSION® Synthetic microRNA expression of pluripotency markers . Representative photo Inhibitor ), Human hsa -miR - 195 - 5p or inhibitory hsa -miR micrographs showing IPS clones expressing embryonic 195 - 5p miRNA/ microRNA Lentivector ( sold by ABM Good stem cells (ESC ) specific markers Oct3 / 4 , Sox2 , Nanog and ( abmgood . com )) . The compositions when applied in an endogenous Oct3 / 4 . Nuclei were stained with DAPI. effective amount to bone marrow stem cells or IPS cells [ 0027 ] FIG . 2 shows the results of DNA methyltransferase promote or facilitate telemore elongation and rejuvenation (DNMT ) activity assay . DNA methylation analysis showing of aged stem cells . The methods promote cardiac regenera significant (95 % ) inhibition of DNA methyltransferase US 2018 /0273906 A1 Sep . 27 , 2018 activity in small molecule , isoxazole or isoxazole like com ated infarct size expansion and regeneration into fully devel pounds treated IPS cells in comparison to the nontreated IPS oped myofibers in vivo . A total 3 .6x10 $ cells were injected cells . per animal in the infarct and peri infarct regions after [0028 ] FIGS . 3A - 3B show that small molecule , isoxazole coronary artery ligation . The animals were sacrificed 7 days or isoxazole like compounds treatment induces cytoprotec and 6 weeks after transplantation . A significant attenuation tion in vitro and that small molecule treatment prevents of infarct size and growth of new myocytes in scar tissue in oxidant induced apoptosis and increases the proliferation . In hearts from transplanted animals (FIG . 8B ) as compared to FIG . 3A , apoptosis was determined by TUNEL assay . Fewer the control group ( FIG . 8A ) was observed . A significant TUNEL positive cells /microscopic field were observed in regeneration of infarcted heart with new fibers resulted in small molecule , isoxazole or isoxazole like compounds reduced infarct size (FIGS . 8C and 8D ) . There were very pretreated IPS as compared to control non - treated IPS cells . limited number of iPS derived growing cells observed in In FIG . 3B , an increase in mitogenic response of small hearts transplanted with non -treated iPS cells ( not shown ). molecule treated IPS was significantly noted in comparison There w as significant increases in LV chamber dimensions with non - treated IPS cells . All values were expressed as during systole (LVESD ) and diastole (LVEDD ) after myo mean : SEM , * p < 0 .05 vs. control. cardial infarction and these were significantly reduced by [0029 ] FIG . 4 shows cytochrome c translocation to cyto iPS cell treated with isoxazole or isoxazole like compounds plasm ( a sign of cell injury ) . Immunostaining of cytochrome ( FIG . 8E ) . Similarly cardiac fraction shortening and ejection c in non -treated and small molecule , isoxazole or isoxazole fraction were increased by the treatment (FIG . 8F ) . like compounds treated IPS cells after exposure to H , O , [0034 ] FIGS. 9A -91 show the characterization of small ( 100 umol) , (merged images with DAPI) , (original magni juvenile stem cells (SJSCs ) from heterogeneous bone mar fications ; 200x ) . Small molecule , isoxazole or isoxazole like row - derived stem cells (BMSCs ) from young and old mouse compounds treated IPS cells show a significant decrease in described in Experiment No. 2 . FIGS . 9A and 9B show that cytochrome c translocation to cytoplasm as compared to the both aged and young SJSCs were positive for CD29 , CD44 , non - treated IPS cells . CD59 , and CD90 but negative for CD45 and CD117 B . In [0030 ] FIGS. 5A -5B show small molecule , isoxazole or FIG . 9C , cell growth curves show that the cell proliferation isoxazole like compounds mediated cardiac differentiation rate was higher in SJSCs than in BMSCs. Compared with of IPS cells in vitro . FIG . 5A shows immunostaining of aged BMSCs, aged SJSCs showed higher expression of cardiac specific gene Nkx - 2 . 5 and a Sarcomeric actin in pluripotency markers such as octamer- binding transcription small molecule treated IPS cells (merged images with factor 4 (Oct - 4 ), Nanog , sex -determining region Y box 2 DAPI) , (original magnifications ; 400x ) . FIG . 5B is RT- PCR (Sox - 2 ), Kruppel- like factor 4 (Klf - 4 ), and Rex - 1 . FIG . 9D analysis of cardiomyocyte specific marker, Nkx - 2 . 5 , in small shows that cardiogenic differentiation markers such as molecule treated IPS cells in comparison with non - treated Gata - 4 and myocyte -specific enhancer factor 2C (Mef2c ). IPS cells . Small molecule , isoxazole or isoxazole like com FIG . 9E shows that antiaging markers such as sirtuin 1 pounds treated IPS cells shows significant upregulation of ( Sirt1 ) and telomerase reverse transcriptase ( Tert) are Nkx - 2 . 5 as isoxazole or isoxazole like compounds compared expressed . In FIG . 9F , marker expression as examined by to non - treated IPS cells . RT- PCR . In FIG . 9G , SJSCs from aged bone marrow [ 0031 ] FIG . 6 shows Affymetrix array - based gene expres showed less senescence - associated B - galactosidase ( ß - gal) sion profiling of small molecule , isoxazole like compounds expression compared with other cells in aged BM . FIG . 9H treated IPS vs non - treated IPS cells further confirmed 2 - 3 is confocal microscopy showing that aged SJSCs had a folds downregulation of Dnmt1 , Dnmt3b and Max gene higher density of telomeres compared with aged BMSCs . associated protein which were associated with global DNA FIG . 91 is quantitative fluorescence in situ hybridization hypomethylation and myc dependent cell transformation . In ( FISH ) analysis demonstrated that SJSCs maintain longer addition , there was 2 - 3 folds concomitant upregulation of telomeres compared with BMSCs. Telomere shortening was CCL7 , CXCR2 , CXCR5, integral membrane protein 2A , delayed in aged SJSCs compared with aged BMSCs. and EphrinA3. [0035 ] FIGS . 10A - 10D show characterization of Sca - 17 [0032 ] FIGS. 7A - 7E show miR -microarray analysis of CSCS. FIG . 10A is a brief description of the procedure of small molecule , isoxazole like compounds treated IPS vs CSCs isolation . FIG . 10B shows Sca - 1 * expression in iso non - treated IPS cells . Microarray analysis showing miR lated CSCs was validated by immunocytochemistry and expression profile in non - treated IPS cells was quite differ flowcytometry . ( light grey = Sca - 1 " ; dark grey = DAPI) . In ent from small molecule treated IPS cells . (FIGS . 7A - 7D ) FIG . 10C , Discoidin domain receptor 2 (DDR2 ) and prolyl Critical miRs known for reprogramming and differentiation 4 -hydroxylase beta (P4HB ) antibodies were rarely positive as observed in non - treated and small molecule treated IPS in CSC cultures. FIG . 10D shows that expression of a cells . Microarray analysis showed upregulation of cardiac hematopoietic progenitor marker ( c -kit ) , pluripotency mark specific miR - 133 , miR - 762 and down regulation of pluri ers (Oct - 4 , Sox2 , Nanog ) , a stem cell side population marker potency associated miR - 290 - 295 cluster and let - 7 family in (Bcrpl ), early cardiac lineage markers (Nkx - 2 . 5 , GATA4 , small molecule treated IPS cells . FIG . 7E shows GPCR MEF2C ) , and a vascular progenitor marker ( Fiki) in iso signaling in small molecule , isoxazole , or an isoxazole lated Sca - 1 + cell colonies as analyzed by RT- PCR . “ C ” similar compound treated SIPs cells. Western blot analysis notation above the columns stands for cultured colonies and shows significant upregulation of Ga (pan ) in small mol their identity numbers . They maintained about 80 - 90 % of ecule treated IPS cells as compared to the non -treated IPS Sca - 17 positive cells from passage 5 to 30 , measured by cells which was blocked by the concomitant use of GPCR flowcytometry . All experiments were performed within this blocker , pertussis toxin . passage. Bar = 100 um . [0033 ] FIGS. 8A - 8F show transplantation of iPS cell [0036 ] FIGS . 11A - 11D show that connective tissue growth treated with isoxazole or isoxazole like compounds attenu factor (“ CTGF” ) is a major downstream factor for electri US 2018 /0273906 A1 Sep . 27 , 2018 cally stimulated - induced focal adhesion kinase (FAK ) - AKT progenitor cells from hiPS cells and subsequent generation ( “ FAK / AKT” ) pathways . CTGF is a major downstream of cells of multiple cell lineages therefrom . FIG . 14D shows factor for EleS - induced FAK / AKT pathways . FIG . 11A the characterization of cardiac progenitor cells with expres shows the results of real- time PCR -based gene expression sion of the markers of Nkx2. 5 and GATA4 by immunos profiling performed for extracellular matrix (“ ECM ” ) and taining (merged images with DAPI) . FIG . 14E shows the cell adhesion molecules . Four of each upregulated and differentiation of human iPS cells into beating cardiomyo downregulated genes were selected based on fold change . cytes. FIG . 14F shows in vitro differentiation of human iPS In FIGS . 11B and 11C , mRNA expression of connective cells into vascular progenitor cells as demonstrated by the tissue growth factor (" CTGF " ) was confirmed by conven - expression of VE - cadherin and CD31 by immunofluores tional RT- PCR and real- time PCR ( n = 5 ) . FIG . 11D shows cence analysis . FIG . 14G shows tube formation by vascular CTGF positivity by immunocytochemistry was higher in progenitor cells labeled with calcein - AM dye . FIG . 14H electrically stimulated cardiac stem cells ( " EleS CSCs” ) shows in vitro differentiation of human iPS cells into smooth when compared with non - electrically stimulated cardiac muscle progenitor cells as shown by a - SMA and calpopnin stem cells ( “ Non - EleS CSCs” ) . immunostaining . [0037 ] FIGS . 12A - 12D show that “ Tert ” is a direct target [0041 ] FIGS . 15A - 15B show the effects of CXCR4 of miR - 195 in stem cell aging. FIG . 12A shows computa expression on muscle progenitor cell ( “MPC ” ) migration tional analysis predicted “ mTert ” as a potential target of and a schematic representation of chemotaxis experiments mmu -miR - 195 . for cell migration . FIG . 15A shows how MPC invading the [0038 ] FIGS . 12B and 12C show transfection of OMSCs collagen gel were quantified by counting 30 optical fields with anti -miR - 195 restored expression of Tert mRNA as per well. FIG . 15B shows MPC cell migration . Null indi well as telomere specific protein TRF2 , as examined by cates GFP / dystrophin expressing control MPC ; MPCCX4 reverse transcriptase polymerase chain reaction and Western indicates GFP /dystrophin expressing MPC that overexpress blot , respectively . FIG . 12D shows cotransfection with CXCR4; MPCsi - CXCR4 indicates MPC treated with siRNA PEZX -Luc vector containing mTert 3 ' UTR and a plasmid targeting CXCR4 (MPCsi -CXCR4 ) , for knock -down of encoding miR - 195 showed decreased luciferase activity CXCR4 gene. The data show increased migration of MPC ( p < 0 .01 vs. PEZX -miR - SC transfected cells ) . Transfection overexpressing CXCR4 . n = 4 , * p < 0 .05 vs . control. efficiency was normalized by Renilla luciferase activity . [0042 ] FIGS . 16A - 16D show that cardiac fibrobrast Acronyms: OMSCs, old mesenchymal stem cells ; Tert , derived human iPS cell colonies were dissociated with telomerase reverse transcriptase ; UTR , untranslated region . accutase and plated in the presence of Y27632 . FIG . 16A [0039 ] FIGS. 13A - 13G show abrogation ofmiR - 195 reju schematically shows cell culture conditions for the genera venates OMSCs by telomere relengthening and antiaging tion of cardiac fibroblasts from human iPSCs. Briefly , to markers reactivation . FIG . 13A shows the structures of generate muscle progenitor cells (MPCs ) from hiPSC in Lenti- miR - 195 inhibitor and Lenti - scramble vectors which vitro , human Induced Pluripotent Stem (iPS ) Cells (ATCC® contain mCherry reporter gene ( red signal) . FIG . 13B shows ACS - 1021TM ) induced from human cardiac fibroblasts were abrogation of miR - 195 significantly reduced senescence cultured with mTeSRTM1 (STEMCELL Technologies Inc . ) associated b - gal expression in OSMCs as compared to on Vitronectin XF ( STEMCELL Technologies Inc. ) coated scramble transfected OMSCs . FIG . 13C shows significant 6 -well plates . iPS Cells were passaged every 4 to 6 days with telomere relengthening of OMSCs upon transfection with ReLeSRTM (STEMCELL Technologies Inc. ) . For differen miR - 195 inhibitor. FIG . 13D shows that miR - 195 inhibition tiation of iPS Cells into MPCs, iPS Cells were dissociated markedly reduced terminal deoxynucleotidyl transferase into single cells with ACCUTASETM (STEMCELL Tech DUTP nick end labeling -positive apoptotic cell death in nologies Inc .) into single cells and seeded at 1x109 cells / cm² OMSCs. FIG . 13E shows that expressions of TERT (not with mTeSRTM1 supplemented with 5 UM RHO /ROCK shown ) , SIRT1 , and Bcl- 2 as well as phosphorylation of pathway inhibitor ( Y -27632 , STEMCELL Technologies p - FOXO1 and p - Akt were significantly increased by trans Inc .) . After 24 hr, the medium was changed to fresh fection of anti- miR - 195 in OMSCs whereas expression of mTeSRTM1. mTeSRTM1 was refreshed daily during first 3 p53 and cleaved caspase 3 was reduced by miR - 195 abro days . After 3 days , culture medium was changed to gation . FIGS . 13F and 13G show knockdown of miR - 195 mTeSRTM1 supplemented with 20 UM ISX - 9 (MedChem significantly restored cell proliferative abilities in OMSCsas Express ) . The medium was refreshed every other day . After examined by cell proliferation assay and colony formation 6 days , the medium was switched to RPMI 1640 Medium assay . Acronyms: OMSCs , old mesenchymal stem cells ; ( Thermo Fisher Scientific ) supplemented with N - 2 Supple SIRT1, sirtuin ( silent mating type information regulation 2 ment ( Thermo Fisher Scientific ) and 20 ?M ISX - 9 and homolog ) 1 ; Bcl- 2 , B cell lymphoma 2 protein , FOX01, refreshed every other day for another 3 to 6 days . Small forkhead box protein 01 also known as forkhead in rhab molecules ( Isx9 & GIV ) were applied to initiate differen domyosarcoma is a protein , and p - FOX01 is phospho tiation and analysed at day 9 . FIG . 16B shows relative forkhead box protein 01, Akt , protein kinase B (PKB ) , also skeletal muscle gene expression by the treatment of Isx9 & known as Akt, p -Akt is phospho - Akt. Giv . FIGS. 16C and 16D show the muscle genes ( PAX3, 10040 ] FIGS. 14A - 14H show isoxazole or isoxazole like PAX7, MYF5 , MYOG , MYOD ) , overexpression superiority compounds mediated generation of cardiac progenitor cells in particular of ISX - 9 . from human induced pluripotent stem cells in vitro . FIGS . [0043 ] FIGS. 17A - 17F show generation and characteriza 14A - 14B show the characterization of human iPS cells for tion of hiPSC derived cardiac progenitor cells (CPCs ). FIG . the expression of pluripotency markers of Oct4 , Sox2 , 17A is a schematic outline of generation of hiPSC -CPCs . Tra - 1 -60 , Tra - 1 -81 , and SSEA4 by immunostaining (merged FIG . 17B showsbright field of hiPSC treated with DMSO or images with DAPI) . FIG . 14C shows an exemplary brief ISX - 9 in RPMI/B27 minus insulin at Dayo , Day3 and Day7 . description of the procedure of the generation of cardiac FIG . 17C show time dependent expression of Nkx2 .5 , US 2018 /0273906 A1 Sep . 27 , 2018

GATA4, ISL - 1 and Mef2c .* vs. Dayo , P < 0 .05 ; # vs . Day3 , staining in Mock , DMSO and ISX - 9 treated groups after 24 P < 0 . 05 , from 3 biological repeated experiments . FIG . 17D hypoxic stresses . (FIG . 21C ) Semi quantitative estimate of shows ISX - 9 upregulated expression of Nkx2 . 5 , GATA4 , TUNEL positive cells . * vs. DMSO group , P < 0 .05 . (FIG . ISL - 1 and Mef2c at day7 compared with DMSO group , * vs . 21D ) Heat map comparing concentrations of paracrine fac DMSO group , P < 0 .05 , from 3 biological repeated experi tors released from cells with different treatments . Each row ments . FIG . 17E shows fluorescence activated cell sorting represents a cytokine and each column represents an inde analysis (FACS ) showing 96 . 5 + 2 . 3 % Nkx2 . 5 positive cells pendent condition . The heat map color scale corresponds to after ISX - 9 treatment . In FIG . 17F , immunofluorescence the absolute concentration ( log 10 , pg /ml ) of cytokines with staining showed that transcription factors ( Nkx2 . 5 , GATA4, minimum and maximum of all values is shown on the right. and ISL - 1 ) were highly upregulated in hiPSC treated with ( FIG . 21E ) Representative images of TUNEL staining 3 ISX - 9 . Scale bar represents 200 um . * * * days post- MI . The host cardiomyocytes were identified by [ 0044 ] FIGS . 18A - 18G : In vitro differentiation of CPCs into three cardiac lineage cells . According to treatment a - sarcomeric actinin . (FIG . 21F ) Quantitation of TUNEL outline , the CPCs expressed CM , EC , SMC specific pro positive cells in the border area of infarct with different teins. (FIG . 18A ) CM progenitors expressed a - sarcomeric treatments . * vs . DPBS group , P < 0 .05 ; # vs. hiPSC group , actinin , cTni, MLC2v , cTnT and Cx43 , scale bar = 50 um . P < 0 .05 . ( FIG . 21G ) Quantitation of TUNEL positive car (FIG . 18B ) Under TEM , these cells were rich in endoplas diomyocytes in the border area of infarct hearts from mice mic reticulum studded with ribosomes, developing myofila in different groups . * vs . DPBS group , P < 0 . 05 ; # vs . hiPSC ments , mitochondria and glycogen particles. Chromatin group, P < 0 .05 . materialwas uniformly distributed in the nucleoplasm . ( FIG . [ 0048 ] FIGS. 22A - 22H : Transplantation of CPCs reversed 18C ) Endothelial progenitors expressed VE - cadherin and cardiac remodeling after MI. Temporal Changes in LVESD CD31, scale bar = 200 um . LDL uptake by these cells was ( FIG . 22A ) , LVEDD ( FIG . 22B ) , FS ( FIG . 22C ) and EF observed ( FIG . 18E ) and formed tube like structures ( FIG . (FIG . 22D ) in mice post transplantation . * P < 0 .05 Vs. DPBS 18D ). (FIG . 18F ) Smooth muscle progenitors expressed treated group at the same time point, # P < 0 .05 vs . hiPSC a - SMA and calponin , scale bar = 200 um . ( FIG . 18G ) FACS treated group at the same time point. DPBS = 13, hiPSC = 16 analysis revealed 95 . 2 + 2 . 1 % CMS, 90 . 3 + 2 . 5 % ECs and and CPC = 16 . (FIG . 22E ) Representative echographs of 92 . 3 + 1. 8 % SMCs in basal differentiation medium . M -mode and B mode echocardiography following 3M post [0045 ] FIGS . 19A - 19C : Global mRNA and miRNA MI. (FIG . 22F ) Cardiac fibrosis was evaluated at seven expression profile in CPCs. ( FIG . 19A ) Heat map of differ levels by Masson ' s trichrome staining at 3M post- MI . (FIG . entially expressed mRNA ( Q value < 10 - 15 ) among undif 22G ) Sections of representative hearts are shown . ( FIG . ferentiated hiPSCs, DMSO or ISX - 9 treated hiPSCs 2211 ) Quantification of scar tissue size . * P < 0 .05 Vs. DPBS detected by mRNA - seq . (FIG . 19B ) Heat map of differen tially expressed miRNAs that exhibited significant increase treated group at the same time point, # P < 0 .05 vs . hiPSC or decrease individually between DMSO and ISX - 9 treated treated group (n = 5 ) . hiPSCs. (FIG . 19C ) Pathway enrichment analysis of upregu [0049 ] FIGS . 23A - 23J: Differentiation of transplanted lated and downregulated genes in CPCs induced by ISX - 9 in CPCs into cardiac lineage cells in the infarcted heart. ( FIG . comparison with DMSO treated hiPSCs . 23A - FIG . 23B ) Engrafted CPCs were identified by PKH - 26 [0046 ] FIGS. 20A - 20G : Potential key signaling pathways fluorescence ( red fluorescence ) ; muscle fibers were visual mediated by ISX - 9 treatment. (FIG . 20A ) Time dependent ized via immunostaining for human specific cTnT or a -sar expression of Wnt5 , Wnt11 and Wnt 3a with ISX - 9 treat comeric actinin (Green fluorescence ) at 3M post -MI . ( FIG . ment. * vs. Dayo , P < 0 .05 ; # vs. Day3, P < 0 .05 , n = 6 from 3 23C - FIG . 23D ) Immunofluorescence images of PKH - 26 and biological repeated experiments . (FIG . 20B ) Continuous EC and SMC makers in the infarcted heart at 2M post- MI . treatment for 7 days with ISX - 9 increased expression of Scale bar = 50 um . ( FIG . 23E - FIG . 23G ) Representative Wnt5 , Wnt11 and Wnt 3a compared with DMSO group . * vs . immunofluorescence images and quantification of trans DMSO group , P < 0 .05 , from 3 biological repeated experi planted CPCs (FIG . 23E ) and hiPSCs (FIG . 23F ) in infarct ments . Continuous treatment with ISX - 9 for 7 days and border area 72 h post- MI using human mitochondria increased expression of Wnt5 , Wnt11 ( FIG . 20C ) and car antigen (human -mit ) tracking . * P < 0 .05 vs. hiPSCs treated diac transcription factors (Nkx2 . 5 , Mef2c , GATA4 and ISL group , n = 3 . (FIG . 23H -FIG . 23J) Representative immuno 1 ) (FIG . 20D ) compared with treatment only for 3 days. * vs. fluorescence images and quantification of differentiated 3 day treatment group , P < 0 .05 , from 3 biological repeated CMs in infarct and border area 3M post -MI from CPCs experiments . (FIG . 20E ) Schematic description of the pro (FIG . 23H ) and hiPSCs ( FIG . 231) treated mice using cTnl tocol to study signaling pathways of cardiac differentiation and human -mit tracking . * P < 0 .05 vs . hiPSCs treated group , induced by ISX - 9 . Inhibition of TGFB signaling pathway n = 3 . with LY2109761 or canonical Wnt signaling pathway with [0050 ] FIGS . 24A -24D show characterization of exo XAV939 and IWP2 in the initial differentiation stage (FIG . somes from iPSC and CPCISX - 9 . FIG . 24A shows exo 20F ) or blockade of non - canonical Wnt signaling in late somes isolated from iPSC and CPCISX - 9 visualized by differentiation stage ( FIG . 20G ) with siWnt5 , siWnt11 or transmission electron microscopy ( TEM ) . Scale bar = 200 WIF - 1 along treatment with ISX - 9 significantly decreased nm . FIG . 24B shows representative western blots showing expression of cardiac transcription factors * vs . ISX - 9 group , that exosomes from iPSC and CPCISX - 9 were enriched in P < 0 .05 , from 3 biological repeated experiments . Exosome (Exo ) - specific markers Tsg101. Other common [0047 ] FIGS. 21A - 21G : Cytoprotective effects of ISX - 9 exosome markers included CD9, Hsp70 and Flotillin - 1. on CPCs. (FIG . 21A ) Morphology of hiPSCs in RPMI/B27 Calnexin was absent in exosomes. FIG . 24C is a represen medium treated with DMSO or ISX - 9 under 1 % 02 for 12 h tative graph of size distribution of exosomes from iPSC and or 24 h . ( FIG . 21B ) Representative images of TUNEL CPCISX - 9 detecting by TRPS . FIG . 24D shows average size US 2018 /0273906 A1 Sep . 27 , 2018 of exsomes as measured by TRPS . No significance differ- Masson ' s trichrome- stained sections from hearts after dif ence in size between exosomes from iPSC and CPCISX - 9 ferent treatments . ( 286 ) Quantitative estimate of fibrosis in was observed . mice after different treatments . * vs . PBS group , P < 0 . 05 ; # [0051 ] FIGS. 25A - 25B shows miRNA expression profil vs. Exo - iPSC group , P < 0 .05 , n = 4 . ing and validation of microarray data . FIG . 25A is heatmap [0055 ] FIG . 29 is a chart depicting Personalized Cell- Free analysis of microarray data showing significantly upregu Therapy with exosomes generated from iPSC - derived car lated miRNAs in Exo -CPCISX - 9 compared with those in diac progenitor cells . Exo - iPSC , Exo -EB or Exo -CPCcomm . Red or green colors [0056 ] FIGS. 30A and 30B show the effect of three small indicate differentially up - or downregulated miRNAs, molecules ( ISX9 , Danzol, Givinostat) on expression of respectively ( P < 0 . 05 ) . n = 3 . FIG . 25B is validation of cardiac and skeletal muscle genes . Real time PCR on microarray data using real- time PCR . Quantitative data dystrophin on small molecule ( ISX9, GIV ) expression in showing significant overexpression of miR - 373, miR - 367 , IPS cells . miR -520 , miR -548ah , and miR - 548q in Exo - CPCISX - 9 . RNA samples were from three biological repeat experi DETAILED DESCRIPTION ments. * , P < 0 . 001. [0052 ] FIGS. 26A - 26E show CPC ISX - 9 - derived exo Definitions somes enriched with miR - 373 reversed TGFB stimulation of [0057 ] The practice of the present invention will employ, fibroblasts . In FIG . 26A , PKH26 labeled exosomes from unless otherwise indicated , conventional techniques of tis CPC ISX - 9 (red ) were incubated together with fibroblasts sue culture, immunology, molecular biology , microbiology , and were observed within the fibroblasts (green , Calcein cell biology and recombinant DNA , which are within the AM ) , mostly located at the perinuclear region . The white skill of the art . See , e . g. , Sambrook , Fritsch and Maniatis arrows show the uptaken exosomes. FIG . 26B show real (1989 ) Molecular Cloning : A Laboratory Manual, 2nd edi time PCR results showing anti -miR -373 ( miR - 373 inhibitor ) tion ; F . M . Ausubel, et al . eds. ( 1987 ) Current Protocols In at a concentration of 25 nM and 50 nM effectively down - Molecular Biology ; the series Methods in Enzymology regulated miR - 373 expression in CPCs ISX - 9. FIG . 26C (Academic Press , Inc . ) : PCR 2 : A Practical Approach ( 1995 ) show expression of exosomal miR - 373 was significantly ( M . J . MacPherson , B . D . Hames and G . R . Taylor eds. ) ; inhibited in CPCs ISX - 9 after treatment with 25 nM anti Harlow and Lane , eds. ( 1988 ) Antibodies, A Laboratory miR - 373 . FIG . 26D show expression of miR - 373 in fibro Manual; Harlow and Lane , eds . ( 1999 ) Using Antibodies, A blasts treated with Exo - CPC ISX - 9 or anti -miR -373 - Exo Laboratory Manual; and R . I. Freshney, ed . ( 1987 ) Animal CPC ISX - 9 . Results were tabulated from three independent Cell Culture . experiments , n = 3 , P < 0 .001 . FIG . 26E show expression of [ 0058 ] All numerical designations , e . g . , pH , temperature , fibrotic genes in fibroblasts stimulated with TGFB after time, concentration , and molecular weight, including ranges , treatment with Exo -CPC ISX -9 or anti- miR -373 - Exo - CPC are approximations which are varied ( + ) or ( - ) by incre ISX - 9 . * vs . Exo - CPC ISX - 9 group , n = 3 , P < 0 .05 . ments of 1 . 0 or 0 . 1 , as appropriate . It is to be understood , [0053 ] FIGS. 27A - 27D show CPCISX - 9 -derived exo although not always explicitly stated that all numerical somes promoted cardiomyocyte proliferation in mice after designations are preceded by the term “ about” . It also is to myocardial infarction (MI ) . FIG . 27A is a representative be understood , although not always explicitly stated , that the image of ki67 positive cardiomyocyte ( cTnT positive ) in reagents described herein are merely exemplary and that Exo - CPCISX - 9 treated mouse 30 days after MI. Bar = 50 um . equivalents of such are known in the art . FIG . 27B is semi quantitative data of proliferating cardio [0059 ] As used in the specification and claims, the singu myocytes in peri - infarct region 30 days after myocardial lar form “ a , " " an ” and “ the ” include plural references unless infarction as demonstrated by positivity of ki67 . PBS group : the context clearly dictates otherwise . n = 940 cardiomyocytes from 3 hearts ; Exo - iPSC group : [0060 ] The terms autologous transfer , autologous trans n = 950 cardiomyocytes from 3 hearts ; Exo - CPCISX - 9 plantation , autograft and the like refer to treatments wherein group , n = 951 cardiomyocytes from 3 hearts . * vs . PBS the cell donor is also the recipient of the cell replacement group , P < 0 . 05 ; # vs . Exo - iPSC group , P < 0 . 05 . FIG . 27C are therapy . The terms allogeneic transfer, allogeneic transplan representative images of arterioles in peri- infarct area from tation , allograft and the like refer to treatments wherein the mice 4 weeks after MI. Arterioles were identified by Q -SMA cell donor is of the same species as the recipient of the cell positive staining ( green ) in vascular structures . Bar = 100 um . replacement therapy, but is not the same individual. A cell FIG . 27D are quantitative estimates of arterioles from MI transfer in which the donor ' s cells and have been histocom mice treated with exosomes derived from different iPSC . * patibly matched with a recipient is sometimes referred to as vs. PBS group , P < 0 .05 ; # vs. Exo - iPSC group , P < 0 .05 , n = 3 . a syngeneic transfer. The terms xenogeneic transfer, xeno [0054 ] FIGS. 28A - 28G show CPC ISX - 9 -derived exo geneic transplantation , xenograft and the like refer to treat somes reversed cardiac remodeling in infarcted mice . FIG . ments wherein the cell donor is of a different species than the 28A shows representative M mode echocardiography recipient of the cell replacement therapy. images from three groups at 30 days after MI. LVDs ( 28B ) [0061 ] A “ biocompatible scaffold ” refers to a scaffold or and LVDd ( 28C ) were significantly decreased 30 days matrix for tissue - engineering purposes with the ability to post -MI in mice with Exo - CPCISX - 9 treatment. Exo -CP perform as a substrate that will support the appropriate CISX - 9 treatment significantly enhanced EF ( 28D ) and FS cellular activity to generate the desired tissue , including the ( 28E ) . * vs. PBS group , P < 0 . 05 ; # vs. Exo -iPSC group , facilitation of molecular and mechanical signaling systems, P < 0 . 05 , PBS group : n = 10 , Exo -iPSC group , n = 9 , Exo without eliciting any undesirable effect in those cells or CPCISX - 9 , n = 11. EF, ejection fraction ; FS , fractional short inducing any undesirable local or systemic responses in the ening ; LVDd , diastolic left ventricular dimensions; LVDs eventual host. In other embodiments, a biocompatible scaf systolic left ventricular dimensions . (28F ) Representative fold is a precursor to an implantable device which has the US 2018 /0273906 A1 Sep . 27 , 2018 ability to perform its intended function , with the desired [ 0067 ] “ Clonal proliferation ” refers to the growth of a degree of incorporation in the host , without eliciting an population of cells by the continuous division of single cells undesirable local or systemic effects in the host. Biocom into two identical daughter cells and /or population of iden patible scaffolds are described in U . S . Pat. No . 6 ,638 , 369. tical cells . [0068 ] CTGF, also known as CCN2 or connective tissue [0062 ] As used herein , a “ cardiac patch ” or “ cardiac growth factor, is a matricellular protein of the CCN family progenitor patch embedded in fibrin ” or “ Epicardial patch ” of extracellular matrix -associated heparin -binding proteins is a bioengineered 2D or 3 - dimensional ( 3D ) tissue patch ( see also CCN intercellular signaling protein ). comprising or containing iPS cells or iPS cells derived [0069 ] Telomerase reverse transcriptase (“ TERT” ) is a cardiac lineage or cardiac progenitor cells . catalytic subunit of the enzyme telomerase , which , together [0063 ] A “ cardiomyocyte ” or “ cardiac myocyte ” is a spe with the telomerase RNA component ( TERC ) , comprises the cialized muscle cell which primarily forms the myocardium most important unit of the telomerase complex . of the heart. Cardiomyocytes have five major components : [0070 ] The miR - 290 - 295 cluster is a pluripotent cluster 1 . cell membrane ( sarcolemma ) and T - tubules , for impulse codes for a family of microRNAs (miRNAs ) that are conduction , 2 . sarcoplasmic reticulum , a calcium reservoir expressed de novo during early embryogenesis and are needed for contraction , 3 . contractile elements , 4 . mitochon specific for mouse embryonic stem cells ( ESC ) and embry dria , and 5 . a nucleus . Cardiomyocytes can be subdivided onic carcinoma cells (ECC ) . Such are known in the art and into subtypes including , but not limited to , atrial cardiomyo described , for example , in Lichner et al. ( 2011 ) Differentia cyte , ventricular cardiomyocyte , SA nodal cardiomyocyte , tion , January 81 ( 1 ) : 11 - 24 . peripheral SA nodal cardiomyocyte , or central SA nodal [0071 ] Chemokine ( C — C motif ) ligand 7 (CCL7 ) is a cardiomyocyte . Stem cells can be propagated to mimic the small cytockine previously known as monocyte -specific physiological functions of cardiomyocytes or alternatively, chemokine 3 (MCP3 ) . The protein sequence is available differentiate into cardiomyocytes . This differentiation can be under Accession number NP _ 006264 and the murine detected by the use of markers selected from , but not limited sequence is available under NP _ 038682 ( see also ncbi. nlm . to , myosin heavy chain , myosin light chain , actinin , tro nih .gov / gene /6354 , last accessed on Apr. 16 , 2014 ). An ponin , tropomyosin , GATA4 , Mef2c , and Nkx - 2 . 5 . antibody and kit to detect CCL7 is available from Sino 100641. The cardiomyocyte marker “ myosin heavy chain ” Biological Inc . and “ myosin light chain " are part of a large family ofmotor [0072 ] CXCR2 chemokine receptor 2 ( CXCR2 ) is a pro proteins found in muscle cells responsible for producing tein encoded by this gene is a member of the G -protein contractile force . These proteins have been sequenced and coupled receptor family . This protein is a receptor for characterized , see for example GenBank Accession Nos . interleukin 8 ( ILS ) . It binds to IL8 with high affinity , and transduces the signal through a G - protein activated second AAD29948 , CAC70714 , CAC70712 , CAA29119 , P12883 , messenger system . This receptor also binds to chemokine NP _ 000248 , P13533 , CAA37068 , ABR18779 , AAA59895 , ( C — X — C motif ) ligand 1 (CXCL1 /MGSA ) . Information AAA59891 , AAA59855 , AAB91993 , AAH31006 , regarding the protein and its gene is found on nchbi. nlm . NP _ 000423, and ABC84220 . The genes for these proteins nih .gov / gene/ 3579 (last accessed on Apr. 16 , 2014 ) . has also been sequenced and characterized , see for example [0073 ] Integral membrane protein 2A is a stem cell GenBank Accession Nos. NM _ 002472 and NM _ 000432 . marker. The sequence of the human gene is reported at 10065 ]. The cardiomyocyte marker " actinin ” is a mircro UniProtKB (043736 ) and the murine sequence is reported at filament protein which are the thinnest filaments of the Q61500 (uniprot . org / uiprot, last accessed on Apr. 16 , 2014 ) . cytoskeleton found in the cytoplasm of all eukaryotic cells . [0074 ] DNA ( cytosine -5 )- methyltransferase 1 is an Actin polymers also play a role in actomyosin - driven con enzyme that is encoded by the DNMT1 gene . The complete tractile processes and serve as platforms for myosin ' s ATP sequence of the protein and its gene is available at gen hydrolysis - dependent pulling action in muscle contraction . ecards .org / cgi -bin / carddisp .pl ? gene = DNMT1, last accessed This protein has been sequenced and characterized , see for on Apr. 16 , 2014 . Antibodies to detect the protein are example GenBank Accession Nos . NP _ 001093 , commercially available , e . g. , from Cell Signaling Technolo NP _ 001095, NP _ 001094 , NP _ 004915 , P35609 , gies (DNMT1 ( D63A6 ) XP® Rabbit mAb # 5032 ) . DNA NP _ 598917 , NP _ 112267 , AAI07534 , and NP _ 001029807 . ( cytosine - 5 )- methyltransferase 3 is an enzyme that is The gene for this protein has also been sequenced and encoded by the DNMT3 gene . characterized , see for example GenBank Accession Nos . [0075 ] EFNA3 or ephrin A3 is a protein receptor. The NM _ 001102 , NM _ 004924 , and NM _ 001103 . human protein sequence is reported at ncbi. nlm .nih . gov / [0066 ] The cardiomyocyte marker “ troponin ” is a com gene / 1944 . Antibodies useful for the detection and analysis plex of three proteins that is integral to muscle contraction of the protein are available from R & D Systems and Santa in skeletal and cardiac muscle . Troponin is attached to the Cruz Biotechnology . protein “ tropomyosin ” and lies within the groove between [0076 ] “ Let- 7 ” refers to a family of microRNAs. The actin filaments in muscle tissue . Tropomyosin can be used as sequences are reported at the miRBase at mirbase . org / cgi a cardiomyocyte marker. These proteins have been bin /mirna _ summary. pl ? fam = MIPF000002 , last accessed on sequenced and characterized , see for example GenBank Apr. 16 , 2014 . Methods for detecting such are known in the Accession Nos. NP _ 000354 , NP _ 003272 , P19429 , art, e. g. , U . S . Patent Application Publication No . 2014 / NP _ 001001430 , AAB59509 , AAA36771, and 0005251 . NP _ 001018007. The gene for this protein has also been [0077 ] Max is a pluripotency marker that bindsMYC . See sequenced and characterized , see for example GenBank Chappell et al. ( 2013 ) Genes & Dev. 27: 725 - 733. Accession Nos . NM _ 000363, NM _ 152263 , and [0078 ] The protein encoded by the Tsg101 gene belongs to NM _ 001018007. a group of apparently inactive homologs of ubiquitin - con US 2018 /0273906 A1 Sep . 27 , 2018 jugating enzymes . The gene product contains a coiled - coil [ 0093 ] mir - 30c1 encodes a microRNA annotated as domain that interacts with stathmin , a cytosolic phosphop ELEnsembl : ENSG00000207962 and miRBase : has -mir - 30c - 1 , rotein implicated in tumorigenesis . The protein may play a which may be involved in ECM maintenance and cancer . role in cell growth and differentiation and act as a negative [0094 mir - 30c2 encodes a microRNA annotated as growth regulator. In vitro steady - state expression of this Ensembl: ENSG00000199094 and miRBase : has- mir - 30c - 2 . tumor susceptibility gene appears to be important for main Similar to miR - 30c1 , it may be involved in ECM mainte tenance of genomic stability and cell cycle regulation . nance and cancer. Mutations and alternative splicing in this gene occur in high [0095 ] Meox1 encodes the mesenchyme homeobox 1 pro frequency in breast cancer and suggest that defects occur tein , and is annotated as Ensembl: ENSG00000005102 and during breast cancer tumorigenesis and / or progression . Uniprot: P50221. This protein plays a role in somite devel [0079 ] CD9 encodes a member of the transmembrane 4 opment. Genetic mutations are associated with Klippel- Feil superfamily , also known as the tetraspanin family . Tetras Syndrome. panins are cell surface glycoproteins with four transmem [0096 ] Meox2 encodes the mesenchyme homeobox 2 pro brane domains that form multimeric complexes with other tein , and is annotated as Ensemble : ENSG00000106511 and cell surface proteins. The encoded protein functions in many Uniprot: P50222 . Based on homology to the mouse , this cellular processes including differentiation , adhesion , and protein is thought to play a role in myogenesis and limb signal transduction , and expression of this gene plays a development. Mutations are associated with craniofacial and critical role in the suppression of cancer cell motility and skeletal abnormalities as well as Alzheimer ' s . metastasis . [0097 ] Pax3 is annotated as Ensembl: ENSG00000135903 [0080 ] As used herein , the term “microRNAs ” or “miR and Uniprot : P23760. This gene encodes a member of the NAs ” refers to post -transcriptional regulators that typically paired box family of transcription factors , which regulates bind to complementary sequences in the three prime proliferation and migration during neural development and untranslated regions ( 3 ' UTRs) of target messenger RNA myogenesis . Mutations in this gene are associated with transcripts (mRNAs ) , usually resulting in gene silencing . craniofacial - deafness -hand syndrome, Rhabdomyosarcoma , Typically , miRNAs are short , non -coding ribonucleic acid and Waardenburg syndrome. (RNA ) molecules , for example, 21 or 22 nucleotides long . [0098 ] Pax7 is annotated as Ensembl: ENSG00000009709 The terms " microRNA ” and “ miRNA ” are used inter and Uniprot : P23759 . This gene encodes a member of the changeably . paired box family of transcription factors , which regulates [0081 ] mir -373 is annotated as ENSG00000199143 and proliferation of muscle precursor cells. It is vital for embry miRBase : has -mir - 373 . onic development and implicated in cancer, including Rhab 10082 ] mir - 210 is annotated as ENSG00000199038 and domyosarcoma. miRBase : has -mir - 210 and is associated with Sudden Infant [0099 ] MyoD1 is annotated as Ensembl: Death Syndrome susceptibility . ENSG00000129152 and Uniprot : P15172 . This gene [0083 ] Tcf15 encodes a basic helix -loop - helix transcrip encodes a myogenic helix - loop -helix transcription factor tion factor expressed early in development, which is believe that regulates myocyte differentiation via inhibition of the to participate in patterning of the mesoderm and its deriva cell cycle . This protein is known to interact with other key tive cell types . This gene is annotated as Ensembl: muscle factors , Myf5 , Myf6 , and MyoG . ENSG00000125878 and Uniprot Q12870 . [0100 ] Myog encodes the muscle - specific basic helix [0084 ] mir - 377 is annotated as ENSG00000199015 and loop -helix transcription activator , myogenin . mirBase: has -mir -377 . [0101 ] Myh2 encodes a class II or conventional myosin [0085 ) mir -367 is annotated as ENSG00000199169 and heavy chain . As a motor protein , it functions in skeletal muscle contraction , and mutations are associated with inclu miRBase : has -mir - 367 . sion - body myopathy. Myh2 is annotated as Ensembl : [0086 ] mir- 520c is annotated as ENSG00000207738 and ENSG00000125414 and Uniprot: Q9UKX2. Numerous miRBase : has -mir - 520c . splice variants have been reported . [0087 ) mir -548ah is annotated as ENSG00000283682 and [0102 ] Myhá encodes a motor protein that forms the alpha mirBase: has -mir -548ah . heavy chain subunit of cardiac myosin . This gene is anno [0088 ] Dystrophin intends a protein encoded by the gene tated as Ensemble : ENSG00000197616 and Uniprot: Dmd , annotated as Ensembl: ENSG00000198947 and Uni P13533 , and mutations are associated with atrial septal prot: P11523 . This protein is a component of the dystrophin defects and hypertrophic cardiomyopathy. glycoprotein complex , which anchors the cytoskeleton to the [0103 ] Tbxl encodes a member of the developmentally extra -cellular matrix . Mutations are associated with Duch important T -box transcription factor family . It is annotated as enne muscular dystrophy , Becker muscular dystrophy and Ensembl : ENSG00000184058 and Uniprot : Q43435 . Muta cardiomyopathy, as well as equivalents thereof. tions in this gene are associated with neural- crest defects , [0089 ] mir -548q is annotated as ENSG00000221331 and DiGeorge syndrome, and velocardiofacial syndrome. miRBase : has -mir -548q . [0104 ] Mespl encodes a basic helix - loop -helix transcrip [0090 ] mir -548q is annotated as ENSG00000221331 and tion factor that is involved in development of the somatic miRBase : has -mir - 548q . and cardiac mesoderm , and rostrocaudal patterning of the [0091 ] mir - 335 encodes microRNA -335 , annotated as somites . Mesp1 is annotated in Ensembl : ENSG00000199043 and miRBase : has -mir - 335 . ENSG00000166823 and Uniprot: QOBRJ9 . [0092 ] mir - 21 encodes microRNA - 21, annotated as [0105 ] Des encodes the protein Desmin , and is annotated ENSG00000284190 and miRBase : has- mir -21 . This as Ensemble : ENSG00000175084 and Uniprot: P17661. miRNA is expressed in stem cells and plays a role in cancer . Desmin is a muscle -specific class III intermediate filament US 2018 /0273906 A1 Sep . 27 , 2018 that forms a fibrous network for myofibrils . Mutations in zinc - finger transcription factors. This protein , Uniprot: Des are associated with cardiac and skeletal muscle myo - P43694 , is key to embryogenesis , cardiac development, and pathies . myocardial function . Mutations are associated with septal [0106 ] Cnntbl is a well- known gene that encodes the defects and various forms of cancer. protein , B -catenin , a key component of the canonical Wnt [0116 ] Tbx5 is a member of the T -box gene family , which signaling pathway . In the presence of Wnt, ß - catenin trans contains a conserved DNA -binding domain . Numerous tran locates to the nucleus as acts as a transcriptional regulator. scripts of Tbx5 are curated in RefSeq and the Ensembl gene This protein is also involved in regulation of contact inhi identifier is ENSG00000089225 . The protein product, Uni bition . Mutations in this gene are associated with mental prot: Q99593 , is important for heart and limb development, retardation and colorectal cancer. The Cnntbl gene is anno and mutations in this gene are associated with Holt -Oram tated as Ensemble : ENSG00000168036 and Uniprot: syndrome. P35222 . [0117 ] TnnT2 is a gene encoding cardiac troponin T2 [ 0107 ] Pax7 is annotated as Ensembl: ENSG00000009709 (Uniprot : P45379 ) the tropomyosin -binding unit of the tro and Uniprot: P23759. This gene encodes a member of the ponin complex . In response to changes in intracellular paired box family of transcription factors , which regulates calcium levels , Tnnt2 regulates muscle contraction . Muta proliferation of muscle precursor cells . It is vital for embry tions in this gene , annotated as Ensembl: onic development and implicated in cancer, including Rhab ENSG00000118194 , are associated with familiar hypertro domyosarcoma. phic cardiomyopathy and dilated cardiomyopathy. [0108 ] Myf5 gene, Ensemble : ENSG00000111049 , encodes a master transcriptional regulator of muscle differ 10118 ] My17 is a gene encoding the calcium binding motor entiation , that binds and promotes transcription of numerous protein myosin light chain 7 . This gene is annotated in myogenic factors (Uniprot : P13349 ) . Mutations in Myf5 are Ensembl: ENSG00000106631 and UniProt: 201449 . Muta associated with skeletal muscle cancer and Rhabdomyosar tion at this locus are associated with Fechtner Syndrome and coma . Familial Atrial Fibrillation . [0109 ] MyoD1 is annotated as Ensembl: [0119 ] MLC2v gene (more commonly denoted as My12 , ENSG00000129152 and Uniprot: P15172 . This gene in humans ) , curated as Refseq : NM _ 00432 and Uniprot: encodes a myogenic helix - loop -helix transcription factor P10916 , encodes the motor protein myosin light chain 2 . that regulates myocyte differentiation via inhibition of the Calcium dependent phosphorylation of this protein results in cell cycle . This protein is known to interact with other key generation of contractile forces. This protein functions in muscle factors , Myf5 , Myf6 , and MyoG . heart development and cardiac contractility , and mutations [ 0110 ] XESI myogenic genes intend Myogenic regulatory are associated with mid - left ventricular chamber hypertro factors (MRF ) which are basic helix - loop - helix ( bHLH ) phic cardiomyopathy. Antibodies are available through transcription factors that regulate myogenesis : MyoD , Invitrogen and Santa Cruz Biotechnology . Myf5 , myogenin , and MRF4 . These proteins contain a [0120 ] Mef2c is a gene ( Ensembl ID ENSG00000081189 ) conserved basic DNA binding domain that binds the E box that produces more than 8 alternatively spliced transcripts DNA motif. [ 2 ] They dimerize with other HLH containing curated in RefSeq . The protein product (Uniprot : Q06413 ) is proteins through an HLH -HLH interaction . a member of the MADS box transcription enhancer factor 2 [0111 ] Pitx2 is annotated as ENSG00000164093 and Uni family , and plays a role in vascular development, cardiac prot: Q99697 . This gene encodes Paired - like homeodomain morphogenesis , myogenesis , and maintenance of the differ transcription factor 2 , which belongs to the bicoid family of entiated state. Genomic aberrations within this gene locus homeodomain proteins . This protein regulates the hormone , are associated with mental retardation , cerebral malforma Prolactin , and is important for development of eyes , teeth , tion , epilepsy, and arrhythmogenic right ventricular dyspla and abdominal organs . Mutations in this gene associate with sia 5 . Cell Signaling Technologies, Novus Biologicals , and Axenfeld - Rieger Syndrome. Invitrogen all provides products for detection and study of [0112 ] ISL1 is a gene (Ensembl : ENSG00000016082 ) that this protein . encodes the transcription factor, ISL LIM homeobox 1 [0121 ] Cdh4 gene produces three transcript variants (Uniprot : P61371 ) . This protein is implicated in motor encoding the protein , Cadherin 4 . A member of the cadherin neuron and retinal ganglion cell specification , and regulating superfamily , Chd4 functions as a calcium - dependent cell expression of the Insulin gene . Mutations are associated adhesion molecule important for brain segmentation and with maturity -onset diabetes and bladder exstrophy. neuronal outgrowth . This protein is also implicated in kid [0113 ] Nkx2. 5 encodes a master transcription factor ney and muscle development. Cdh4 is annotated in Refseq : involved in cardiac development. Annotated as Ensembl: NM _ 001252399 and Uniprot: P55283 . Purified protein , ENSG00000183072 and Uniprot P52952 , mutations this antibodies, and other detection kits are widely available gene can result in atrial septal defects , and a form of through sources including Invitrogen , Abcam , and R & D congenital hypothyroidism . systems. [0114 ] Handl is a basic helix - loop - helix transcription fac [0122 ] Lhx2 encodes the Lim homeobox 2 protein , a tor annotated as Ensembl: ENSG00000113196 and Uniprot : member of the LIM domain family , which carry a cysteine 096004 . During heart development, Handl is expressed rich zinc binding domain . Lhx2 is curated in Refseq : asymmetrically with another Hand protein to direct cardiac NM 004789 and UniProt: P50458 , and believed to function morphogenesis and formation of the right ventricle and as a transcriptional activator involved in cellular differen aortic arch arteries . Mutations in genes encoding Hand tiation and development of the lymphoid and neural lin proteins are associated with congenital heart disease . eages . Antibodies and ELISA detection kits for Lhx2 are [0115 ] GATA4 is annotated as ENSG000001366574 in commercially available from Origene, Santa Cruz Biotech Ensembl, and encodes a member of the gata family of nology and Invitrogen . US 2018 /0273906 A1 Sep . 27 , 2018

[0123 ] Gai is a heterotrimeric G protein subunit that verts phosphatidylinositol 4 ,5 -biphosphate (PI ( 4 ,5 )P2 ) to inhibits the product of cAMP from ATP . An exemplary PIP3 [ 6 ]. P?P3 is the second messenger that activates diverse sequence is provided under GenBank Ref. : NM _ 002069 and signal cascades, including PDK and AKT pathway. Phos UnProt P63096 . Antibodies that recognize this marker are phatase PTEN acts as a negative regulator for the PI3K / AKT commercially available from Santa Cruz Biotechnology signaling pathway, converting PI( 3 ,4 , 5 ) P3 into PI( 4 ,5 )P2 . [0124 ] Cytoskeletal remodeling intends remodeling AKT and PDK phosphorylate diverse proteins that mediate intends the dynamic reorganization of microfilaments ( ac various insulin - and growth factor - induced cellular tins ) , microtubules ( tubulin ) , and intermediate filaments ( i . e . responses such as glycogen synthesis, protein synthesis , cell vimentin , keratin , desmin ), which comprise the eukaryotic cycle initiation , and promotion of cell survival by regulation cytoskeleton . Though complex , this process occurs within of apoptosis factors such as BAD and Bcl -x ( L ) . minutes and facilitates biological functions such as cell [0128 ] As used herein , the term “ microRNAs" or " miR migration , cytokinesis , and muscle contraction . NAs” refers to posttranscriptional regulators that typically [0125 ) Promoting TGF - B induced emt ( epithelial- mensen bind to complementary sequences in the three prime chymal transition ) signaling intends transdifferentiation of untranslated regions ( 3 ' UTRs ) of target messenger RNA cells with epithelial- like properties into cells with mesen transcripts (mRNAs ) , usually resulting in gene silencing . chymal- like properties , as mediated by the signaling mol Typically , miRNAs are short , non - coding ribonucleic acid ecule TGF - B . Non - limiting biological roles for this process , (RNA ) molecules , for example , 21 or 22 nucleotides long . referred to as EMT, include cancer, fibrosis , heart develop The terms " microRNA ” and “ miRNA ” are used inter ment, and cardiac differentiation . Transforming growth fac changeably. tor- ß ( TGF - B ) is a potent inducer of EMT both during [0129 ] miR - 133 refers to a microRNA thathas been linked development and in cancer . In TGF- B induced EMT, acti to an immature or undifferentiated phenotype . Methods to vation of Smad proteins results in their nuclear transloca detect such include , for example , microarray -RT - PCR and tion , DNA binding , and upregulation of EMT transcription RNA - seq. Commercially available kits to miR - 133 is avail factors . Non - limiting examples of EMT transcription factors able from EMD Millipore (SmartFlareTM Detection Probes ) include Snail, Twist , and Zeb . EMT requires cytoskeletal which allow for the detection of miRNA in live cells . remodeling and cardiac differentiation intends efficient dif [0130 ] miR - 762 is a non - coding RNA that has been linked ferentiation of human pluripotent stem cells (PSCs ) such a to posttranscriptional regulation of gene expression in mul IPS cells to contracting cardiomyocytes . ticellular organisms. The miR - 762 human sequence is [ 0126 ] Promoting expression of genes for development of reported under Accession No . MI0003892 ( last accessed on pip3 signaling in cardiomyocytes, muscle contraction and Apr. 16 , 2014 ) . The murine sequence is reported under nf -at hypertrophy signaling pathways intends activate down NR _ 030428 . 1 (see ncbi. nlm .nih . gov / gene / 79103 , last stream signaling components , the most notable one being accessed on Apr . 16 , 2014 ) . Methods to detect such are the protein kinase AKT, which activates downstream ana known in the art and kits are commercially available from , bolic signaling pathways required for cell growth and sur for example , Origene (miR - 762, see origene. com , last vival. Phosphatidylinositol ( 3 , 4 , 5 ) - trisphosphate (PtdIns ( 3 , accessed on Apr. 16 , 2014 ) . 4 ,5 ) P3 ) , abbreviated PIP3 , is the product of the class I [0131 ] miR - 133 refers to a microRNA that has been linked phosphoinositide 3 -kinases ( PI 3 -kinases ) phosphorylation to an immature or undifferentiated phenotype . Methods to of phosphatidylinositol ( 4 , 5 ) - bisphosphate ( PIP2 ) . It is a detect such include, for example , microarray -RT -PCR and phospholipid that resides on the plasma membrane . PIP3 RNA -seq . Commercially available kits to miR - 133 is avail signaling in cardiac myocytes. able from EMD Millipore ( SmartFlareTM Detection Probes ) [0127 ] Phosphoinositide 3 -kinase (PI3K ) can be activated which allow for the detection of miRNA in live cells . in cardiac myocytes by the receptors with intrinsic tyrosine [0132 ] miR - 762 is a non - coding RNA that has been linked kinase activity , such as insulin receptor (INSR ) , growth to post - transcriptional regulation of gene expression in mul factor receptors ( IGF1 receptor and HGF receptor ) , and by ticellular organisms. The miR - 762 human sequence is the G protein -coupled receptors (GPCRs ) . INSR and IGF1 reported under Accession No . MI0003892 ( last accessed on receptor engagement triggers receptor activation and auto Apr. 16 , 2014 ). The murine sequence is reported under phosphorylation . The activated receptor can then phospho NR _ 030428 . 1 (see ncbi. nlm .nih . gov / gene / 79103 , last rylate several intracellular protein substrates , most notably accessed on Apr. 16 , 2014 ) . Methods to detect such are the insulin receptor substrate (IRS1 - 4 ) proteins . Tyrosine known in the art and kits are commercially available from , phosphorylated IRS1 can recruit and activate the down for example , Origene (miR - 762 , see origene . com , last stream effector, PI3K , which generates phosphatidylinositol accessed on Apr. 16 , 2014 ) . 3 , 4 , 5 - trisphosphate (PIP3 ) using inositol- containing phos [0133 ] miR - 195 is an RNA gene , and is reported to be pholipids resident in the plasma membrane as substrates . affiliated with the miRNA class . Diseases associated with IRS proteins also recruit adaptors Shc and Grb - 2 . The miR - 195 include tongue squamous cell carcinoma and pri protein tyrosine phosphatase PTP1B is responsible for nega mary peritoneal carcinoma. Among its related pathways are tively regulating INSR signaling by dephosphorylating the microRNAs in cancer and microRNAs in cardiomyocyte phosphotyrosine residues of this receptor. Hepatocyte hypertrophy . It is also known as MIRN195 , Has -MIR - 195 growth factor receptor ( HGF receptor ) activation induces the and MiRNA 195 . The sequence and homologs are reported tyrosine phosphorylation of GAB1 and its association with in the genecards web page . Nucleic acid sequences are PI3K via the recruitment of its regulatory subunit (PI3KR reported under GenBank Accession No . AK098506 , last class 1A ) that stimulates its catalytic subunit (PI3KC class accessed on Nov . 18 , 2015 . 1A ). Activated adaptors Shc and Grb - 2 recruit exchange [0134 ] ILS1 refers to an insulin gene enhancer protein , factor SOS that activates H - RAS [ 4 ] . H -RAS directly stimu which plays an important role in regulating insulin gene lates PI3K catalytic subunit ( PI3KC class 1A ) . PI3K con - expression . ISL1 is also found central to the development of US 2018 /0273906 A1 Sep . 27 , 2018 pancreatic cell lineages and may also be required for motor Non - limiting examples of such stem cells include adult stem neuron generation . ISL1 is identified as a marker for cardiac cells , embryonic stem cells , embryonic - like stem cells , non progenitor cells . embryonic stem cells, or induced pluripotent stem cells . 10135 ] Tbx - 5 is a cardiac transcription factor, also known (0140 ] As used herein , the terms " overexpress ," " overex as T -box transcription factor (“ TBX5” ) is a protein that in pression ,” and the like are intended to encompass increasing humans is encoded by the TBX5 gene. As indicated on the the expression of a nucleic acid or a protein to a level greater GeneCards human gene database , this gene is a member of than the exosome or microvesicle naturally contains . It is a phylogenetically conserved family of genes that share a intended that the term encompass overexpression of endog common DNA - binding domain , the T - box . T -box genes enous, as well as heterologous nucleic acids and proteins . encode transcription factors involved in the regulation of [0141 ] As used herein , the term “ homogeneous” in refer developmental processes . This gene is closely linked to ence to a population of e cell -derived exosomes or microve related family member T - box 3 (ulnar mammary syndrome) sicles refers to population of cell - derived exosomes or on human chromosome 12 . The encoded protein may play a microvesicles that have a similar amount of an exogenous role in heart development and specification of limb identity . nucleic acid , a similar amount of an exogenous protein , are Mutations in this gene have been associated with Holt -Oram of a similar size , or combinations thereof. A homogenous syndrome, a developmental disorder affecting the heart and population is one wherein about 65 % , about 70 % , about upper limbs . Several transcript variants encoding different 75 % , about 80 % , about 85 % , about 90 % , about 95 % , about isoforms have been described for this gene . The accession 98 % , or 100 % of the cell -derived exosomes or microvesicles for the protein is 099593 or alternatively A6ND77 , or share at least one characteristic . Another example of a alternatively 015301, or alternatively Q96TBO . Antibodies homogenous population is one wherein about 90 % of the to the protein are commercially available from R & D Sys exosomes or microvesicles are less than 50 nm in diameter. tems, Browse EMD , OriGene Antibodies , and Novus Bio 10142 ]. As used herein , the term " heterogeneous ” in ref logicals . erence to a population of cell- derived exosomes or microve [0136 ] As used herein , the term “ a protein that facilitates sicles refers to population of cell- derived exosomes or regeneration and /or improves function of a tissue” intends a microvesicles that have differing amounts of an exogenous protein that can either regenerate or regrow or improve the nucleic acid , differing amounts of an exogenous protein , are tissue function or bone function is a potential cytokine of a different size , or combinations thereof. which improves tissue regeneration or bone function . The [ 0143 ] The term “ substantially ” refers to the complete or gel - forming property makes certain protein polymer highly nearly complete extent or degree of a characteristic and in suitable for biomedical applications, such as tissue regen some aspects, defines the purity of the isolated or purified eration in operations and wounds . Non - limiting examples of population of exosomes or microvesicles . such include IGFBP5 protein which enhances periodontal [0144 ] The term “ purified population , ” relative to cell tissue and PPAR , which activates liver regeneration . populations , cell -derived exosomes or microvesicles or [0137 ] As used herein , " lyophilization ” intends low tem miRNA , as used herein refers to plurality of such that have perature drying or freeze drying . undergone one or more processes of selection for the enrich [0138 ] Cell - derived exosomes or microvesicles, also ment or isolation of the desired exosome or microvesicle or referred to as extracellular exosomes or microvesicles , are miRNA population relative to some or all of some other membrane surrounded structures that are released by cells in component with which cell -derived exosomes or microve vitro and in vivo . Extracellular exosomes or microvesicles sicles are normally found in culture media . Alternatively , can contain proteins , lipids, and nucleic acids and can " purified ” can refer to the removal or reduction of residual mediate intercellular communication between different undesired components found in the conditioned media ( e. g . , cells , including different cell types , in the body. Two types cell debris , soluble proteins, etc .) . A “ highly purified popu of extracellular exosomes or microvesicles are exosomes or lation ” as used herein , refers to a population of cell - derived microvesicles and microvesicles . Exosomes or microve exosomes or microvesicles in which at least 65 % , at least sicles are small lipid -bound , cellularly secreted exosomes or 70 % , at least 75 % , at least 80 % , at least 85 % , at least 90 % , microvesicles that mediate intercellular communication via at least 95 % , at least 98 % , at least 99 % or 100 % of cell cell- to - cell transport of proteins and RNA (El Andaloussi, S . debris and soluble proteins ( e . g ., proteins derived from fetal et al. (2013 ) Nature Reviews: Drug Discovery 12 ( 5 ): 347 bovine serum and the like ) in the conditioned media along 357 ) . Exosomes or microvesicles range in size from with the cell - derived exosomes or microvesicles or miRNA approximately 30 nm to about 200 nm . Exosomes or are removed . The cells , populations, exosomes or microve microvesicles are released from a cell by fusion of multi sicles and miRNA as described herein can be provided in vesicular endosomes (MVE ) with the plasma membrane . isolated , purified , highly purified forms, homogeneous, sub Microvescicles , on the other hand , are released from a cell stantially homogeneous and heterogenous forms. upon direct budding from the plasma membrane (PM ) and [0145 ] As used herein the terms " culture media ” and are packaged with different factors. Microvesicles are typi “ culture medium ” are used interchangeably and refer to a cally larger than exosomes or microvesicles and range from solid or a liquid substance used to support the growth of cells approximately 200 nm to 1 um and have different function ( e . g . , stem cells ) . Preferably , the culture media as used alities . herein refers to a liquid substance capable of maintaining [0139 ] Cell -derived exosomes or microvesicles can be stem cells in an undifferentiated state . The culture media can isolated from eukaryotic cells using commercially available be a water -based media which includes a combination of kits as disclosed herein and available from biovision .com ingredients such as salts , nutrients , minerals , vitamins, and novusbio . com , or using the methods described herein . amino acids, nucleic acids, proteins such as cytokines, Non - limiting examples of cells that cell -derived exosomes growth factors and hormones , all of which are needed for or microvesicles can be isolated from include stem cells . cell proliferation and are capable of maintaining stem cells US 2018 /0273906 A1 Sep . 27 , 2018 in an undifferentiated state . For example , a culture media can phenotype ) , and a negative control ( a subject or a sample be a synthetic culture media such as , for example, minimum from a subject lacking the altered expression or phenotype ). essential media a (MEM -a ) (HyClone Thermo Scientific , Additionally , when the purpose of the experiment is to Waltham , Mass . , USA ) , DMEM /F12 , GlutaMAX (Life determine if an agent effects the differentiation of a stem cell Technologies , Carlsbad , Calif ., USA ) , Neurobasal Medium or expression of an exosome or microvesicle or miRNA , it (Life Technologies, Carlsbad , Calif ., USA ), KO -DMEM is preferable to use a positive control ( a sample with an (Life Technologies, Carlsbad , Calif ., USA ), DMEM /F12 aspect that is known to affect differentiation or altered (Life Technologies, Carlsbad , Calif ., USA ) , supplemented expression ) and a negative control (an agent known to not with the necessary additives as is further described herein . In have an affect or a sample with no agent added ) . some embodiments , the cell culture media can be a mixture [0150 ] The term " culturing” refers to the in vitro propa of culture media . Preferably, all ingredients included in the gation of cells or organisms on or in media of various kinds . culture media of the present disclosure are substantially pure It is understood that the descendants of a cell grown in and tissue culture grade. “ Conditioned medium ” and “ con culture may not be completely identical ( i . e . , morphologi ditioned culture medium ” are used interchangeably and refer cally , genetically , or phenotypically ) to the parent cell. By to culture medium that cells have been cultured in for a “ expanded ” is meant any proliferation or division of cells . period of time and wherein the cells release / secrete compo [0151 ] As used herein , the term “ detectably labeled ” nents ( e . g . , proteins, cytokines , chemicals , etc . ) into the means that the agent (biologic or small molecule ) is attached medium . to another molecule , compound or polymer that facilitates [0146 ] “ composition ” is also intended to encompass a detection of the presence of the agent in vitro or in vivo . combination of a cell, a cell population , an exosome or 0152 ] A “ detectable label ” intends a directly or indirectly microvesicle, an miRNA , or populations of such , or an detectable compound or composition that is conjugated active agent, and another carrier, e . g ., compound or com directly or indirectly to the composition to be detected , e. g. , position , inert ( for example, a detectable agent or label ) or N -terminal histadine tags (N -His ), magnetically active iso active , such as an adjuvant, diluent, binder , stabilizer, buf topes , e . g . , 115Sn , 117Sn and 119Sn, a non - radioactive iso fers , salts , lipophilic solvents , preservative , adjuvant or the topes such as 13C and SN , polynucleotide or protein such as like. Carriers also include biocompatible scaffolds , pharma an antibody so as to generate a " labeled " composition . The ceutical excipients and additives proteins, peptides, amino term also includes sequences conjugated to the polynucle acids , lipids, and carbohydrates ( e . g ., sugars, including otide that will provide a signal upon expression of the monosaccharides , di- , tri - , tetra -, and oligosaccharides ; inserted sequences, such as green fluorescent protein (GFP ) derivatized sugars such as alditols , aldonic acids, esterified and the like . The label may be detectable by itself ( e . g . sugars and the like ; and polysaccharides or sugar polymers ) , radioisotope labels or fluorescent labels ) or, in the case of an which can be present singly or in combination , comprising enzymatic label, may catalyze chemical alteration of a alone or in combination 1 - 99 . 99 % by weight or volume. substrate compound or composition which is detectable . The Exemplary protein excipients include serum albumin such as labels can be suitable for small - scale detection or more human serum albumin (HSA ) , recombinant human albumin suitable for high - throughput screening . As such , suitable ( PHA ) , gelatin , casein , and the like . Representative amino labels include, but are not limited to magnetically active acid /antibody components, which can also function in a isotopes , non - radioactive isotopes , radioisotopes, fluoro buffering capacity , include alanine, glycine , arginine, chromes , luminescent compounds , dyes , and proteins , betaine, histidine, glutamic acid , aspartic acid , cysteine , including enzymes. The label may be simply detected or it lysine , leucine , isoleucine , valine , methionine , phenylala may be quantified . A response that is simply detected nine , aspartame, and the like . Carbohydrate excipients are generally comprises a response whose existence merely is also intended within the scope of this invention , examples of confirmed , whereas a response that is quantified generally which include but are not limited to monosaccharides such comprises a response having a quantifiable ( e . g ., numeri as fructose , maltose , galactose , glucose , D -mannose , sor cally reportable ) value such as an intensity , polarization , bose , and the like ; disaccharides, such as lactose , sucrose , and / or other property . In luminescence or fluorescence trehalose , cellobiose , and the like ; polysaccharides , such as assays, the detectable response may be generated directly raffinose , melezitose , maltodextrins , dextrans , starches , and using a luminophore or fluorophore associated with an assay the like ; and alditols , such as mannitol , xylitol, maltitol, component actually involved in binding , or indirectly using lactitol , xylitol sorbitol ( glucitol ) and myoinositol . a luminophore or fluorophore associated with another ( e . g . , [0147 ] A preservative intends a composition that enhances reporter or indicator ) component. the viability of an agent in a composition . Non - limiting [0153 ] Examples of luminescent labels that produce sig examples include Benzoates ( such as sodium benzoate , nals include , but are not limited to bioluminescence and benzoic acid ), Nitrites (such as sodium nitrite ) and Sulphites chemiluminescence . Detectable luminescence response gen ( such as sulphur dioxide ). erally comprises a change in , or an occurrence of, a lumi 10148 ] A cryoprotective is a compound that protects the nescence signal. Suitable methods and luminophores for agent during freezing and thawing procedures . Non - limiting luminescently labeling assay components are known in the examples of such include DMSO , Glycerol, PEG . art and described for example in Haugland , Richard P . [ 0149 ] A " control” is an alternative subject or sample used ( 1996 ) Handbook of Fluorescent Probes and Research in an experiment for comparison purpose . A control can be Chemicals (6th ed .) . Examples of luminescent probes " positive” or “ negative ” . For example , where the purpose of include , but are not limited to , aequorin and luciferases . the experiment is to determine a correlation of an altered [0154 ] Examples of suitable fluorescent labels include, but expression level of a gene with a particular phenotype , it is are not limited to , fluorescein , rhodamine , tetramethylrhod generally preferable to use a positive control ( a sample from amine, eosin , erythrosin , coumarin , methyl- coumarins , a subject , carrying such alteration and exhibiting the desired pyrene , Malacite green , stilbene , Lucifer Yellow , Cascade US 2018 /0273906 A1 Sep . 27 , 2018 14

BlueTM , and Texas Red . Other suitable optical dyes are such as a heart , liver, or muscle cell. “ Directed differentia described in the Haugland , Richard P . ( 1996 ) Handbook of tion ” refers to the manipulation of stem cell culture condi Fluorescent Probes and Research Chemicals (6th ed . ) . tions to induce differentiation into a particular cell type . [ 0155 ] As used herein , " expression ” refers to the process “ Dedifferentiated " defines a cell that reverts to a less com by which polynucleotides are transcribed into mRNA and / or mitted position within the lineage of a cell . As used herein , the process by which the transcribed mRNA is subsequently the term “ differentiates or differentiated " defines a cell that being translated into peptides , polypeptides , or proteins . If takes on a more committed ( “ differentiated ” ) position within the polynucleotide is derived from genomic DNA , expres the lineage of a cell. As used herein , “ a cell that differentiates sion may include splicing of the mRNA in an eukaryotic into a mesodermal (or ectodermal or endodermal ) lineage” cell . defines a cell that becomes committed to a specific meso [0156 ] “ Differentially expressed ” intends an up - or down dermal, ectodermal or endodermal lineage , respectively . ward expression of a gene, exosome or microvesicle , or Examples of cells that differentiate into a mesodermal marker, for example , as compared to a control. In one aspect, lineage or give rise to specific mesodermal cells include , but a control is a differentiated cell as compared to a pluripotent are not limited to , cells that are adipogenic , leiomyogenic , or stem cell . “ Differentially expressed ” as applied to a gene , chondrogenic , cardiogenic , dermatogenic , hematopoetic , protein , cell, population , exosome or microvesicle , miRNA , hemangiogenic , myogenic , nephrogenic , urogenitogenic , or marker , refers to the differential production of the product osteogenic , pericardiogenic , or stromal. as compared to a control such as expression level found in [0159 ] As used herein , the term “ differentiates or differ the native environment . Differently expressed is mRNA entiated " defines a cell that takes on a more committed transcribed from the gene or the protein product encoded by (" differentiated " ) position within the lineage of a cell . the gene . A differentially expressed gene may be overex “ Dedifferentiated " defines a cell that reverts to a less com pressed or underexpressed ( a .k . a . inhibited ) as compared to mitted position within the lineage of a cell. Induced pluri the expression level of a normal , non - treated , native or potent stem cells are examples of dedifferentiated cells. control cell. In one aspect, it refers to overexpression that is [0160 ] As used herein , the “ lineage ” of a cell defines the 1 . 5 times , or alternatively, 2 times, or alternatively , at least heredity of the cell, i. e. its predecessors and progeny . The 2 . 5 times , or alternatively , at least 3 . 0 times , or alternatively , lineage of a cell places the cell within a hereditary scheme at least 3 . 5 times, or alternatively, at least 4 . 0 times , or of development and differentiation . alternatively , at least 5 times , or alternatively 10 times higher [ 0161 ] A “ multi - lineage stem cell ” or “ multipotent stem ( i. e ., and therefore overexpressed ) or lower than the expres cell” refers to a stem cell that reproduces itself and at least sion level detected in a control sample . The term “ differen two further differentiated progeny cells from distinct devel tially expressed ” also refers to nucleotide sequences in a cell opmental lineages . The lineages can be from the same germ or tissue which are expressed where silent in a control cell layer ( i . e . mesoderm , ectoderm or endoderm ) , or from or not expressed where expressed in a control cell . different germ layers . An example of two progeny cells with [0157 ] The term " stem cell” refers to a cell that is in an distinct developmental lineages from differentiation of a undifferentiated or partially differentiated state and has the multilineage stem cell is a myogenic cell and an adipogenic capacity for self -renewal and / or to generate differentiated cell (both are of mesodermal origin , yet give rise to different progeny . Self -renewal is defined as the capability of a stem tissues ) . Another example is a neurogenic cell ( of ectoder cell to proliferate and give rise to more such stem cells , mal origin ) and adipogenic cell ( of mesodermal origin ) . while maintaining its developmental potential ( i. e . , totipo [0162 ] A “ precursor” or “ progenitor cell ” intends to mean tent, pluripotent, multipotent, etc . ) . The term “ somatic stem cells that have a capacity to differentiate into a specific type cell” is used herein to refer to any stem cell derived from of cell. A progenitor cell may be a stem cell. A progenitor non -embryonic tissue , including fetal , juvenile , and adult cell may also be more specific than a stem cell. A progenitor tissue . Natural somatic stem cells have been isolated from a cell may be unipotent or multipotent. Compared to adult wide variety of adult tissues including blood , bone marrow , stem cells , a progenitor cell may be in a later stage of cell brain , olfactory epithelium , skin , pancreas , skeletalmuscle , differentiation . An example of progenitor cell includes , and cardiac muscle . Exemplary naturally occurring somatic without limitation , a progenitor nerve cell. stem cells include , but are not limited to , mesenchymal stem [0163 ] A “ parthenogenetic stem cell ” refers to a stem cell cells (MSCs ) and neural stem cells (NSCs ) . In some embodi arising from parthenogenetic activation of an egg . Methods ments , the stem or progenitor cells can be embryonic stem of creating a parthenogenetic stem cell are known in the art . cells . As used herein , " embryonic stem cells” refers to stem See , for example , Cibelli et al . (2002 ) Science 295 (5556 ): cells derived from tissue formed after fertilization but before 819 and Vrana et al. ( 2003 ) Proc . Natl. Acad . Sci. USA 100 the end of gestation , including pre - embryonic tissue ( such ( Suppl. 1 ) 11911 - 6 . as, for example , a blastocyst ) , embryonic tissue , or fetal [0164 ] As used herein , a “ pluripotent cell” defines a less tissue taken any time during gestation , typically but not differentiated cell that can give rise to at least two distinct necessarily before approximately 10 - 12 weeks gestation . ( genotypically and / or phenotypically ) further differentiated Most frequently, embryonic stem cells are pluripotent cells progeny cells . In another aspect , a " pluripotent cell ” derived from the early embryo or blastocyst. Embryonic includes an Induced Pluripotent Stem Cell ( iPSC ) which is stem cells can be obtained directly from suitable tissue , an artificially derived stem cell from a non - pluripotent cell , including , but not limited to human tissue , or from estab typically an adult somatic cell , that has historically been lished embryonic cell lines . “ Embryonic - like stem cells " produced by inducing expression of one or more stem cell refer to cells that share one or more , but not all character specific genes . Such stem cell specific genes include , but are istics , of an embryonic stem cell . not limited to , the family of octamer transcription factors , [0158 ] “ Differentiation ” describes the process whereby an i. e . Oct- 3 / 4 ; the family of Sox genes, i. e ., Sox1 , Sox2 , Sox3 , unspecialized cell acquires the features of a specialized cell Sox 15 and Sox 18 ; the family of Klf genes, i. e . Klfi , Klf2 , US 2018 /0273906 A1 Sep . 27 , 2018 15

K1f4 and K1f5 ; the family of Myc genes , i. e . c -myc and such are commercially available from , for example , EMD L -myc ; the family of Nanog genes , i . e ., OCT4 , NANOG and Millipore (MILLIPLEX® Map Kit ) . REX1; or LIN28 . Examples of iPSCs are described in [ 0176 ] A “ skeletal myoblast (SM )” is an immature cell Takahashi et al. ( 2007 ) Cell advance online publication 20 that can be isolated from between the basal lamina and Nov . 2007 : Takahashi & Yamanaka (2006 ) Cell 126 :663 - 76 ; sarcolemma . They account for 2 - 5 % of sub - laminar nuclei Okita et al. (2007 ) Nature 448 :260 -262 ; Yu et al. ( 2007 ) of mature skeletal muscle . Skeletal myoblasts are activated Science advance online publication 20 Nov. 2007 ; and in response to muscle damage or disease - induced muscle Nakagawa et al. ( 2007 ) Nat. Biotechnol. Advance online degeneration . Skeletal myoblasts express desmin , CD56 , publication 30 Nov. 2007. Pax3, Pax7 , c -met , myocyte nuclear factor, M - cadherin , [0165 ] “ Embryoid bodies or EBs” are three -dimensional VCAM1, N - CAM , CD34 , Leu - 19 , and syndecan 3 and 4 . ( 3D ) aggregates of embryonic stem cells formed during Activated skeletal myoblasts first express Myf- 5 and/ or culture that facilitate subsequent differentiation . When MyoD , and finally myogenin and MRF4 as the cells differ grown in suspension culture, EBs cells form small aggre entiate into multinucleated myotubes. gates of cells surrounded by an outer layer of visceral [0177 ] As used herein , the term “ small juvenile stem cells endoderm . Upon growth and differentiation , EBs develop ( SJSCs ) ” intends stem cells isolated from aged bone mar into cystic embryoid bodies with fluid - filled cavities and an row - derived stem cells (BMSCs ) with high proliferation and inner layer of ectoderm - like cells . differentiation potential. See Igura et al. ( 2013 ) 305 ( 8 ) : [ 0166 ] An “ induced pluripotent cell” intends embryonic H1354 -62 . SJSCs express mesenchymal stem cell markers , like cells reprogrammed to the immature phenotype from CD29 ( + )/ CD44 ( + ) /CD59 ( + ) /CD90 ( + ) , but are negative for adult cells . Various methods are known in the art, e . g. , “ A CD45 ( - ) /CD117 ( - ) as examined by flow cytometry analy simple newway to induce pluripotency : Acid .” Nature , 29 sis . SJSCs show higher proliferation , colony formation , and Jan . 2014 and available at sciencedaily . com / releases /2014 / differentiation abilities compared with BMSCs. They also 01/ 140129184445, last accessed on Feb . 5 , 2014 and U . S . are reported to significantly express cardiac lineage markers Patent Application Publication No. 2010 /0041054 . Human (Gata - 4 and myocyte -specific enhancer factor 2C ) and pluri iPSCs also express stem cell markers and are capable of potency markers (octamer - binding transcription factor 4 , generating cells characteristic of all three germ layers . sex - determining region Y box 2 , stage -specific embryonic [0167 ] . As used herein , the term “ a cardiac progenitor ” antigen 1 , and Nanog ) as well as antiaging factors such as intends a dynamic progenitor that is able to differentiate into telomerase reverse transcriptase and sirtuin 1. terminally derived cardiac cell types . Cardiac progenitor [0178 ] A “ marrow stromal cell ” also referred to as “ a bone cells (CPCs ) represent the earliest stages of mesodermal marrow stromal cell ” or a " mesenchymal stromal cell ” is a commitment to the cardiac lineage and show a classical CPC multipotent stem cell that can differentiate into a variety of marker pro le of KDR /PDGFR - apos/ CKITneg and are cell types. Cell types that MSCs have been shown to responsive to permissive conditions for proliferation as a differentiate into in vitro or in vivo include osteoblasts , progenitor population and /or differentiation into terminal chondrocytes , myocytes, and adipocytes . Mesenchyme is cardiac cell . embryonic connective tissue that is derived from the meso [0168 ] As used herein , the term “ a skeletal myogenic derm and that differentiates into hematopoietic and connec progenitor ” intends cells which are characterized by the tive tissue , whereas MSCs do not differentiate into expression of Pax3 and Pax7 and also give rise to the hematopoietic cells . Stromal cells are connective tissue cells satellite cells of postnatal muscle . that form the supportive structure in which the functional [0169 ] As used herein , a “ fibroblast ” intends a cell cells of the tissue reside . While this is an accurate descrip expressing the following markers Vimentin , CollA1, FSP - 1 . tion for one function of MSCs, the term fails to convey the [0170 ] As used herein , a " skeletal myoblast” intends a cell relatively recently -discovered roles of MSCs in repair of expressing the following markers MyOG , Desmin , m -cal tissue . Methods to isolate such cells, propagate and differ pain , human alpha - skeletal actin . entiate such cells are known in the technical and patent [0171 ] As used herein , a " pluripotent cell ” defines a less literature , e . g . , U . S . Patent Application Publication Nos . differentiated cell that can give rise to at least two distinct 2007 /0224171 , 2007 /0054399 , 2009 /0010895 , which are ( genotypically and /or phenotypically ) further differentiated incorporated by reference in their entireties . progeny cells. [0179 ] Adipose stem cells are also known as adipose [0172 ] A juvenile or young stem cells intends from 2 tissue - derived stem cells (ADSC ) that are routinely isolated months or younger mice possessing antiaging genes . In from the stromal vascular fraction (SVF ) of homogenized vitro , the cells average from about 3 -5 um in size and adipose tissue . Similar to other types ofmesenchymal stem express the embryonic stem cell marker , OCT4 and surface cells (MSC ), ADSC remain difficult to define due to the lack markers , CD29 , CD44 , and CD90 . of definitive cellular markers . Adipose -derived stem cells [0173 ] As used herein , a “ fibroblast ” intends a cell (ASCs ) are a mesenchymal stem cell source with self expressing the following markers Vimentin , CollA1, FSP - 1 . renewal property and multipotential differentiation . [0174 ] As used herein , a " skeletal myoblas? ” intends a cell 0180 ] Hematopoietic stem cells are defined as a stem cell expressing the following markers Myog , Desmin , m - cal that gives rise to all red and white blood cells and platelets . pain , human alpha -skeletal actin . They are commonly isolated by use of the markers CD34 + . [ 0175 ] As used herein , the term " pluripotent gene or In another aspect , the hematopoietic stem cell is an adult marker ” intends an expressed gene or protein that has been stem cell comprising the marker profile of: CD34 + and /or correlated with an immature or undifferentiated phenotype , CD34 + / Thy - 1 - HSC ) . See also Andrews, R . G . et al. ( 1990 ) e . g . , Oct 3 /4 , Sox2 , Nanog , c -Myc and LIN - 28 . Methods to J. Exp . Med . 172 ( 1 ) : 355 - 358 , incorporated herein by refer identify such are known in the art and systems to identify ence . US 2018 /0273906 A1 Sep . 27 , 2018 16

[0181 ] Mesenchymal stem cells , or MSCs, are defined as ring of the sulfanilamide derivatives may be replaced by an multipotent stromal cells that can differentiate into a variety alkyl and /or corresponding alkoxy alkyl radical to generate . of cell types , including : osteoblasts (bone cells ) , chondro Non -limiting examples of “ isoxazole - like compound ” or cytes (cartilage cells ), myocytes (muscle cells ) and adipo “ similar compound ” comprise 1 , 2 -oxazole , 4 - deuterio - 1 , 2 cytes ( fat cells ) . oxazole , 1 ,2 -oxazole ; potassium , hydron ; 1, 2 -oxazole , [0182 ] As used herein , the term chemically induced or 1 -oxido - 1, 2 -oxazol - 1- ium , 1, 2 -oxazole ; hydrobromide , 1, 2 oxazole ; hydrochloride, ethane ; 1 , 2 - oxazole , potassium ; 1 , 2 chemically modified pluripotent stem cell ( iPSC ) is intended oxazole ; hydroxide , 1, 2 -oxazole ; hydrate ; hydrochloride , to include an iPSC treated with a small molecule such as ethane ; 1 , 2 -oxazole , 1 , 2 -oxazole ; cyanate , 2 - oxido - 1 , 2 -ox isoxazole or a derivative thereof, or an isoxazole similar azol - 2 - ium , carbon monoxide ; chromium ; 1 , 2 - oxazole , eth molecule . ane ; 1 , 2 -oxazole , ethane ; 1 , 2 -oxazole ; propane , 1 , 2 - oxazol [018 ] “ Isoxazole ” is a class of compounds found in some 2 - ium - 2 -sulfonate , carbonyl dichloride ; 1 , 2 - oxazole , natural products , such as ibotenic acid , as well as a number isocyanic acid ; 1, 2 -oxazole , ethoxyethane ; 1, 2 -oxazole , 2, 2 of drugs , including a COX - 2 inhibitor, and furoxan , a nitric dimethylpropane ; ethane ; 1 , 2 - oxazole , ethane ; methoxy oxide donor. Isoxazoles are useful isosteres of pyridine, and have been found to inhibit voltage - gated sodium channels to ethane ; 1 , 2 -oxazole , ethane ; 2 -methylpropane ; 1, 2 -oxazole , control pain , enable the construction of tetracycline antibi 1 , 2 - oxazole ; urea , ethanol ; 1 , 2 - oxazole , carbonic acid ; 1 , 2 otic derivatives , and as treatments for depression . Com oxazole , 1 , 2 -oxazol - 1 - ium - 1 - sulfonic acid , 1 , 2 - oxazol- 2 pounds of this class available from Sigma- Aldrich and ium ; iodide . methods to synthesize such are known in the art as described [0184 ] “ Isoxazole 9 ” (ISX - 9 ) is a small molecule inducer for example in U . S . Pat. Nos . 5 ,059 , 614 and 8 , 318 , 951 and of adult neural stem cell differentiation both in vitro and in PCT Publication No. WO 1999 /002507 . Structurally , isox vivo ( Schneider et al. ) . It has been shown to act through a azole is a five membered heterocyclic compound containing calcium - activated signaling pathway dependent on myo oxygen and nitrogen atoms in the 1 , 2 positions. Its partially cyte - enhancer factor 2 (MEF2 ) -dependent gene expression saturated analogs are called isoxazolines and completely (Schneider et al. , Petrik et al .) . Compounds are also avail saturated analog is isoxazolidine . Examples of isoxazole able from Sigma- Aldrich and StemCell Technologies . The like compounds include derivatives, non - limiting examples molecular formula is CuH N2O S , and the chemical name of such include sulfamethoxazole , sulfisoxazole , oxacillin , is N -cyclopropyl - 5 - thiophen - 2 -yl - 1 , 2 - oxazole - 3 - carboxam cycloserine and acivicin . isoxazoles , isoxazolines and isox ide . The two dimensional structure is : azolidines may be considered as useful synthons in organic 0 - N synthesis . Isoxazoles may be efficiently transformed in to various classes of medicinally important molecules. For example , Anthracen - 9 - ylmethylene- ( 3 , 4 - dimethylisoxazol 5 - yl) aminemay be synthesized in high yield by reaction of anthracene - 9 - carbaldehyde and 5 -amino - 3 , 4 - dimethylisox azole in ethanol. In an embodiment, all the derivatives of [0185 ] " Isoxazole 1” ( ISX - 1 ) is a small molecule having isoxazole may be considered as “ isoxazole - like compound ” the structure : or " similar compound ” . In an embodiment, the isoxazole derivatives such as 5 - Amino - 3 -methyl - 4 - isoxazolecarbox ylic acid semicarbazides and thiosemicarbazides may be synthesized . The reaction of 5 - amino - 3 -methyl - 4 - isoxazole carboxylic acid hydrazide with isocyanates and isothiocya nates may be designed and conducted . The isocyanates , in the reaction of nucleophilic addition with compounds con [0186 ] As used herein , the term “ isoxazole - like com taining the primary amino group , form urea derivatives and pound ” or “ similar compound ” intends an agent or small isothiocyanates the thiourea derivatives. Only the hydrazide molecule that has the same functional property of the terminal group ( NH2) participates in this reaction . The isoxazole as disclosed herein . Non - limiting examples amino group in position 5 of isoxazole ring remains not include Cardionogen ; CDNG1/ vuc230 , CDNG2/ vuc198 , reactive under the reaction conditions. The mechanism of and CDNG3/ vuc247 ( see Terri et al . ( 2011 ) Chem Biol. , the reaction consists in nucleophilic attack of the nitrogen December 23 18 (12 ) : 1658 - 1668 ) . Non - limiting examples atom in the hydrazide group ( - NH2 ) on the carbon atom of further include sulfisoxazole as described herein below . Yet isocyanate or isothiocyanate . The intermediate forms appear a further example is leflunomide (Arava ) , also known as which undergo amidoiminole tautomerization leading to 5 -methyl - N -[ 4 -( trifluoromethyl ) phenyl] - 1 ,2 -oxazole -4 -car formation of substituted 5 - amino - 3 -methyl - 4 - isoxazolecar boxamide . boxylic acid semicarbazides and thiosemicarbazides. In an embodiment, examples of isoxazole derivatives may com [0187 ] An isoxazole compound or derivative thereof can prise 5 - sulfanilamido -isoxazoles of the general formula also be a compound of the formula : wherein R and R are lower alkyl and / or lower alkoxy alkyl R2 groups. Sulfanilamide derivatives with the isoxazole ring attached in N -position of the sulfanilamide molecule may be R3 OS generated . For example , sulfanilamide radical in 4 -position of the isoxazole ring . Further, a sulfanilyl derivative of N - RI 5 -amino - isoxazole namely , 5 - sulfanilamido - 3 -methyl - isox azole may also be considered as an isoxazole derivative . In an embodiment, both the 3 - and 4 -positions of the isoxazole US 2018 /0273906 A1 Sep . 27 , 2018 17 wherein R , and R2 are both hydrogen or R , is hydrogen and boxylate , methyl 5 - ( 4 - chlorophenyl) isoxazole - 4 - carboxy R2 is selected from the group consisting of substituted or late , methyl 5 - ( 4 -bromophenyl ) isoxazole - 4 - carboxylate , unsubstituted C . - C . alkyl, Cz- Co cycloalkyl, C2- C . alkenyl , 5 - ( 4 -methoxyphenyl ) isoxazole - 3 -carboxylic acid , 5 - ( 3 C2- C , alkynyl, and benzyl, or where R , and R2 may be methoxy -phenyl ) - isoxazole - 3 - carboxylic acid , 3 - ( 2 joined together to form a ring selected from azetidinyl, methoxyphenyl ) isoxazole - 5 - carboxylic acid , 5 - ( 4 -methoxy pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl; R ,' , phenyl) isoxazole -3 -carboxaldehyde , 3 -( 4 -methoxyphenyl ) R3 and R4 are independently selected from the group con isoxazole -5 -carbaldehyde , 3 -( 2 -methoxyphenyl ) isoxazole sisting of hydrogen , halogen , C . - C . alkyl, C3 -Co cycloalkyl, 5 -carbaldehyde , 5 -( 4 -methoxyphenyl ) isoxazole , 3 -( 4 substituted or unsubstituted aromatic or heteroaromatic ring , methoxyphenyl ) isoxazole , 3 - ( 2 -methoxy - phenyl) - 4 , 5 cyano , nitro , and acyl; and Y is 0 , NH or S . dihydro - isoxazole - 5 - carboxylic acid , 3 -methoxy - isoxazole [ 0188 ] In one aspect , an isoxazole compound has the 5 -carboxylic acid , isoxazole - 5 - carboxylic acid , isoxazole - 4 formula : carboxylic acid , isoxazole -5 -carbothioamide , isoxazole - 5 carbonyl chloride , isoxazole - 3 - carbonitrile , isoxazole - 3 carbaldehyde , isoxazole - 4 -boronic acid , isoxazole , 5 -cyclopropyl - 4 -[ 2 - (methylsulfonyl ) -4 - (trifluoromethyl ) benzoyl] isoxazole , 6 -( 5 -( thiophen -2 - yl) isoxazole - 3 -carbox amido )hexyl 5 -( ( Zas , 4s, bar) - 2 - oxohexahydro - 1h - thieno [ 3 , 4 -djimidazol - 4 - yl) pentanoate , isocarboxazid 5 -methyl - 3 R3 isoxazole - carboxylic acid 2 -benzylhydrazide , 5 - isobutyl Rs isoxazole - 3 -carboxylic acid , 4 - iodo -5 -methyl - isoxazole , 3 ,3 '- iminobis ( n ,n - dimethylpropylamine ), 3 -( 3 -hydroxy wherein R1 and R2 is each selected from C1 - C4 alkyl, phenyl ) - isoxazole - 5 - carboxylic acid methyl ester , 5 - ( 4 - hy phenyl, benzyl, trifluoromethyl or halogen , R3 is selected droxy - phenyl) - isoxazole - 3 - carboxylic acid , 5 - ( 3 -hydroxy from hydrogen , hydroxy , C1- C4 alkyl or alkoxy, R4 , in phenyl ) - isoxazole - 3 - carboxylic acid , 5 - hydroxymethyl) - 3 position 3 or 5 , is selected from hydrogen , trifluoromethyl, methylisoxazole , 3 -hydroxy - 5 -methylisoxazole , 5 - ( 1 C1 - C4 alkoxy, C1 - C4 alkyl, or C1 - C4 hydroxyalkyl, R5 is hydroxyethyl) - 3 - ( 4 - trifluoromethylphenyl) isoxazole , 3a , 4 , selected from hydrogen or C4 - C4 alkyl or R4 and R5 5 , 6 , 7 , 7a - hexahydro -benzo [ d ] isoxazole -3 - carboxylic acid , together form a tetramethylene group , Z at position 3 or 5 on 5 - ( 2 - furyl) isoxazole - 3 - carbaldehyde, 5 - furan - 2 - yl- isox the heterocycle is selected from : — N (R6 ) -CO , CO azole - 3 -carboxylic acid , 6 - fluoro - 3 - ( 4 - piperidinyl) ben N (R6 ) - , _ N ( R6 ) CO _ N ( R6 )- , CH ( RF )- NH - CO , zisoxazole , 5 -( 4 - fluorophenyl) isoxazole - 3 -methanol , 3 -( 2 or — NH - CO - CH (R6 ) , in which R6 is selected from fluoro -phenyl ) - isoxazole - 5 -carboxylic acid , 5 - ( 4 hydrogen or C1 - C4 alkyl. Non - limiting examples include fluorophenyl) isoxazole - 3 -carboxaldehyde , 3 - ( 4 5 - ( trifluoromethyl) - 3 - ( 4 -methoxyphenyl ) isoxazole - 4 -car fluorophenyl) isoxazole - 5 - carbaldehyde, 3 - ( 3 - fluorophenyl) boxylic acid , 5 - ( trifluoromethyl ) - 3 - ( 4 - fluorophenyl) isox isoxazole - 5 -carbaldehyde , 3 - ( 2 - fluorophenyl) isoxazole - 5 azole - 4 -carboxylic acid , 5 - ( thiophen - 2 - yl) isoxazole - 3 - car carbaldehyde , 5 - (4 - fluorophenyl) isoxazole , 3 -( 4 boxaldehyde, 5 ,6 , 7 , 8 - tetrahydro - 4h - cyclohepta [ d ] fluorophenyl) isoxazole , 5 - ( 3 - fluoro - 4 -methoxy -phenyl ) isoxazole - 3 - carboxylic acid , 4 , 5 , 6 , 7 - tetrahydro -benzo [ d ] isoxazole- 3 -carboxylic acid , ethyl 5 -( trifluoromethyl ) - 3 - ( 4 isoxazole - 3 -carboxylic acid , 3 - amino - 5 -methylisoxazole , methoxyphenyl ) isoxazole - 4 - carboxylate , ethyl - 5 4 - amino - n - ( 5 -methyl - 3 - isoxazolyl )benzenesulfonamide , (tributylstannyl ) isoxazole- 3 - carboxylate , ethyl 5 - ( thiophen 3 -phenyl - isoxazole - 5 -boronic acid pinacol ester , 5 - phe 2 -yl ) isoxazole - 3 -carboxylate , 5 -ethyl - isoxazole - 4 nylisoxazole , l - phenyl- 1 - cyclopentanecarboxylic acid , carboxylic acid , 5 - ethyl- isoxazole - 3 -carboxylic acid , ethyl 3 -phenyl -benzo [c ] isoxazole - 5 -carboxylic acid , 5 -methyl - 3 5 -( 4 - fluorophenyl) isoxazole -4 -carboxylate , ethyl 5 - (4 - fluo phenylisoxazole - 4 - carboxylic acid , 3a , 4 , 5 ,6 , 7 , 8 , 9 , 9a - octa rophenyl) isoxazole - 3 - carboxylate , ethyl 5 - ( 2 , 3 - dihyd hydro - cycloocta [ d ] isoxazole - 3 -carboxylic acid , 5 - ( 3 -nitrop robenzo [ b ] [ 1 , 4 ] dioxin - 7 -yl ) isoxazole - 3 - carboxylate , ethyl henyl) isoxazole , 3 - ( 4 -nitrophenyl ) isoxazole, 3 -hydroxy - 5 3 -( 4 -chlorophenyl ) - 5 -( trifluoromethyl ) isoxazole - 4 -car aminomethyl- isoxazole , 5 - morpholinomethyl) isoxazole - 3 boxylate , ethyl 5 - ( 4 -chlorophenyl ) isoxazole - 3 -carboxylate , carboxylic acid hydrochloride , 5 -( morpholinomethyl ) ethyl 3 - ( 4 -bromophenyl ) - 5 - ( trifluoromethyl) isoxazole - 4 isoxazole - 3 - carbaldehyde, 3 -methyl - 5 - ( trifluoromethyl ) carboxylate , ethyl 5 - ( 4 - bromophenyl) isoxazole - 3 -carboxy isoxazole- 4 -carboxylic acid , methyl 5 - ( thiophen - 2 -yl ) late , ethyl 5 - amino - 4 - ( 4 - chlorophenyl) isoxazole - 3 - carboxy isoxazole - 3 -carboxylate , 3 - (methylsulfonyl ) - 5 - ( 2 - thienyl ) late , ethyl 5 -amino -4 - (4 -bromophenyl ) isoxazole - 3 isoxazole - 4 - carbonitrile , 5 -methyl - 3 - ( 2 -pyrrolidinyl ) carboxylate , ethyl 6b -acetyl - 2 -( acetyloxy ) -4a ,6a - dimethyl isoxazole , 3 -methyl - 5 - ( 2 - pyrrolidinyl ) isoxazole , 3 - ( 1 2 , 3 , 4 ,4a , 4b , 5 ,6 , 6a ,6b , 9a , 10 , 10a , 10b , 11 - tetradecahydro - 1h methyl- 1h - pyrazol - 4 - yl) - isoxazole - 5 - carboxylic acid , 3 - ( 1 naphtho[ 2 ', 1 ": 4 , 5 ] indeno [ 2 , 1 - d ] isoxazole - 9 - carboxylate , methyl- 1h -pyrazol - 4 - yl) - 4 , 5 - dihydro - isoxazole - 5 3 ,5 -dimethyl - 4 -( tributylstannyl ) isoxazole , 5 -( 1 ,5 - dimethyl carboxylic acid , 5 - ( 4 -methylphenyl ) isoxazole - 3 - carboxylic 1h - pyrazol- 4 - yl) - isoxazole - 3 -carboxylic acid , 5 - ( 1 , 3 -dim acid , 5 -methyl - 3 - phenylisoxazole - 4 - carboxylic acid , 5 - ( 4 ethyl- 1h -pyrazol - 4 - yl) - isoxazole - 3 - carboxylic acid , 5 - (1 , 5 methylphenyl) isoxazole -3 -carboxaldehyde , 5 -methyl - 3 -( 4 dimethyl - 1h -pyrazol - 4 -yl ) - isoxazole , 3 , 5 phenoxyphenyl) isoxazole - 4 - carboxylic acid , 3 -methyl - 5 - ( 4 dimethylisoxazole - 4 -boronic acid pinacol ester, 3, 5 methyl- 1 ,2 , 3 - thiadiazol- 5 - yl) isoxazole - 4 - carboxylic acid , dimethylisoxazole , 3 -( dimethylamino )- 1- ( 2 -pyridyl ) - 2 3 -methyl - 5 -( 5 -methylisoxazol - 3 -yl ) isoxazole -4 -carboxylic propen - 1 - one , 5 - ( 3 , 5 - difluorophenyl) isoxazole, [ 2 ,6 acid , methyl 5 - ( 4 -methoxyphenyl ) isoxazole -4 -carboxylate , dichloro - 4 - ( trifluoromethyl ) phenyl] hydrazine , 5 - ( 2 , 5 methyl 5 -( 4 -methoxyphenyl ) isoxazole -3 -carboxylate , dichlorophenyl) isoxazole - 3 - carboxylic acid , danazol , 3 - ( 4 5 -methylisoxazole , methyl 5 - ( 4 - fluorophenyl) isoxazole - 4 chlorophenyl) - 5 - ( trifluoromethyl )isoxazole - 4 - carboxylic carboxylate , methyl 5 -( 4 - fluorophenyl) isoxazole - 3 -car acid , 5 - ( 4 - chlorophenyl) isoxazole - 3 - propionic acid , 5 - (4 US 2018 /0273906 A1 Sep . 27 , 2018 chlorophenyl) isoxazole - 4 - carboxylic acid , 5 - ( 4 - chlorophe pyrrolo ( 3 , 4 - d ) isoxazole- 4 ,6 (3h , 5h ) dione, 5 - ( p - tolyl) isox nyl) isoxazole - 3 -carboxylic acid , 3 - ( 4 - chlorophenyl) isox azole , 5 -( 4 -methylphenyl ) - 3 - ( 4 -nitrophenyl )- 2 -phenyldi azole - 5 - carboxylic acid , 3 - ( 3 - chlorophenyl) isoxazole - 5 hydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , 5h )- dione, 5 - ( 4 carboxylic acid , 5 - ( 4 - chlorophenyl) isoxazole - 3 methoxyphenyl ) - 2 -phenyl - 3 - ( 4 -pyridinyl ) dihydro - 2h carboxaldehyde , 3 -( 4 - chlorophenyl) isoxazole - 5 pyrrolo [ 3 ,4 - d ] isoxazole - 4 ,6 (3h ,5h )- dione , 5 - (4 carbaldehyde , 3 -( 3 - chlorophenyl) isoxazole -5 -carbaldehyde , methoxyphenyl ) - 2 - phenyl - 3 - ( 3 - pyridinyl) dihydro - 2h 3 -( 2 -chlorophenyl ) isoxazole -5 -carbaldehyde , 5 - (4 - chloro pyrrolo [ 3, 4 -d ]isoxazole -4 ,6 ( 3h ,5h )- dione , 5 -( 4 -methoxy phenyl) isoxazole , 3 - ( 4 - chlorophenyl ) isoxazole , 5 - ( chlorom ph ) - 2 , 3 - diphenyldihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 ethyl) isoxazole - 4 - carboxylic acid , 3 - (chloromethyl ) - 5 - ( 2 ( 3h , 5h )- dione, 5 - ( 4 - fluorophenyl) - 3 - ( 4 -methoxyphenyl ) - 2 furyl ) isoxazole , 4 - chloromethyl - 3 , 5 - dimethylisoxazole, phenyldihydro - 2h -pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h ,5h ) 5 - ( chloromethyl) - 3 - ( 4 - chlorophenyl) isoxazole , 5 - ( 3 - chloro dione , 5 - (4 - fluorophenyl) - 2 -( 2 -methylphenyl ) - 3 - ( 4 4 -methoxy - phenyl) - isoxazole - 3 - carboxylic acid , 3 - chloro pyridinyl) dihydro - 2h -pyrrolo [3 ,4 -d ] isoxazole- 4 ,6 (3h ,5h ) 4 - fluorobenzaldehyde , 3 - ( 5 - chloro - 2 ,4 - dimethoxy - phenyl) dione , 5 - (4 - ethoxyphenyl) - 3 - ( 4 -nitrophenyl ) - 2 4 , 5 -dihydro - isoxazole - 5 -carboxylic acid , 5 - tert -butyl - 4 , 5 , 6 , phenyldihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) 7 - tetrahydro -benzo [ d ]isoxazole -3 -carboxylic acid , 3 -( 4 dione , 5 - ( 4 - ethoxyphenyl) - 3 - ( 4 - fluorophenyl) - 2 bromophenyl ) - 5 - ( trifluoromethyl ) isoxazole - 4 - carboxylic phenyldihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 (3h ,5h ) acid , 5 - ( 4 - bromophenyl) isoxazole - 3 - propionic acid , 5 - ( 4 dione , 5 -( 4 - ethoxyphenyl) - 2 -methyl - 3 - (4 -nitrophenyl ) bromophenyl) isoxazole - 3 - carboxylic acid hydrazide, 5 - ( 4 dihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) -dione , 5 - ( 4 bromophenyl) isoxazole - 4 - carboxylic acid , 5 - ( 4 -bromophe ethoxy - ph ) - 2 - ph - 3 - thiophen - 2 -yl - 4h - pyrrolo ( 3 , 4 - d ) nyl) isoxazole - 3 - carboxylic acid , 3 - ( 4 - bromophenyl ) isoxazole -4 ,6 -dione , 5 -( 4 -cl - ph )- 3 -( 3 -nitro -ph )- 2 -phenyl isoxazole - 5 -carboxylic acid , 3 - ( 4 - bromophenyl) isoxazole tetrahydro - pyrrolo ( 3 , 4 - d )isoxazole - 4 , 6 - dione , 5 - ( 4 5 -carboxaldehyde , 5 -( 4 -bromophenyl ) isoxazole , 5 -( 3 bromophenyl) - 3 - ( 4 -methoxyphenyl ) - 2 -phenyldihydro - 2h bromophenyl) isoxazole , 3 - ( 4 - bromophenyl) isoxazole , pyrrolo [ 3 , 4 - d ]isoxazole - 4 ,6 ( 3h , 5h ) - dione, 5 - (4 5 -( bromomethyl ) -3 - (4 -methoxyphenyl ) isoxazole , 4 -( bro bromophenyl) -3 - ( 2 - furyl) - 2 -( 2 -methylphenyl ) dihydro -2h momethyl) isoxazole , 5 - bromomethyl) - 3 - ( 4 -fluorophenyl ) pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 (3h ,5h ) -dione , 5 - (4 isoxazole , 5 - (bromomethyl ) - 3 - ( 4 - chlorophenyl ) isoxazole, bromophenyl) - 2 -phenyl - 3 - ( 2 - pyridinyl) dihydro - 2h -pyrrolo 5 - ( bromomethyl) - 3 - ( 4 -bromophenyl ) isoxazole , 6 -bromo - 3 [ 3 , 4 - d ] isoxazole- 4 , 6 ( 3h ,5h ) - dione , 5 - ( 4 -br - ph ) 2 -ph - 3 - ( 2 -ph methylbenzo [ d ] isoxazole , 5 -bromo - 3 -methylbenzo [ d ] isox vinyl) dihydro - 2h - pyrrolo ( 3 , 4 - d ) isoxazole- 4 , 6 ( 3h , 5h )dione , azole , 4 - bromo- 5 - ( 4 -methoxyphenyl ) isoxazole , 3 -bromo 5 - ( 2 - cl- ph ) - 3 - ( 4 - dimethylamino - ph ) 2 -0 - tolyl- 4h -pyrrolo ( 3 , isoxazole , 3 -bromo -5 - ( 2 -hydroxyethyl ) isoxazole , 4 -bromo 4 - d ) isoxazole - 4 , 6 -dione , 5 - ( 2 - chlorophenyl) - 3 - [ 4 - ( dimeth 5 - ( 4 - fluorophenyl) isoxazole , 3 -bromo - 5 - ( 4 - fluorophenyl) ylamino )phenyl ] - 2 - phenyldihydro - 2h - pyrrolo [ 3 , 4 - d ] isox isoxazole, 4 -bromo -5 - (4 -chlorophenyl ) isoxazole , 4 -bromo azole - 4 ,6 ( 3h , 5h ) - dione, 5 - ( 2 - chlorophenyl) - 3 - ( 4 5 -( 4 -bromophenyl ) isoxazole , 6 -bromo -benzo [ d ] isoxazole methoxyphenyl) - 2 - phenyldihydro -2h -pyrrolo [3 ,4 - d ] 3 - carboxylic acid , benzo [ d ] isoxazole- 3 - carboxylic acid , isoxazole - 4 ,6 (3h ,5h )- dione , 4 - (( 3 -( 2 - cl -ph )- 5 -methyl 3 -amino - 5 -methylisoxazole , 5 -amino -3 -( 4 -methoxyphenyl ) isoxazole - 4 -carbonyl ) - amino ) -benzoic acid ethyl ester, 4 , 5 , isoxazole , 3 - aminoisoxazole , 3 - amino - 5 - ( 4 - fluorophenyl) 6 ,6a - tetrahydro -3ah - cyclopenta [ d ]isoxazole - 3 - carboxylic isoxazole , 5 -amino -3 - (4 -chlorophenyl ) isoxazole , 5 - amino acid , 3 -phenyl - 3a , 6a - dihydrothieno [2 , 3 -d ] isoxazole 4 ,4 -di 4 - ( 4 - bromophenyl) isoxazole, 3 - amino - 5 - ( 4 - bromophenyl) oxide , 3 -methyl - 5 - ( 3 - phenylpropyl) isoxazole , 3 -methyl - 4 isoxazole , 5 - acetyl- 3 - (4 - fluorophenyl) isoxazole , 5 - acetyl- 3 nitro - 5 -[ ( e )- 2 -phenylethenyl ] isoxazole , 3 -methyl - 4 ,5 ,8 , 9 ( 3 - fluorophenyl) isoxazole , 3 -methyl - 5 - [( 2S ) - 1- methyl - 2 tetrahydrocycloocta ( d ) isoxazole , 3 -methyl - 4 , 5 , 5a ,6a , 7 , 8 pyrrolidinyl] isoxazole hydrochloride , 7 -methoxy - 5 -methyl hexahydrooxireno ( 2 ', 3 : 5 ,6 ) cycloocta ( 1 , 2 - d ) isoxazole , 4 ,5 -dihydronaphtho [2 , 1- d ] isoxazole , 5 -methyl - 3 -phenyl 3 -methyl - 3a , 4 , 5 , 8 , 9 , 9a -hexahydrocycloocta ( d ) isoxazole , isoxazole - 4 -carboxylic acid methylamide , 5 -methyl - 3 3 - furan - 2 - yl- 2 -phenyl - 5 - p - tolyl -tetrahydro -pyrrolo ( 3 , 4 - d ) phenylisoxazole - 4 - carbothioic acid methylamide, isoxazole - 4 , 6 - dione , 3 - chloro - 4 , 5 - dihydro ( 1 ) - benzothiepino 5 - methyl- 3 - phenyl- 4 - ( 1h -pyrazol - 5 - yl) isoxazole , 5 -benzyl ( 5 , 4 - c ) isoxazole , 3 - [ 4 - ( dimethylamino ) phenyl] - 5 - ( 4 3 - furan - 2 - yl- 2 - phenyl- tetrahydro -pyrrolo ( 3 , 4 - d ) isoxazole methoxyphenyl) - 2 - ( 2 -methylphenyl ) dihydro - 2h -pyrrolo [ 3 , 4 ,6 -dione , 5 -benzyl - 3 - [4 - (dimethylamino )phenyl ] - 2 -phe 4 -d ] isoxazole -4 ,6 (3h ,5h ) -dione , 3 - (5 -br - 2 -ho -phenyl ) -2 , 5 nyldihydro - 2h - pyrrolo [ 3 , 4 - d ]isoxazole - 4 , 6 ( 3h , 5h )- dione , diphenyl- tetrahydro -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 - dione , 5 -benzyl - 3 - (5 -br - 2 -ho -phenyl ) -2 -ph -tetrahydro -pyrrolo (3 , 4 3 - ( 5 - br- 2 -ho - ph ) - 5 - ( 2 - cl - ph )- 2 - ph - tetrahydro -pyrrolo ( 3 , 4 - d ) d ) isoxazole - 4 , 6 -dione , 5 -benzyl - 3 - ( 4 -nitro -ph ) - 2 -ph -di isoxazole- 4 ,6 -dione , 3 -( 4 -meo -phenyl ) - 5 -phenyl -2 - 0 -tolyl hydro -2h -pyrrolo (3 , 4 - d )isoxazole - 4 ,6 (3h , 5h )- dione, 5 -ben tetrahydro -pyrrolo (3 ,4 -d ) isoxazole -4 ,6 -dione , 3 - (4 - fluoro zyl- 3 - ( 4 -methoxy - phenyl) - 2 -ph - tetrahydro - pyrrolo ( 3 , 4 - d ) phenyl) - 5 - ( 4 - nitrophenyl) - 2 -phenyldihydro - 2h -pyrrolo [ 3 , 4 isoxazole - 4 , 6 -dione , 5 -benzyl - 3 - ( 4 - fluorophenyl) - 2 - 2 d ]isoxazole -4 , 6 (3h ,5h ) -dione , 3 - ( 4 -fluorophenyl ) - 5 - ( 4 methylphenyl ) dihydro - 2h -pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h , methylphenyl ) - 2 - phenyldihydro - 2h -pyrrolo [ 3 , 4 - d ] 5h ) - dione , 5 -benzyl - 3 - ( 3 -nitro -ph ) - 2 -phenyl - tetrahydro isoxazole - 4 , 6 ( 3h , 5h ) - dione, 3 - ( 4 -dimethylamino -ph ) - 5 -ph pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 -dione , 5 -benzyl - 2 -ph - 3 - ( 2 2 -o - tolyl -4h -pyrrolo ( 3, 4 - d )isoxazole -4 ,6 -dione , 3 - ( 4 pyridinyl) dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 ( 3h , 5h ) fluorophenyl) - 5 -( 4 -methoxyphenyl ) - 2 -phenyldihydro - 2h dione, 5 - benzyl- 2 -ph - 3 - ( 2 - ph - vinyl) dihydro - 2h -pyrrolo ( 3 , pyrrolo [ 3 , 4 - d ]isoxazole - 4 ,6 ( 3h , 5h ) - dione, 3 - ( 4 - br- ph ) - 2 - ph 4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) - dione , 5 -benzyl - 2 - ( 4 5 -( 2 - trifluoromethyl- ph ) -4h -pyrrolo ( 3 , 4 -d ) isoxazole - 4 ,6 chlorophenyl) - 3 - (2 -thienyl ) dihydro - 2h -pyrrolo [3 ,4 -d ] dione , 3 - (3 -nitro -phenyl ) - 2, 5 -diphenyl - tetrahydro -pyrrolo isoxazole -4 ,6 (3h ,5h )- dione , 5 -benzyl - 2 ,3 -diphenyldihydro ( 3 ,4 -d ) isoxazole - 4 ,6 -dione , 3 - (3 -br - phenyl ) -2 , 5 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) - dione , 5 -benzyl - 2 ( 4 diphenyldihydro -2h -pyrrolo ( 3 ,4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) cl- ph ) 3 - ( 2 - furyl) dihydro - 2h - pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 dione , 3 - ( 3 - br- ph )- 5 - ( 2 -meo -ph ) - 2 - 0 - tolyl- tetrahydro (3h ,5h )dione , 5 -benzyl - 2 (4 -cl - ph )- 3 -( 4 -f - ph )dihydro - 2h pyrrolo ( 3 , 4 - d ) isoxazole- 4 ,6 - dione , 3 - ( 2 - furyl ) - 5 - [ 4 - (4 US 2018 /0273906 A1 Sep . 27 , 2018 morpholinyl) phenyl] - 2 - phenyldihydro - 2h - pyrrolo [ 3 , 4 - d ] apoptotic pathway, inducing apoptosis in hepatoma cells and isoxazole - 4 ,6 ( 3h , 5h ) - dione, 3 - ( 2 -furyl ) - 2 - ( 2 - me- ph ) - 5 - ph leukemic cells . This agent may also exhibit anti - angiogenic dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 ( 3h , 5h ) - dione, 3 - ( 2 activity , inhibiting the production of angiogenic factors such furyl) -2 , 5 - diphenyldihydro -2h -pyrrolo ( 3 ,4 - d ) isoxazole - 4 ,6 as IL -6 and vascular endothelial cell growth factor (VEGF ) (3h ,5h )- dione , 3 -( 2 -cl - phenyl ) -5 -methyl -isoxazole - 4 by bone marrow stromal cells . carboxylic acid ( 2 , 5 -dichloro -phenyl ) - amide , 3 - ( 2 - cl - ph ) - 5 10190 Rho - associated kinase (ROCK ) inhibitors intend me- isoxazole - 4 -carboxylic acid ( 4 , 5 - dihydro - thiazol- 2 -yl ) Rho - associated protein kinase (ROCK ) is a kinase belonging anamide , 3 - ( 2 - chloro -phenyl ) - 5 -methyl - isoxazole - 4 to the AGC (PKA / PKG /PKC ) family of serine - threonine carboxylic acid cyanomethyl- amide , 3 -( 2 ,4 kinases . It is involved mainly in regulating the shape and dichlorophenyl) - 5 -( 4 -methoxyphenyl ) - 2 -phenyldihydro -2h movement of cells by acting on the cytoskeleton . Non pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h ,5h ) - dione, 3 - ( 2 , 4 -di - cl- ph ) 2 , 5 -diphenyldihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 ( 3h , limiting examples of such include Thiazovivin or Y27632 , 5h ) -dione , 3 - (2 ,2 -dichloro -vinyl ) - 5 -phenyl - isoxazole , 3, 5 which both can be purchased from Stemcell Technologies diphenyl - isoxazole , 3 , 5 -dimethyl - 4 - ( 1 -pyrrolidinylsulfonyl ) and respectively ; SR3677 , which can be purchased from isoxazole , 3 ( 4 -dimethylamino -ph ) - 5 - ( 4 - eto -ph ) 2 - o - tolyl- 4h tocris. com ; and GSK429286 , which can be purchased from pyrrolo (3 ,4 - d ) isoxazole - 4 ,6 -dione , 2 -( 4 -cl -ph ) 5 -ph - 3 -( 2 tocris . com . thienyl )dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) [0191 ] A TGF- beta type - l receptor inhibitor intends ( ac dione, 2 - ( 4 -cl - ph ) - 5 - ( 3 -meo -ph ) - 3 - ( 3 -nitro - ph ) - 4h -pyrrolo tivin A receptor type II- like kinase , 53 kDa ) is an inhibitor ( 3 ,4 -d ) isoxazole- 4, 6 -dione , 2 -( 4 - cl -ph )- 3 -( 4 -meo - ph ) - 5 -p for membrane -bound receptor protein for the TGF beta tolyl - tetrahydro - pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 - dione , 2 - ( 4 superfamily of signaling ligands . TGFBR1 is its human chlorophenyl) -5 - (4 -methylphenyl )- 3 -( 2 - thienyl) dihydro - 2h gene . Non - limiting examples of such include SB431542 and pyrrolo [ 3 , 4 - d ]isoxazole - 4 , 6 ( 3h ,5h ) -dione , 2 -( 4 A8301 that can be purchased from www . tocris . com _ and chlorophenyl) -3 - [4 - (dimethylamino )phenyl ] - 5 _ www .esibio .com _ , respectively ; LY2157299 , which can be phenyldihydro -2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h , 5h ) purchased from www .selleckchem . com ; and LY2109761, dione, 2 -( 4 -chlorophenyl ) - 3 -( 4 - fluorophenyl) - 5 -( 4 which can be purchased from www .selleckchem .com . nitrophenyl )dihydro - 2h -pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , [0192 ] A DNA methyltransferase inhibitor is a small mol 5h ) -dione , 2 - ( 4 - chlorophenyl) - 3 - ( 2 - thienyl) - 5 - [ 3 ecule or other agent the ability to inhibit hypermethylation , ( trifluoromethyl) phenyl ] dihydro -2h -pyrrolo [ 3 ,4 - d ] restore suppressor gene expression and exert antitumor isoxazole - 4 , 6 ( 3h , 5h ) - dione , 2 - ( 4 - chlorophenyl) - 3 - ( 2 , 4 effects in in vitro and in vivo laboratory models . Goffin and dichlorophenyl) - 5 -phenyldihydro - 2h -pyrrolo [ 3, 4 - d ] Eisenhauer (2002 ) Ann . Oncol. November 13 ( 11 ) : 1699 isoxazole - 4 , 6 (3h ,5h ) -dione , 2 , 3 -di - ph - 5 - ( 3 - ( tri- f -me ) ph ) 16716 . One non - limiting example of such an inhibitor is dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) - dione, N - phthalyl- L - tryptopha (C19H14N204 , sold under the danazol, and n - cyclopropyl- 5 - (thiophen - 2 - yl) isoxazole - 3 tradename RG108 , Sigma- Aldrich ) . Additional examples carboxamide . In a further aspect, the isoxazole compound is include 5 ' -azacytidine , 5 -azacytidine , antisense oligonucle danazol or n -cyclopropyl - 5 - ( thiophen - 2 -yl ) isoxazole -3 - car otides to methyltransferase 1 , e .g ., MG98 ( see Amato (2007 ) boxamide . Additional non - limiting examples include com Clin . Gentourin Cancer, December , 5 ( 7 ) :422 -426 and 1 - (ß pounds of the isoxazole compound has the general formula D -Ribofuranosyl ) - 2 ( 1H ) -pyrimidinone ( a nucleoside analog ( I ) : of cytidine, sold under the name Zebularine ( Abcam® ) . [0193 ] DNA hypomethylation intends a lower than normal level of DNA methylation . A B [0194 ] Methods of determining the level of DNA meth ylation are known in the art , some of which are described herein . [0195 ] “ Sulfisoxazole ” is a sulfonamide antibacterial with an oxazole substituent. It has antibiotic activity against a wide range of Gram - negative and Gram -positive organisms. wherein A is a heterocyclic group optionally substituted with Compounds of this class available from Sigma- Aldrich and G or phenyl optionally substituted with G , wherein G is methods to synthesize such are known in the art as described halogen , C1 - C6 alkyl, or optionally substituted phenyl; B is for example in U . S . Pat . No . 2 ,721 , 200 . Non - limiting phenyl substituted with halogen , or a heterocyclic group examples of sulfisoxazole include FDA approved drugs of substituted with halogen , C1 - C6 alkyl; and R is hydrogen , or AZO GANTRISIN , ERYTHROMYCIN ETHYLSUCCI C1- C6 alkyl. NATE and SULFISOXAZOLE ACETYL , ERYZOLE , [0189 ] Givinostate (GIV ) is an orally bioavailable GANTRISIN ( with effective ingredients as SULFISOX hydroxymate inhibitor of histone deacetylase (HDAC ) with AZOLE ) , GANTRISIN (with effective ingredients as potential anti - inflammatory , anti- angiogenic , and antine SULFISOXAZOLE ACETYL ) , GANTRISIN (with effec oplastic activities . Givinostat inhibits class I and class II tive ingredients as SULFISOXAZOLE ACETYL ) , GAN HDACs, resulting in an accumulation of highly acetylated TRISIN PEDIATRIC , ILOSONE SULFA , LIPO GANTRI histones, followed by the induction of chromatin remodeling SIN , PEDIAZOLE , SOSOL , SOXAZOLE , SOXAZOLE , and an altered pattern of gene expression . At low , nonapop SULFISOXAZOLE , SULFISOXAZOLE DIOLAMINE , totic concentrations, this agent inhibits the production of and SULSOXIN . (Drugs @ FDA : FDA Approved Drug Prod pro - inflammatory cytokines such as tumor necrosis factor ucts at the website of accessdata . fda .org ) . ( TNF - ) , interleukin - 1 ( IL - 1 ) , IL - 6 and interferon - gamma. [0196 ] Danazol ( also known as 17a -Ethynyl - 17ß -hy Givinostat has also been shown to activate the intrinsic droxyandrost - 4 - en - [2 , 3 - d ] isoxazole ) is a a synthetic steroid US 2018 /0273906 A1 Sep . 27, 2018 20 that is used primarily in the treatment of endometriosis . The conversion into other materials or breakdown and elimina compound is commercially available and manufactured by a tion through natural pathways . variety of vendors. [0200 ] A population of cells intends a collection of more [0197 ] The term “ isolated as used herein refers to mol than one cell , exosome or microvesicle, or miRNA that is ecules or biological or cellular materials being substantially identical ( clonal) or non - identical in phenotype and /or geno free from other materials, e . g ., greater than 70 % , or 80 % , or type. The population can be purified , highly purified , sub 85 % , or 90 % , or 95 % , or 98 % . In one aspect , the term stantially homogenous or heterogeneous as described herein . “ isolated ” refers to nucleic acid , such as DNA , RNA , [0201 ] The term “ propagate ” means to grow or alter the miRNA , exosome or microvesicle , protein or polypeptide , phenotype of a cell or population of cells . The term “ grow or cell or cellular organelle , or tissue or organ , separated ing ” refers to the proliferation of cells in the presence of from other DNA , RNA , miRNA , exosome or microvesicle , supporting media , nutrients , growth factors, support cells , or protein or polypeptide, or cell or cellular organelle , or tissue any chemical or biological compound necessary for obtain or organ , respectively , that are present in the natural source ing the desired number of cells or cell type . In one embodi and which allow the manipulation of the material to achieve ment, the growing of cells results in the regeneration of results not achievable where present in its native or natural tissue. In yet another embodiment, the tissue is comprised of state , e . g . , recombinant replication or manipulation by muta cardiac progenitor cells or cardiac cells . tion . The term “ isolated ” also refers to a nucleic acid or 10202 ] The term “ effective amount” refers to a concentra peptide that is substantially free of cellular material, viral tion or amount of a reagent or composition , such as a material, or culture medium when produced by recombinant composition as described herein , cell population or other DNA techniques, or chemical precursors or other chemicals agent, that is effective for producing an intended result , when chemically synthesized . Moreover, an “ isolated including cell growth and / or differentiation in vitro or in nucleic acid ” is meant to include nucleic acid fragments that vivo , or for the treatment of a condition as described herein . are not naturally occurring as fragments and would not be It will be appreciated that the number of cells to be admin found in the natural state . The term “ isolated ” is also used istered will vary depending on the specifics of the disorder herein to refer to polypeptides which are isolated from other to be treated , including but not limited to size or total cellular proteins and is meant to encompass both purified volume/ surface area to be treated , as well as proximity of the and recombinant polypeptides , e . g . , with a purity greater site of administration to the location of the region to be than 70 % , or 80 % , or 85 % , or 90 % , or 95 % , or 98 % . The treated , among other factors familiar to the medicinalbiolo term “ isolated ” is also used herein to refer to cells , exosomes gist. or microvesicles , miRNA , or tissues that are isolated from 10203 ] The terms effective period ( or time) and effective other cells , exosomes or microvesicles , miRNA , or tissues conditions refer to a period of time or other controllable and is meant to encompass both cultured and engineered conditions (e . g. , temperature , humidity for in vitro meth cells or tissues and products produced or isolated from such . ods) , necessary or preferred for an agent or composition to achieve its intended result , e . g ., the differentiation or dedi [0198 ] The term “ phenotype ” refers to a description of an fferentiation of cells to a pre - determined cell type . individual 's trait or characteristic that is measurable and that [0204 ] A “ subject, " “ individual” or “ patient" is used inter is expressed only in a subset of individuals within a popu changeably herein , and refers to a vertebrate , preferably a lation . In one aspect of the invention , an individual' s phe mammal, more preferably a human . Mammals include , but notype includes the phenotype of a single cell, a substan are not limited to , murines , rats , simians , bovines , canines , tially homogeneous population of cells , a population of felines, humans , farm animals , sport animals and pets . differentiated cells , or a tissue comprised of a population of [0205 ) “ Substantially homogeneous” describes a popula cells . tion of cells in which more than about 50 % , or alternatively [0199 ] The term pharmaceutically acceptable carrier ( or more than about 60 % , or alternatively more than 70 % , or medium ) , which may be used interchangeably with the term alternatively more than 75 % , or alternatively more than biologically compatible carrier or medium , refers to 80 % , or alternatively more than 85 % , or alternatively more reagents , cells , compounds, materials , compositions, and /or than 90 % , or alternatively, more than 95 % , of the cells are dosage forms that are not only compatible with the cells and of the same or similar phenotype. Phenotype can be deter other agents to be administered therapeutically , but also are , mined by a pre - selected cell surface marker or other marker. within the scope of sound medical judgment, suitable for use 0206 ] As used herein , the terms “ treating , " " treatment” in contact with the tissues of human beings and animals and the like are used herein to mean obtaining a desired without excessive toxicity , irritation , allergic response, or pharmacologic and /or physiologic effect . The effect can be other complication commensurate with a reasonable benefit / prophylactic in terms of completely or partially preventing risk ratio . Pharmaceutically acceptable carriers suitable for a disorder or sign or symptom thereof, and / or can be use in the present invention include liquids, semi- solid ( e . g ., therapeutic in terms of a partial or complete cure for a gels ) and solid materials ( e . g ., cell scaffolds and matrices , disorder and /or adverse effect attributable to the disorder. tubes sheets and other such materials as known in the art and Examples of " treatment” include but are not limited to : described in greater detail herein ) . These semi- solid and preventing a disorder from occurring in a subject that may solid materials may be designed to resist degradation within be predisposed to a disorder, but has not yet been diagnosed the body (non -biodegradable ) or they may be designed to as having it ; inhibiting a disorder, i . e . , arresting its devel degrade within the body (biodegradable , bioerodable ) . A opment; and / or relieving or ameliorating the symptoms of biodegradable material may further be bioresorbable or disorder , e. g . , cardiac arrhythmia . As is understood by those bioabsorbable , i .e . , it may be dissolved and absorbed into skilled in the art , “ treatment " can include systemic amelio bodily fluids (water -soluble implants are one example ), or ration of the symptoms associated with the pathology and /or degraded and ultimately eliminated from the body , either by a delay in onset of symptoms such as chest pain . Clinical and US 2018 /0273906 A1 Sep . 27 , 2018 21 sub - clinical evidence of “ treatment” will vary with the juvenile stem cell (SJST ) , a marrow stromal cell , a mesen pathology, the individual and the treatment. chymal stem cell , a hematopoietic stem cell, or a mononu [0207 ] “ Administration ” or “ delivery ” of a cell , exosome clear cell. or microvesicle , miRNA , therapeutic or other agent and [0211 ] Methods to derive or prepare an iPSC from these compositions containing same can be effected in one dose , parent cell types are known in the art . In one aspect, the iPSC continuously or intermittently throughout the course of was created by a method comprising contacting the parent treatment. Methods of determining the most effective means cell with an effective amount of a DNA methyltransferase and dosage of administration are known to those of skill in inhibitor to upregulate Oct4 . Non - limiting examples of the art and will vary with the composition used for therapy , DNA methyltransferease inhibitors include 5 ' -azacytidine , the purpose of the therapy , the target cell being treated , and 5 - aza - 2 '- deoxycytidine, MG98 , zebularine and RG108 . In the subject being treated . Single or multiple administrations another aspect, the iPS cell was created by a method that can be carried out with the dose level and pattern being excludes the insertion of exogenous genes into the parent selected by the treating physician or in the case of animals , cell. by the treating veterinarian . Suitable dosage formulations [ 0212 ] In a yet further embodiment, the parent cells are and methods of administering the agents are known in the progenitor cells , e . g . , cardiac progenitor cells , prepared by art . Route of administration can also be determined and preconditioning the cells with electrical stimulation . In one method of determining the most effective route of adminis aspect, the parent cell is a stem cell that expresses Sca 1 and tration are known to those of skill in the art and will vary pluripotency and cardiac genes as described herein . The with the composition used for treatment , the purpose of the cells are subsequently contacted with isoxazole or an isox treatment, the health condition or disease stage of the subject azole similar compound as described herein . being treated , and target cell or tissue . Non - limiting [0213 ] In one aspect , the parent cell is a small juvenile examples of route of administration include oral adminis stem cell or young stem cell that is not modified or pro tration , intraperitoneal, infusion , nasal administration , inha grammed to an iPSC . In this aspect, the SJST is treated with lation , injection , and topical application . the isoxazole or isoxazole similar compound and used diagnostically , in research or therapeutically . In another Descriptive Embodiments aspect, the disclosure provides a method to direct an imma ture or progenitor cell , e . g. , a SJST cell, a circulating blood Induced Pluripotent Stem Cells or heart derived stem cell ) to a cardiac progenitor phenotype by contacting the cell with an effective amount of an [0208 ] This disclosure provides an induced pluripotent isoxazole or isoxazole similar compound as described stem cell ( iPSC ) characterized by DNA hypomethylation . In herein . In another aspect, the parent cells are juvenile or one aspect, the cell is additionally characterized by overex young stem cells that can be identified by low expression of pression of one or more cardiac gene or marker selected miR - 195 as compared to a cell that does not express the from the group of Gai, mir - 133 , mir - 762 , CCL7 , CXCR2, characteristics of a young or juvenile cell as described CXC5 , integral membrane protein 2A , and ephrin A3 . Nkx herein . In a further aspect, the parent cell is further charac 2 . 5 , ISL1, GATA4 , AMHC , Sarcomeric actin , Gai, mir - 133 , terized by low expression of one or more marker selected mir- 762, CCL7 , CXCR2 , CXC5 , integral membrane protein from miR - 29b , miR - 205 , miR - 378 , and miR - 542 - 3p as 2A , and ephrin A3. In another aspect, the cell under compared to a cell that does not express the characteristics expresses one or more pluripotent genes or markers , non of a young or juvenile cell as described herein . In a further limiting examples of such include one or more of miR - 290 or alternative aspect, the parent cell has been pre - condi 295 cluster , let - 7 family , Max and under expresses one or tioned with an effective amount of electrical stimulation . In more DNA methyltransferase genes Dnmt1 , Dnmt3b . In one another aspect, isoxazole or isoxazole similar compound is aspect , the one or more cardiac gene or marker is Gai. In one injected therapeutically in the ischemic heart where BMSC , aspect, the one or more cardiac gene or marker is overex skeletal myoblasts , peripheral blood -derived endothelial pressed at least 1 . 5 fold , or alternatively at least 2 fold , or progenitor cells (EPC ) , resident cardiac stem / progenitor alternatively at least 3 fold , over that of a control cell . In cells and fibroblasts are mobilized to the injury area by the another aspect, the cell is characterized by underexpression art described (Konoplyannikov M I, Haider K H , Lai V K , of one or more other cardiac gene or marker selected from Ahmed RP, Jiang S , Ashraf M Activation of diverse the group of miR - 290 cluster, miR -574 -5P , let - 7 family , signaling pathways by ex -vivo delivery of multiple cytok Dnmtl , Dnmt3b , and Max . In one aspect , the one or more ines for myocardial repair. Stem Cells Dev . 2013 Jan . 15 ; cardiac gene or marker is not expressed or underexpressed 22 ( 2 ) :204 - 15 ) prior to treatment for maximum drug contact by at least 1 . 5 fold , or alternatively at least 2 fold , or to induce regeneration . alternatively at least 3 fold , under that of a control cell . [0214 ] The chemically modified iPS cell as described Methods to identify and quantitate genes and markers are herein is prepared by contacting the cell with an effective known in the art and described herein to supplement well amount of an isoxazole or isoxazole similar compound , e . g ., known methods . an amount that is selected from the group of from about 0 . 3 [ 0209 ] The cell can be from any animal species , such as a to about 30 uM ; from about 0 . 5 to about 25 uM ; from about mammal, e . g . , an equine , a murine , a bovine , a canine , a 12 to about 25 uM and from about 0 . 5 uM to about 20 UM . feline , or a human patient. See also FIG . 14C . [0210 ] The iPS cell is derived from any suitable parent [ 0215 ] While methods to prepare an iPS cell are known in cell . Non - limiting examples of such include, without limi the art, Applicant has determined that an iPS cell created by tation , a cell selected from the group consisting of a bone a method comprising contacting a parent cell with an marrow cell , a myoblast , a skin fibroblast, a cord blood cell , effective amount of a DNA methyltransferase inhibitor, e . g . , an adult peripheral blood , a cardiac progenitor cell, a small 5 '- azacytidine or RG108 (Sigma - Aldrich ) to upregulate US 2018 /0273906 A1 Sep . 27 , 2018

Oct4 is particularly useful. In one aspect , the iPS cells are compound . In another aspect, the parental cell is selected for additionally prepared by a method that excludes the inser chemical modification because it is characterized by low tion of exogenous genes into the parent cell thereby enhanc expression of miR - 195 and the cell may or may not be ing the safety of the cells for clinical use . In another aspect, precondition by application of an effective amount electri the cells are modified to an iPS cell by insertion of genes and cal stimulation . other factors known in the art. [0220 ] In a particular aspect , the method comprises, or [ 0216 ] This disclosure also provides a population of cul alternatively consists essentially of, or yet further consists tured cells as described herein , by culturing the chemically of, contacting the cells with an effective amount of the modified cells to expand the cells as described herein . In one isoxazole or isoxazole similar compound for at least 3 days , aspect the population is substantially homogenous, e .g ., at or alternatively at least 4 days, or alternatively at least 5 least 70 % identical in phenotype , or alternatively at least days , or alternatively at least 6 days , or alternatively at least 75 % identical in phenotype, or alternatively at least 80 % 7 days, in DMEM F12 supplemented with from about 10 % identical in phenotype , or alternatively at least 85 % identical to about 30 % , or alternatively 20 % Knockout Serum in phenotype , or alternatively at least 90 % identical in Replacement (KSR ; Invitrogen , USA ) , and from about 0 . 05 phenotype , or alternatively at least 95 % identical in pheno mM to about 0 . 2 mM , or about 0 . 1 mM MEM Non - Essential type , or alternatively at least 98 % identical in phenotype or Amino Acids solution ( Invitrogen , CA , USA ), and from alternatively , a clonal population of cells . In one aspect, the about 0 . 1 mM to about 0 . 3 mM or about 0 . 2 mM L - gluta population is a clonal population . In one aspect , the cells are mine ( Invitrogen , USA ) ; and from about 0 . 05 mM to about cultured under conditions that favor differentiation into a 0 . 2 mM or alternatively from about 0 . 1 mM B -mercap particular cell type, e . g . , a cardiac cell. These culturing toethanol ( Invitrogen , CA , USA ) ; and from about 750 U /ml conditions are known in the art . to about 1250 U /ml or alternatively from about 1000 U /ml [0217 ] The cells and populations of cells can be further LIF (Millipore ) ; and an effective amount of 0 . 5 % penicillin modified for therapeutic or research use , for by example , and streptomycin . The cells are then kept in the media further comprising a detectable label or exogenous poly without drug for five days . The media is changed every day nucleotide or polypeptide . Methods and appropriate poly otherwise due to increase in cell number the pH of the media nucleotides for therapeutic use are described in Durrani et al. get changed and effect the cell survival and gene expression . ( 2010 ) Regen . Med . 5 (6 ) :919 - 932 . An effective amount of the isoxazole or isoxazole similar compound comprises , or alternatively consists essentially Method for Preparing the iPS Cells of, or yet further consists of from about 0 . 3 to about 30 uM ; [0218 ] This disclosure also provides a method for prepar or from about 0 . 5 to about 25 uM ; or from about 12 to about ing a cardiac lineage cell from a stem cell, comprising 25 uM ; or from about 0 . 5 uM to about 20 uM . As used contacting the stem cell with an effective amount of an herein , isoxazole or isoxazole similar intends a class of isoxazole or isoxazole similar compound . Stem cells useful compounds as described herein . in the method includes , without limitation , a one or more of 0221 ] In another particular aspect, the method comprises , an iPS cell , such as an iPS cell derived from a bone marrow or alternatively consists essentially of, or yet further consists cell, bone marrow stomal cell , a mesenchymal stem cell, a of, contacting the cells with an effective amount of the hematpoietic stem cell , a myoblast , a skin fibroblast , a cord isoxazole or isoxazole similar compound for at least 3 days , blood cell, an adult peripheral blood , a SJSC , a cardiac or alternatively at least 4 days , or alternatively at least 5 progenitor cell, or mononuclear cell . Methods to derive or days , or alternatively at least 6 days , or alternatively at least prepare an iPSC from these parent cell types are known in 7 days , in RPMIF12 without insulin . An effective amount of the art . The cells prepared by this method are characterized the isoxazole or isoxazole similar compound comprises, or in overexpressing one or more cardiac gene or marker, e . g . , alternatively consists essentially of, or yet further consists of Nkx - 2 . 5 , ISL1, Tbx - 5 , GATA4 , AMHC , Sarcomeric actin , from about 0 . 3 to about 30 uM ; or from about 0 . 5 to about Gai, miR - 133 , miR - 762 , CCL7 , CXCR2, CXC5, integral 25 uM ; or from about 12 to about 25 uM ; or from about 0 . 5 membranem protein 2A , or ephrin A3 and /or underexpressing UM to about 20 uM . In yet another particular aspect, an one or more cardiac gene or marker , e . g ., miR -290 - 295 isoxazole is an ISX - 9 as described herein above . As used cluster, let- 7 family, Dnmt1, Dnmt3b , and Max , each as herein , isoxazole or isoxazole similar intends a class of compared to a control cell such as a cell that has not been compounds as described above . contacted with or exposed to the isoxazole or isoxazole [0222 ] The cells are then kept in RPMI F12 with insulin similar compound. In one aspect, the over - or under -expres for another about 7 - 10 days to generate cardiomyocyte sion is at least 1 . 5 fold over - or under - that of the control cell. differentiation . The cells are then kept in EGM - 2 medium [0219 ] In one aspect, the cell is a small juvenile stem cell ( Lonza , Lonza Walkersville Inc . , Walkerswille Md. 21793 (SJSC ) that is not modified or programmed to an iPSC . In 0127 ) for about 10 days to generate endothelial cell differ this aspect, the SJST is treated with the isoxazole or isox entiation . The cells are kept in TGFB ( 2 ng /ml ) and azole similar compound and used diagnostically, in research PDGFBB (long / ml , R & D Systems, Inc ,Minneapolis ,Minn . or therapeutically . In another aspect , the disclosure provides 55413 ) about 10 days to generate smooth muscle cell a method to direct an immature or progenitor cell , e . g . , a differentiation . SJST cell, ( contained with a population of circulating blood [0223 ] Methods to identify and quantitate genes and mark or heart derived stem cells ) to a cardiac progenitor pheno ers are known in the art and described herein to supplement type by contacting the cell with an effective amount of an well known methods . isoxazole or isoxazole similar compound as described [0224 ] One can determine if the methods of this disclosure herein . An effective amount of electrical stimulation can be have been effective by molecular, clinical and other tech applied to the cell or tissue in need of the treatment, alone niques. In one aspect, Nkx2. 5 , GATA4 , Tbx5 , ISL - 1 and or in combination with the isoxazole or isoxazole similar Mef2c upregulation is started after 3 days after contacting US 2018 /0273906 A1 Sep . 27 , 2018 and expression of these markers can be determined by differentiation into a particular cell type , e . g ., a cardiac cell . application histochemistry and / or PCR , as appropriate . These culturing conditions are known in the art . Additional lineage identifying markers are shown in FIG . 0229 ] The methods can be further modified by inserting 14C . The cells can be further evaluated for 7 day treatment into the cells and populations of cells a detectable label or by qPCR . Immunofluorescence staining also showed that exogenous polynucleotide or polypeptide . Methods and transcription factors (Nkx2 . 5 , GATA4 and ISL - 1 ) were appropriate polynucleotides for therapeutic use are highly expressed in hiPSC with 7 day small molecule described in Durrani et al . (2010 ) Regen . Med . 5 (6 ): 919 treatment. The purity of Nkx2. 5 positive cells in these small 932 . molecule treated cells by FACS was 96 . 5 + 2 . 5 % cells . To establish that the Nkx2 . 5 + cells are truly committed cardio Exosome or Microvesicle Populations and Exosome or vascular precursors , Applicant found that these Nkx2 . 5 + Microvesicle Compositions cells were multipotent and directly differentiated into all [0230 ] This disclosure provides composition comprising, three cardiovascular lineages , including CMs, ECs and or alternatively consisting essentially of, or yet further SMCs in basal differentiation conditions without any spe consisting of, an isolated population of exosomes or cific induction signaling molecules with percentage of microvesicles overexpressing a microRNA miRNA ) 95 . 2 + 2 . 1 % CMS ( TnT + ), 90 . 3 + 2 . 5 % ECs (CD31 + ) and selected from the group of mir - 373 , mir - 210 , mir - 377 , 92 . 3 + 1 . 8 % SMCs ( Q -SMA + ) . In addition , Applicant deter mir - 367, mir- 520 , mir -548ah , mir -335 , mir - 21, mir - 30c , mined that the differentiated ECs exhibited phenotype and mir - 214 and /or mir - 548q ; and / or one or more of a protein function similar to primary endothelial cells ( ECS ) (FIGS . selected from the group of Tsg101 , CD9, Hsp70 , Flotilline 14A - 14H ) 1 , or GAPDH . In another aspect, the population comprises [ 0225 ] The cell can be from any animal species , such as a two or more , or three or more , or four or more , or five or mammal, e . g ., an equine , a murine, a bovine , a canine, a more , or six or more , or seven or more , or eight or more, or feline , or a human patient. The cell can be autologous or nine or more , or ten ormore , or eleven or more , or all twelve allogeneic to the animal or patient being treated . of the miRNA and /or at least one , at least two , at least three , 0226 ]. The contacting can be performed in vitro , e . g ., in a at least four or all five of the proteins . In an alternative tissue culture dish or plate as described herein or in vivo , by aspect, for the population of exosomes or microvesicles, at administering an effective amount of the isoxazole or isox least 80 % , or alternatively 85 % , or alternatively 90 % , or azole similar compound to a cell culture or in vivo by alternatively 95 % , or alternatively 97 % or alternatively administering an effective amount of the compound or 99 % , or alternatively 100 % , of the exosomes or microve alternatively , compound and iPS cells to a patient or subject sicles overexpress the miRNA and /or the proteins. The in need of such treatment. Methods for administering , sys exosomes or microvesicles of the population are isolated temically or locally , such are described below . The com from a cell selected from a cardiac progenitor cell (“ CPC ” ), pounds and / or cells can be combined with a pharmaceuti a cardiomyocyte, an endothelial cell, a myocyte, or a smooth cally acceptable carrier for ease of use . This treatment can , muscle cell. in one aspect , be combined with the administration of an [0231 ] Also provided is a cell, a population of cells , or a effective amount of electrical stimulation to the tissue in composition comprising , or alternatively consisting essen need of such treatment. The electrical stimulation can be tially of, or yet further consisting of: an isolated population administered prior to , concurrently or subsequent to the of one or more of: a cardiac progenitor cell a (CPSs ) , a administration of the isoxazole or isoxazole similar com cardiomyocyte , a myocyte , an endothelial cell , a smooth pound . muscle cell , a skeletal muscle cell, each generated from a cell selected from the group of an iPSC , an embryonic stem [0227 ] In one aspect, the stem cell contacted with the cell, or a stem cell that was contacted with an isoxazole compound is an iPS cell. Methods to generate iPS cell from compound , a derivative or an equivalent thereof. The cells or terminally differentiated cells are known in the art, e . g . , by population of cells are identified by overexpression of a a method comprising contacting the parent cell with an microRNA (miRNA ) selected from the group of mir - 373 , effective amount of a DNA methyltransferase inhibitor, e. g ., mir -210 , mir - 377, mir -367 , mir -520 , mir - 548ah , mir - 335 , RG108 ( Sigma - Aldrich ) to upregulate Oct4 . In another mir - 21 , mir - 30c , mir - 214 or mir - 548q ; and / or a muscle gene aspect, the iPS cell is created by a method that excludes the selected from the group of paZ3 , PAX7, MYF5 , MYOD , insertion of exogenous genes into the parent cell. MYOG , or dystrophin . In a further aspect, provided herein [ 0228 ] This disclosure also provides an isolated cell pre is a cardiomyocyte or a population of cardiomyocytes that pared by a method as described herein by further comprising express on or more of cTnT, cTnI, MLC2V , and /or CX43 . isolating the cells . Yet further , the method further comprises Further provided is a cell or a population of endothelial cells culturing the cells to prepare a population of cells. In one that expressCD31 and VE - cadherin . Yet further provided is aspect the population is cultured under conditions to prepare a smooth muscle cell or population of these cells that express a substantially homogenous , e . g ., at least 70 % identical in a - smooth muscle actin (SMA ) and calponin . phenotype , or alternatively at least 75 % identical in pheno [ 0232 ] In another aspect , the populations as described type , or alternatively at least 80 % identical in phenotype , or herein comprises two or more , or three or more , or four or alternatively at least 85 % identical in phenotype , or alter more , or five or more , or six or more , or seven or more , or natively at least 90 % identical in phenotype , or alternatively eight or more, or nine or more , or ten or more , or all eleven at least 95 % identical in phenotype , or alternatively at least of the miRNA and /or at least one, at least two , at least three , 98 % identical in phenotype . In one aspect , the method at least four, or at least four, or at least five , or all six five of further comprises culturing the cells under conditions that the muscle genes . In an alternative aspect, for the population favor clonal expansion of the cells to a clonal population . In of exosomes or microvesicles , at least 80 % , or alternatively one aspect, the cells are cultured under conditions that favor 85 % , or alternatively 90 % , or alternatively 95 % , or alterna US 2018 /0273906 A1 Sep . 27 , 2018 tively 97 % or alternatively 99 % , or alternatively 100 % , of tissue, skeletal muscle tissue , a blood vessel , a capillary, or the cells of the population overexpress the miRNA and /or a myocyte ; promoting cardiac regeneration ; promoting car the genes and / or the proteins . diac regeneration in a subject suffering from an acute cardiac [ 0233] Also provided is an isolated population of cells that event; promoting cardiac regeneration in a subject suffering express one or more protein selected from the group : cTnl, from a myocardial infarction ; promoting cardiac regenera MLC2v, cTnT, VE - cadherin , CD31 , A -SMA ( actin ), cal tion in subject suffering from Duchenne Muscular Dystro ponin or Cx43 , wherein optionally at least two , or at least phy (DMD ) or Duchenne Muscular Dystrophy (“ DMD ” ) three , or at least four , or at least five , or at least six , or at least associated cardiomyopathy, promoting cardiac regeneration seven , or all eight of expressed proteins. In one aspect, the in a subject suffering from age - related diseases , such as for population of cells is selected from the group consisting of example , chronic obstructive pulmonary disease (“ COPD " ), a smooth muscle cells , an endothelial cells , a blood vessel or arthritis, osteoporosis , osteoarthritis , diabetes , vascular a cardiomyocyte . dementia , or macular degeneration ; promoting tissue regen [ 0234 ] In an alternative aspect, for the population of cells , eration in tissue damaged from one or more of stroke , at least 80 % , or alternatively 85 % , or alternatively 90 % , or arthritis, Alzheimer' s, memory loss disorders , cystic fibro alternatively 95 % , or alternatively 97 % or alternatively sis , inflammatory disorders and cancer ; decreasing cardiac 99 % , or alternatively 100 % , of the exosomes or microve wall thickness in a tissue damaged from a cardiac infarction ; sicles express the proteins . Methods to identify protein altering gene expression of one or more of protein kinase C , expression are known in the art and include the use of iL - 6 , mmp , PDGF ; reducing or inhibiting the expression of protein - specific antibodies are hybridization techniques that an inflammatory protein that is optionally a chemokine , monitormRNA expression . The populations can be prepared macrophage or a cytokine ; directly or indirectly stimulating by a method comprising, or alternatively consisting essen angiogenesis ; promoting cardiac regeneration in a subject tially of, or yet further consisting of culturing a population suffering from a disease selected from the group of coronary of iPSC in the presence of an effective amount of an artery disease , myocardial infarction , heart failure , hypopla isoxazole , a derivative or an equivalent thereof. In a yet sic left heart syndrome, peripheral artery disease (PAD ) , further , the population is derived from a fibroblast or a cardiac mypertrophy, valvular heart disease (aortic stenosis ) , skeletal myoblast. The populations can be homogenous, myocardial hypertrophy, hypertrophy fibrosis ; and /or heterogeneous, purified , highly purified , homogenous or directly or indirectly inhibit cellular replication . The meth substantially homogeneous. In a further aspect , the exo ods are accomplished by administering an effective amount somes or microvesicles can be detectably labeled and / or of the exosomes or microvesicles, cells , populations or combined with a carrier, preservative or cryprotectant. The compositions containing the same to a subject in need populations and compositions can be lyophilized . Methods thereof . The methods can further comprise administering an to isolate exosome or microvesicle populations and produce effective amount of non - embryonic stem cell or a progenitor isolated population of cells are known in the art , some of cell to the subject that is optionally the same or different type which are described herein . In a further aspect, the popula of tissue that is in need of repair . In one aspect , the tion is derived from a fibroblast or a skeletal myoblast . non - embryonic stem or progenitor cell is autologous to the [0235 ] In a further aspect, the population of cells is subject. In another aspect, the non - embryonic stem or pro selected from the group consisting of smooth muscle cells , genitor cell is allogeneic to the subject. The exosomes or endothelial cells , and cardiomyocytes . As used herein , a microvesicles , cells , populations can be locally or systemi " smooth muscle cell” intends a cell expressing the following cally delivered to the subject, e . g . , via an intramyocardialor markers SMTN (smoothelin ), Cnn1 (calponin 1 ) , telokin . As an intracoronary route . Subjects suitably treated by these used herein , an “ endothelial cell ” intends a cell expressing methods include animals , mammals and human patients . the following markers VE - cadherin (CD144 ) , CD31 , von Methods to determine the effectiveness of the therapy are Willerbrand factor (vWF ). known in the art, some of which are described herein . [ 0236 ] The compositions can further comprise a protein 10239 ] The compositions also are useful in methods for that facilitates regeneration and / or improved function of a one or more of the following therapies: anti -oxidative tissue or a nucleic acid that encodes the protein and / or an therapy ; provding anti -oxidative therapy ; promoting activa agent that inhibits the expression of an inflammatory pro tion of local or resident cardiomyocytes ; promoting the tein , that is optionally a cytokine . In one aspect, the protein release of angiogenesis and /or paracrine factors ; promoting is selected from the group of a transforming growth factor activation of wnt, a BMP, and / or cytoskeleton remodeling ; beta ( TGF -beta ), a WNT protein , a cytokine , or a histon promoting TGF - B induced emt signaling and cardiac differ deacetylase . entiation ; increasing expression of wnt5 and wnt11 or a [0237 ] In one aspect, the cells are cultured in serum - free BMP family protein , optionally BMP4 ; increasing expres media . In another aspect, the effective amount of the isox sion of a cardiac transcription factor selected from the group azole compound , derivative or equivalent thereof , is an consisting of nkx2 . 5 , mef2c , gata4 and isl - 1 ; promoting amount that results in overexpressed of the mir . In one expression of genes for development of pip3 signaling in aspect, the effective amount is from about 5 uM to about 25 cardiomyocytes , muscle contraction and nf- at hypertrophy uM . In another aspect, the effective amount is 8 to 20 or from signaling pathways ; reducing fibrosis and apoptosis ; pro about 10 to about 15 or from about 5 to about 20 uM and moting myoangeneis and muscle differentiation ; promoting from about 10 uM to about 25 uM . the release of a cytokine selected from the group consisting [ 0238 ] These compositions are useful in a method for one of angiopoietin - 2 , il -6nmp , pgfbb , timp 1 or a gene identified or more of: regenerating damaged tissue such as muscle in the Figures as disclosed herein ; promoting upregulation of and / or cardiac tissue ; improving the viability of damaged a gene selected from the group consisting of wnt3a , wnt5a , tissue , such as muscle and / or cardiac tissue ; facilitating the wnt11 ; and /or promoting cytoskeletal remodeling , by formation of new tissue, such as cardiac tissue, muscle administering an effective amount of the exosome or US 2018 /0273906 A1 Sep . 27 , 2018 25 microvesicle , cells , populations or compositions as wherein R1 and R2 is each selected from C1 - C4 alkyl, described above to a subject in need thereof. phenyl, benzyl, trifluoromethyl or halogen , R3 is selected [0240 ] Further provided is an isolated population of cells from hydrogen , hydroxy , C1- C4 alkyl or alkoxy, R4 , in that express one or more protein selected from the group of position 3 or 5 , is selected from hydrogen , trifluoromethyl, cTnl, MLC2v , cTnT, VE - cadherein , CD31, a - SMA (actin ), C1 -C4 alkoxy , C1 - C4 alkyl, or C1- C4 hydroxyalkyl, R5 is calponin or Cx43 . [ 0241] In another aspect , the above noted compositions selected from hydrogen or C4 -C4 alkyl or R4 and R5 further comprise a protein that facilitates regeneration and/ or together form a tetramethylene group , Z at position 3 or 5 on improved function of a tissue or a nucleic acid that encodes the heterocycle is selected from : — N (R6 ) - CO — , - CO the protein . Non - limiting examples of such proteins include N ( RF) -, NORG ) - CO - NRG ) -, CH ( RF )- NHCO , or for example , Bmp7 in kidney, Fgf- 21 in liver , Op- 1 in bone - NH - CO CH (R6 ) , in which R6 is selected from hydro regeneration , etc . In a yet further aspect , the compositions gen or C1 -C4 alkyl . further comprise a agent that inhibits the expression of an [ 0246 ] In a yet further aspect, the isoxazole compound or inflammatory protein . Non - limiting examples of these a derivative thereof is selected from the group : 5 - ( trifluo agents include for example Phospholipase A2 inhibitors, p38 romethyl ) - 3 - ( 4 -methoxyphenyl ) isoxazole - 4 -carboxylic Mitogen - Activated Protein (MAP ) kinase inhibitors , etc . In a further aspect, the protein is a cytokine, non - limiting acid , 5 - ( trifluoromethyl ) - 3 - ( 4 - fluorophenyl) isoxazole - 4 examples of such cytokines include interleukin and inter carboxylic acid , 5 - ( thiophen - 2 - yl) isoxazole - 3 - carboxalde feron . hyde, 5 ,6 ,7 , 8 - tetrahydro - 4h -cyclohepta [ d ] isoxazole - 3 -car [0242 ] In one aspect, the populations as described herein boxylic acid , 4 , 5 , 6 , 7 - tetrahydro -benzo [ d ] isoxazole - 3 further comprise a carrier, wherein in one aspect, a non carboxylic acid , 3 -amino -5 -methylisoxazole , 4 -amino -n -( 5 naturally occurring carrier . In another aspect, the popula methyl - 3 - isoxazolyl) benzenesulfonamide, 3 - phenyl tions further comprise a preservative or cryoprotectant, such isoxazole - 5 -boronic acid pinacol ester , 5 -phenylisoxazole , as polyethylene glycol. 1 -phenyl - 1 -cyclopentanecarboxylic acid , 3 -phenyl - benzo [ c ] [ 0243 ] In a further aspect, the populations as described isoxazole - 5 -carboxylic acid , 5 - methyl- 3 - phenylisoxazole - 4 herein are lyophilized . carboxylic acid , 3a, 4 , 5 ,6 , 7 , 8 , 9 , 9a - octahydro -cycloocta [ d ] [ 0244 ] In a further aspect , the compositions described isoxazole - 3 -carboxylic acid , 5 - ( 3 - nitrophenyl) isoxazole, herein are isolated from a population of stem cells or 3 - (4 -nitrophenyl ) isoxazole , 3 -hydroxy -5 -aminomethyl progenitor cells cultured in the presence of an effective isoxazole , 5 - ( morpholinomethyl) isoxazole - 3 - carboxylic amount of an isoxazole compound or a derivative thereof. In acid hydrochloride, 5 - morpholinomethyl) isoxazole - 3 -carb one aspect , the isoxazole compound is selected from isox aldehyde , 3 -methyl - 5 - ( trifluoromethyl) isoxazole- 4 -carbox azole - 1 ( isx - 1 ) or isoxazole - 9 ( isx - 9 ) . In another aspect, the ylic acid , methyl 5 - ( thiophen - 2 -yl ) isoxazole - 3 -carboxylate , isoxazole derivative is a compound having the formula : 3 - (methylsulfonyl ) - 5 - ( 2 - thienyl) isoxazole - 4 -carbonitrile , 5 - methyl- 3 - ( 2 -pyrrolidinyl ) isoxazole , 3 -methyl - 5 - ( 2 -pyrro lidinyl) isoxazole , 3 - ( 1 -methyl - 1h - pyrazol - 4 - yl) - isoxazole 5 -carboxylic acid , 3 - (1 -methyl - 1h -pyrazol -4 -yl ) - 4 ,5 - di hydro - isoxazole - 5 -carboxylic acid , 5 - ( 4 -methylphenyl ) isoxazole - 3 -carboxylic acid , 5 - methyl- 3 -phenylisoxazole - 4 z carboxylic acid , 5 - ( 4 -methylphenyl ) isoxazole - 3 carboxaldehyde , 5 -methyl - 3 - ( 4 -phenoxyphenyl ) isoxazole R3 4 -carboxylic acid , 3 -methyl - 5 - ( 4 -methyl - 1 , 2 , 3 - thiadiazol- 5 yl) isoxazole -4 -carboxylic acid , 3 -methyl - 5 -( 5 methylisoxazol- 3 - yl) isoxazole - 4 - carboxylic acid , methyl 5 - ( 4 -methoxyphenyl ) isoxazole - 4 -carboxylate , methyl 5 - ( 4 wherein R , and R , are both hydrogen or R , is hydrogen and methoxyphenyl) isoxazole - 3 -carboxylate , 5 -methylisox R , is selected from the group consisting of substituted or azole , methyl 5 - ( 4 - fluorophenyl) isoxazole - 4 - carboxylate , unsubstituted C . - C . alkyl, Cz- Co cycloalkyl, C2- C . alkenyl, methyl 5 - ( 4 - fluorophenyl) isoxazole - 3 - carboxylate , methyl C2 -C6 alkynyl, and benzyl, or where R , and R2 may be 5 - ( 4 -chlorophenyl )isoxazole -4 - carboxylate , methyl 5 - (4 joined together to form a ring selected from azetidinyl, bromophenyl) isoxazole - 4 -carboxylate , 5 - ( 4 -methoxyphe pyrrolidinyl , piperidinyl , piperazinyl , or morpholinyl; R2' , nyl) isoxazole - 3 - carboxylic acid , 5 - ( 3 -methoxy -phenyl ) Rz and R4 are independently selected from the group con isoxazole - 3 - carboxylic acid , 3 - ( 2 - methoxyphenyl ) sisting of hydrogen , halogen , C . - C . alkyl, Cz- Co cycloalkyl, isoxazole - 5 -carboxylic acid , 5 -( 4 -methoxyphenyl ) substituted or unsubstituted aromatic or heteroaromatic ring, isoxazole - 3 - carboxaldehyde, 3 - ( 4 -methoxyphenyl ) cyano , nitro , and acyl; X is 0 , NH or S ; and Y is 0 , NH or isoxazole - 5 - carbaldehyde , 3 - ( 2 -methoxyphenyl ) isoxazole S . 5 - carbaldehyde, 5 - ( 4 -methoxyphenyl ) isoxazole , 3 - (4 [0245 ] In a further aspect , the isoxazole compound has the methoxyphenyl) isoxazole , 3 -( 2 -methoxy -phenyl ) - 4, 5 formula : dihydro - isoxazole - 5 - carboxylic acid , 3 -methoxy - isoxazole 5 -carboxylic acid , isoxazole - 5 - carboxylic acid , isoxazole - 4 carboxylic acid , isoxazole - 5 -carbothioamide , isoxazole - 5 carbonyl chloride , isoxazole - 3 - carbonitrile , isoxazole - 3 O2 carbaldehyde, isoxazole - 4 - boronic acid , isoxazole , 5 -cyclopropyl - 4 -[ 2 - (methylsulfonyl ) -4 - ( trifluoromethyl) benzoyl] isoxazole , 6 - ( 5 - ( thiophen - 2 -yl ) isoxazole - 3 -carbox R3 amido )hexyl 5 - (( 3as, 4s , bar )- 2 - oxohexahydro -lh -thieno [ 3 , 4 - d ]imidazol - 4 - yl ) pentanoate , isocarboxazid 5 -methyl - 3 isoxazole - carboxylic acid 2 -benzylhydrazide , 5 -isobutyl US 2018 /0273906 A1 Sep . 27 , 2018

isoxazole - 3 - carboxylic acid , 4 - iodo - 5 -methyl - isoxazole , nyl ) isoxazole -3 -carboxylic acid , 3 - ( 4 -bromophenyl ) 3 , 3 '- iminobis ( n , n -dimethylpropylamine ), 3 - ( 3 -hydroxy isoxazole- 5 - carboxylic acid , 3 - ( 4 -bromophenyl ) isoxazole phenyl) - isoxazole - 5 - carboxylic acid methyl ester, 5 - ( 4 -hy 5 -carboxaldehyde , 5- ( 4 -bromophenyl ) isoxazole , 5 - ( 3 droxy -phenyl ) - isoxazole - 3 - carboxylic acid , 5 - ( 3 -hydroxy bromophenyl) isoxazole , 3 - ( 4 -bromophenyl ) isoxazole, phenyl) - isoxazole -3 -carboxylic acid , 5 -( hydroxymethyl ) - 3 5 -( bromomethyl ) -3 - (4 -methoxyphenyl ) isoxazole , 4 -( bro methylisoxazole , 3 -hydroxy - 5 -methylisoxazole , 5 -( 1 momethyl) isoxazole , 5 - bromomethyl) - 3 - ( 4 -fluorophenyl ) hydroxyethyl) -3 - (4 - trifluoromethylphenyl) isoxazole , 3a , 4 , isoxazole , 5 -( bromomethyl ) - 3 -( 4 -chlorophenyl ) isoxazole , 5 , 6 , 7 ,7a -hexahydro -benzo [ d ] isoxazole - 3 - carboxylic acid , 5 - ( bromomethyl) - 3 - ( 4 -bromophenyl ) isoxazole , 6 -bromo - 3 5 -( 2 -furyl ) isoxazole - 3 -carbaldehyde , 5 - furan - 2 - yl- isox methylbenzo [ d ] isoxazole , 5 -bromo - 3 -methylbenzo [ d ]isox azole - 3 -carboxylic acid , 6 - fluoro - 3 - ( 4 - piperidinyl) ben azole, 4 -bromo - 5 - ( 4 -methoxyphenyl ) isoxazole , 3 -bromo zisoxazole , 5 - ( 4 - fluorophenyl) isoxazole - 3 - methanol, 3 -( 2 isoxazole , 3 -bromo - 5 - (2 -hydroxyethyl ) isoxazole , 4 -bromo fluoro -phenyl ) - isoxazole - 5 - carboxylic acid , 5 - ( 4 5 -( 4 -fluorophenyl ) isoxazole , 3 -bromo -5 - ( 4 -fluorophenyl ) fluorophenyl) isoxazole - 3 - carboxaldehyde, 3 - ( 4 isoxazole , 4 - bromo - 5 - ( 4 - chlorophenyl) isoxazole , 4 -bromo fluorophenyl) isoxazole - 5 -carbaldehyde , 3 -( 3 - fluorophenyl) 5 - ( 4 -bromophenyl ) isoxazole , 6 - bromo- benzo [ d ] isoxazole isoxazole - 5 - carbaldehyde , 3 - ( 2 - fluorophenyl) isoxazole - 5 3 - carboxylic acid , benzo [ d ]isoxazole - 3 - carboxylic acid , carbaldehyde, 5 - ( 4 - fluorophenyl ) isoxazole , 3 - ( 4 3 -amino - 5 -methylisoxazole , 5 - amino - 3 - ( 4 -methoxyphenyl ) fluorophenyl) isoxazole , 5 - ( 3 - fluoro - 4 -methoxy - phenyl) isoxazole, 3 -aminoisoxazole , 3 -amino - 5- ( 4 -fluorophenyl ) isoxazole - 3 -carboxylic acid , ethyl 5 - ( trifluoromethyl) - 3 - ( 4 isoxazole , 5 -amino - 3 - ( 4 - chlorophenyl) isoxazole , 5 -amino methoxyphenyl) isoxazole - 4 -carboxylate , ethyl- 5 4 - ( 4 -bromophenyl ) isoxazole , 3 - amino - 5 - ( 4 -bromophenyl ) ( tributylstannyl) isoxazole - 3 -carboxylate , ethyl 5 - ( thiophen isoxazole , 5 -acetyl - 3 - ( 4 - fluorophenyl) isoxazole, 5 -acetyl - 3 2 - yl) isoxazole - 3 - carboxylate , 5 -ethyl - isoxazole - 4 ( 3 - fluorophenyl) isoxazole , 3 -methyl - 5 - [ (2s )- 1 -methyl - 2 carboxylic acid , 5 - ethyl- isoxazole- 3 - carboxylic acid , ethyl pyrrolidinyl] isoxazole hydrochloride , 7 -methoxy - 5 -methyl 5 - ( 4 - fluorophenyl) isoxazole - 4 - carboxylate , ethyl 5 - ( 4 - fluo 4 , 5 - dihydronaphtho [ 2 , 1 - d ] isoxazole , 5 -methyl - 3 -phenyl rophenyl) isoxazole - 3 - carboxylate , ethyl 5 - (2 , 3 -dihyd isoxazole- 4 -carboxylic acid methylamide , 5 -methyl - 3 robenzo [ b ][ 1 , 4 ]dioxin - 7 - yl) isoxazole - 3 - carboxylate , ethyl phenylisoxazole - 4 - carbothioic acid methylamide, 3 -( 4 - chlorophenyl) - 5 -( trifluoromethyl) isoxazole -4 - car 5 -methyl - 3 -phenyl - 4 -( 1h - pyrazol- 5 - yl) isoxazole , 5 -benzyl boxylate , ethyl 5 - ( 4 - chlorophenyl) isoxazole - 3 - carboxylate , 3 - furan - 2 - yl- 2 - phenyl- tetrahydro -pyrrolo ( 3 ,4 - d ) isoxazole ethyl 3 - ( 4 - bromophenyl ) - 5 - ( trifluoromethyl) isoxazole - 4 4 ,6 -dione , 5 -benzyl - 3 - [ 4 - ( dimethylamino ) phenyl ] - 2 -phe carboxylate , ethyl 5 - (4 -bromophenyl ) isoxazole- 3 -carboxy nyldihydro - 2h - pyrrolo [3 ,4 -d ] isoxazole -4 , 6 ( 3h ,5h ) -dione , late , ethyl 5 - amino - 4 - ( 4 - chlorophenyl) isoxazole - 3 - carboxy 5 -benzyl - 3 - ( 5 -br - 2 -ho -phenyl ) - 2 - ph - tetrahydro -pyrrolo ( 3 , 4 late , ethyl 5 - amino - 4 - ( 4 - bromophenyl) isoxazole - 3 d ) isoxazole - 4 ,6 - dione, 5 -benzyl - 3 - ( 4 -nitro -ph ) - 2 - ph - di carboxylate , ethyl 6b -acetyl - 2 -( acetyloxy )- 4a ,6a -dimethyl hydro - 2h - pyrrolo ( 3 ,4 - d ) isoxazole -4 , 6 ( 3h ,5h )- dione , 5 -ben 2 , 3 ,4 , 4a ,4b , 5 ,6 ,6a , 6b , 9a, 10 , 102 , 10b , 11 - tetradecahydro - 1 zyl- 3 - (4 -methoxy -phenyl ) - 2 -ph -tetrahydro -pyrrolo ( 3 ,4 - d ) h -naphtho [ 2 ' , 1 ' : 4 , 5 ] indeno [ 2 , 1 - d ] isoxazole - 9 - carboxylate , isoxazole - 4 , 6 - dione, 5 -benzyl - 3 - ( 4 - fluorophenyl) - 2 - ( 2 3 ,5 -dimethyl - 4 -( tributylstannyl ) isoxazole , 5 -( 1 ,5 - dimethyl methylphenyl) dihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole- 4 , 6 (3h , 1h -pyrazol - 4 -yl ) - isoxazole - 3 -carboxylic acid , 5 - ( 1, 3 - dim 5h ) -dione , 5 -benzyl - 3 - ( 3 -nitro - ph ) - 2 - phenyl- tetrahydro ethyl - 1h -pyrazol - 4 - yl) - isoxazole - 3 -carboxylic acid , 5 - ( 1 , 5 pyrrolo ( 3 , 4 - d ) isoxazole- 4 ,6 - dione , 5 -benzyl - 2 - ph - 3 -( 2 dimethyl - 1h -pyrazol - 4 -yl ) - isoxazole , 3 , 5 pyridinyl) dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 ( 3h , 5h ) dimethylisoxazole - 4 -boronic acid pinacol ester, 3 , 5 dione , 5 -benzyl - 2 - ph - 3 - ( 2 - ph - vinyl) dihydro - 2h - pyrrolo ( 3 , dimethylisoxazole , 3 -( dimethylamino )- 1 -( 2 -pyridyl ) - 2 4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) - dione , 5 -benzyl - 2 - ( 4 propen - 1 -one , 5 - ( 3 , 5 -difluorophenyl ) isoxazole , [ 2 ,6 chlorophenyl) - 3 - ( 2 - thienyl) dihydro - 2h -pyrrolo [ 3 , 4 - d ] dichloro -4 - ( trifluoromethyl) phenyl] hydrazine , 5 - (2 , 5 isoxazole - 4 ,6 (3h ,5h )- dione , 5 -benzyl - 2, 3 -diphenyldihydro dichlorophenyl) isoxazole - 3 - carboxylic acid , danazol, 3 - ( 4 2h - pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 ( 3h , 5h )- dione , 5 -benzyl - 2 ( 4 chlorophenyl) -5 - (trifluoromethyl ) isoxazole- 4 -carboxylic cl- ph )3 - ( 2 - furyl) dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 acid , 5 - ( 4 -chlorophenyl ) isoxazole - 3 -propionic acid , 5 - ( 4 (3h ,5h )dione , 5 -benzyl - 2 (4 -cl - ph )- 3 -( 4 -f - ph )dihydro -2h chlorophenyl) isoxazole - 4 -carboxylic acid , 5 - (4 - chlorophe pyrrolo ( 3 ,4 -d )isoxazole -4 ,6 (3h ,5h )dione , 5 -( p -tolyl ) nyl) isoxazole - 3 -carboxylic acid , 3 -( 4 -chlorophenyl ) isox isoxazole , 5 -( 4 -methylphenyl ) - 3 - (4 - nitrophenyl) - 2 azole - 5 - carboxylic acid , 3 - ( 3 - chlorophenyl) isoxazole - 5 phenyldihydro - 2h - pyrrolo [ 3 , 4 - d ]isoxazole - 4 ,6 ( 3h , 5h ) carboxylic acid , 5 - ( 4 - chlorophenyl) isoxazole - 3 dione , 5 - (4 -methoxyphenyl ) - 2 - phenyl- 3 - ( 4 - pyridinyl) carboxaldehyde, 3 - (4 -chlorophenyl ) isoxazole - 5 dihydro - 2h -pyrrolo [ 3 ,4 - d ] isoxazole - 4 ,6 (3h ,5h )- dione , 5 -( 4 carbaldehyde, 3 -( 3 - chlorophenyl) isoxazole - 5 -carbaldehyde , methoxyphenyl) - 2 - phenyl- 3 - ( 3 -pyridinyl ) dihydro - 2h 3 -( 2 - chlorophenyl) isoxazole - 5 - carbaldehyde, 5 - ( 4 -chloro pyrrolo [ 3, 4 -d ]isoxazole -4 ,6 ( 3h ,5h )- dione , 5 -( 4 -methoxy phenyl) isoxazole , 3 - ( 4 - chlorophenyl ) isoxazole , 5 - ( chlorom ph ) - 2 , 3 - diphenyldihydro - 2h -pyrrolo ( 3 ,4 - d )isoxazole -4 , 6 ethyl) isoxazole - 4 -carboxylic acid , 3 - (chloromethyl ) - 5 -( 2 ( 3h ,5h ) - dione , 5 - ( 4 - fluorophenyl) - 3 - ( 4 -methoxyphenyl ) - 2 furyl) isoxazole , 4 - chloromethyl- 3 , 5 - dimethylisoxazole , phenyldihydro - 2h - pyrrolo [ 3 ,4 - d ] isoxazole - 4 , 6 (3h ,5h ) 5 - ( chloromethyl) - 3 - ( 4 - chlorophenyl) isoxazole , 5 - ( 3 - chloro dione, 5 - ( 4 - fluorophenyl ) - 2 - ( 2 -methylphenyl ) - 3 - ( 4 4 -methoxy -phenyl ) -isoxazole - 3 -carboxylic acid , 3 -chloro pyridinyl) dihydro - 2h - pyrrolo [ 3 , 4 - d ]isoxazole - 4 , 6 ( 3h , 5h ) 4 - fluorobenzaldehyde , 3 - ( 5 - chloro - 2 ,4 - dimethoxy - phenyl) dione, 5 - ( 4 - ethoxyphenyl) - 3 - ( 4 - nitrophenyl) - 2 4 , 5 - dihydro - isoxazole - 5 - carboxylic acid , 5 - tert - butyl - 4 , 5 , 6 , phenyldihydro -2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h , 5h ) 7 -tetrahydro -benzo [ d ]isoxazole - 3 -carboxylic acid , 3 - ( 4 dione , 5 - (4 -ethoxyphenyl ) - 3 -( 4 - fluorophenyl) - 2 bromophenyl) - 5 - ( trifluoromethyl ) isoxazole - 4 -carboxylic phenyldihydro - 2h - pyrrolo [3 ,4 - d ] isoxazole -4 ,6 ( 3h ,5h ) acid , 5 - (4 -bromophenyl ) isoxazole - 3 -propionic acid , 5 - ( 4 dione , 5 - ( 4 - ethoxyphenyl) - 2 -methyl - 3 - ( 4 -nitrophenyl ) bromophenyl ) isoxazole - 3 - carboxylic acid hydrazide , 5 - ( 4 dihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) -dione , 5 - ( 4 bromophenyl) isoxazole- 4 -carboxylic acid , 5 - ( 4 -bromophe ethoxy - ph ) - 2 - ph - 3 - thiophen - 2 -yl - 4h - pyrrolo ( 3 , 4 - d ) US 2018 /0273906 A1 Sep . 27 , 2018 27

isoxazole - 4 , 6 -dione , 5 - ( 4 - cl- ph ) - 3 - ( 3 -nitro - ph ) - 2 -phenyl tolyl- tetrahydro -pyrrolo (3 ,4 - d ) isoxazole - 4 ,6 -dione , 2 -( 4 tetrahydro -pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 - dione , 5 - ( 4 chlorophenyl) - 5 - ( 4 -methylphenyl ) - 3 - ( 2 - thienyl) dihydro - 2h bromophenyl ) - 3 - ( 4 -methoxyphenyl ) - 2 - phenyldihydro - 2h pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h , 5h ) - dione, 2 -( 4 pyrrolo [3 ,4 - d ] isoxazole - 4 ,6 (3h ,5h ) -dione , 5 - ( 4 chlorophenyl) - 3 - [ 4 - ( dimethylamino ) phenyl) - 5 bromophenyl ) -3 - (2 - furyl) - 2 -( 2 -methylphenyl ) dihydro -2h phenyldihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 (3h ,5h ) pyrrolo [ 3, 4 -d ]isoxazole - 4, 6 (3h , 5h ) -dione , 5 -( 4 dione , 2 -( 4 -chlorophenyl ) - 3 -( 4 - fluorophenyl) - 5 -( 4 bromophenyl) -2 - phenyl- 3 -( 2- pyridinyl) dihydro -2h -pyrrolo nitrophenyl) dihydro -2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 (3h , [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) -dione , 5 - ( 4 -br - ph ) 2 - ph - 3 - ( 2 - ph 5h ) - dione, 2 - ( 4 -chlorophenyl ) - 3 - ( 2 - thienyl) - 5 - [ 3 vinyl) dihydro - 2h -pyrrolo ( 3 , 4 - d )isoxazole - 4 ,6 ( 3h , 5h )dione , ( trifluoromethyl ) phenyl] dihydro - 2h - pyrrolo [ 3 , 4 - d ] 5 -( 2 - cl -ph )- 3 - (4 - dimethylamino -ph ) 2 - o - tolyl- 4h -pyrrolo (3 , isoxazole - 4 , 6 ( 3h , 5h ) - dione, 2 - ( 4 - chlorophenyl ) - 3 - ( 2 , 4 4 - d ) isoxazole - 4 ,6 - dione , 5 - ( 2 - chlorophenyl) - 3 - [ 4 - ( dimeth dichlorophenyl) - 5 -phenyldihydro - 2h -pyrrolo [ 3 , 4 - d ] ylamino )phenyl ] -2 -phenyldihydro - 2h - pyrrolo [ 3 ,4 -d ] isox isoxazole - 4 , 6 ( 3h , 5h ) - dione, 2 , 3 - di- ph - 5 - ( 3 - ( tri- f -me ) ph ) azole - 4 ,6 ( 3h , 5h ) - dione, 5 - ( 2 - chlorophenyl ) - 3 - ( 4 dihydro - 2h -pyrrolo ( 3 ,4 -d ) isoxazole - 4 ,6 ( 3h ,5h )- dione , methoxyphenyl ) - 2 - phenyldihydro - 2h -pyrrolo [ 3 ,4 - d ] danazol, and n -cyclopropyl - 5 -( thiophen - 2 -yl ) isoxazole - 3 isoxazole - 4 , 6 ( 3h , 5h ) -dione , 4 - ( 3 - ( 2 - cl- ph ) - 5 -methyl carboxamide . isoxazole - 4 -carbonyl ) -amino ) -benzoic acid ethyl ester, 4 , 5 , [0247 ] In one aspect, the effective amount comprises an 6 ,6a - tetrahydro - 3ah - cyclopenta [ d ] isoxazole - 3 - carboxylic amount that results in overexpression of the miRNA , such as acid , 3 - phenyl -3a , 6a - dihydrothieno [ 2 , 3 - d ]isoxazole 4 , 4 - di for example , about 5 uM to about 25 uM . oxide , 3 -methyl - 5 - ( 3 -phenylpropyl ) isoxazole , 3 -methyl - 4 [0248 ] In a further aspect, the cells are cultured in serum nitro - 5 -[ (e ) - 2 -phenylethenyl ] isoxazole , 3 -methyl - 4 ,5 ,8 , 9 free media . tetrahydrocycloocta ( d ) isoxazole , 3 -methyl - 4 , 5 , 5a ,ba , 7 , 8 hexahydrooxireno ( 2 ', 3 ' :5 , 6 ) cycloocta ( 1 , 2 - d ) isoxazole , [0249 ] In one aspect, the stem cell or progenitor cell is 3 -methyl - 3a, 4 , 5 , 8 , 9 , 9a - hexahydrocycloocta ( d ) isoxazole , selected from the group of an iPSC - derived cardiac progeni 3 - furan - 2 - yl- 2 - phenyl - 5 - p -tolyl - tetrahydro -pyrrolo ( 3 ,4 - d ) tor cell or an iPSC - derived embryoid body. Methods to isoxazole -4 ,6 -dione , 3 -chloro - 4, 5 -dihydro ( 1 )- benzothiepino derive iPSC - cardiac progenitor cells from stem cells are ( 5 , 4 - c ) isoxazole , 3 - [ 4 - ( dimethylamino )phenyl ] - 5 - ( 4 known in the art and described herein . Methods to derive methoxyphenyl) - 2 - ( 2 - methylphenyl) dihydro - 2h -pyrrolo [ 3 , stem cells from an iPSC -derived embryoid body are known 4 - d ] isoxazole - 4 ,6 ( 3h , 5h ) - dione , 3 - ( 5 -br - 2 -ho -phenyl ) - 2 , 5 in the art as described for example in Michael W . Nestor , et diphenyl- tetrahydro - pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 -dione , al. , Differentiation of serum - free embryoid bodies from 3 -( 5 -br -2 - ho -ph )- 5 -( 2 -cl - ph )- 2- ph -tetrahydro -pyrrolo ( 3, 4 -d ) human induced pluripotent stem cells into networks, In Stem isoxazole - 4 ,6 - dione, 3 - ( 4 -meo - phenyl) - 5 -phenyl - 2 - 0 - tolyl Cell Research , Volume 10 , Issue 3 , 2013 , Pages 454 - 463 , tetrahydro -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 -dione , 3 - ( 4 - fluoro ISSN 1873 - 5061, https: // doi. org / 10 . 1016 / j . scr. 2013 .02 .001 . ; phenyl) - 5 -( 4 - nitrophenyl )- 2 -phenyldihydro -2h -pyrrolo [ 3 ,4 Steven D . Sheridan , et al ., “ Analysis of Embryoid Bodies dJisoxazole - 4 , 6 (3h , 5h ) -dione , 3 - ( 4 - fluorophenyl) - 5 - ( 4 Derived from Human Induced Pluripotent Stem Cells as a methylphenyl) - 2 - phenyldihydro - 2h - pyrrolo [ 3 , 4 - d ] Means to Assess Pluripotency, ” Stem Cells International , isoxazole- 4 ,6 (3h ,5h )- dione , 3 -( 4 - dimethylamino -ph )- 5 - ph vol. 2012 , Article ID 738910 , 9 : 2012 ; Heming Wei, et al. , 2 -o -tolyl -4h -pyrrolo (3 , 4 -d )isoxazole -4 ,6 -dione , 3 -( 4 " One - step derivation of cardiomyocytes and mesenchymal fluorophenyl) - 5 - ( 4 -methoxyphenyl ) - 2 - phenyldihydro - 2h stem cells from human pluripotent stem cells ” Stem Cell pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h ,5h ) - dione, 3 - ( 4 -br - ph ) - 2 - ph Research , Volume 9 , Issue 2 , 2012 , Pages 87 - 100 , ; and Di 5 - ( 2 - trifluoromethyl- ph ) -4h -pyrrolo ( 3 , 4 - d )isoxazole - 4 , 6 Pasquale E . , et al. , J . Vis Exp . 2013 Jun . 28 ; ( 76 ) . “ Genera dione , 3 - ( 3 - nitro - phenyl) - 2 , 5 - diphenyl- tetrahydro -pyrrolo tion of human cardiomyocytes: a differentiation protocol ( 3 , 4 - d ) isoxazole - 4 , 6 - dione , 3 - ( 3 -br - phenyl ) - 2 , 5 from feeder - free human induced pluripotent stem cells ” diphenyldihydro -2h -pyrrolo ( 3 ,4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) [0250 ] In one aspect, the isoxazole compound is selected dione, 3 - ( 3 -br - ph ) - 5 - ( 2 -meo -ph ) - 2 - 0 - tolyl - tetrahydro from isoxazole- 1 (isx - 1) or isoxazole - 9 (isx - 9 ). In another pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 - dione, 3 - ( 2 - furyl) - 5 - [ 4 - ( 4 aspect, the isoxazole derivative is compound of the formula : morpholinyl) phenyl ] - 2 - phenyldihydro - 2h -pyrrolo [ 3 , 4 - d ] isoxazole -4 ,6 (3h ,5h )- dione , 3 -( 2 - furyl) - 2 -( 2- me - ph )- 5 -ph dihydro -2h -pyrrolo ( 3 ,4 - d ) isoxazole- 4, 6 (3h ,5h )- dione, 3 -( 2 furyl) -2 , 5 - diphenyldihydro -2h -pyrrolo ( 3 ,4 - d ) isoxazole - 4 ,6 R2 ( 3h ,5h ) - dione, 3 - ( 2 - cl- phenyl) - 5 -methyl - isoxazole - 4 carboxylic acid ( 2 ,5 - dichloro -phenyl ) - amide , 3 -( 2 -cl - ph )- 5 z me- isoxazole - 4 -carboxylic acid ( 4 , 5 - dihydro - thiazol - 2 - yl) amide , 3 - ( 2 - chloro -phenyl ) - 5 -methyl - isoxazole - 4 carboxylic acid cyanomethyl- amide , 3 -( 2 ,4 dichlorophenyl) - 5 - ( 4 -methoxyphenyl ) - 2 -phenyldihydro - 2h pyrrolo [ 3, 4 -d ]isoxazole -4 ,6 ( 3h ,5h )- dione , 3 -( 2 ,4 -di - cl - ph ) 2 , 5 -diphenyldihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 ( 3h , wherein R , and R , are both hydrogen or R , is hydrogen and 5h ) -dione , 3 - (2 ,2 -dichloro -vinyl ) - 5 -phenyl - isoxazole , 3, 5 R2 is selected from the group consisting of substituted or diphenyl- isoxazole , 3 , 5 -dimethyl - 4 - ( 1 - pyrrolidinylsulfonyl) unsubstituted C , - C . alkyl, C3 - C . cycloalkyl , C2- C . alkenyl , isoxazole , 3 ( 4 - dimethylamino - ph ) - 5 - ( 4 -eto - ph ) 2 - o - tolyl- 4h C2 - C , alkynyl, and benzyl, or where R1 and R , may be pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 -dione , 2 - ( 4 - cl- ph ) 5 - ph - 3 - ( 2 joined together to form a ring selected from azetidinyl, thienyl )dihydro - 2h -pyrrolo ( 3 , 4 - d )isoxazole - 4 , 6 ( 3h , 5h ) pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl; R2' , dione, 2 -( 4 -cl -ph )- 5 - (3 -meo -ph ) -3 - ( 3 -nitro -ph )- 4h -pyrrolo Rz and R4 are independently selected from the group con ( 3, 4 -d ) isoxazole - 4 ,6 -dione , 2 - (4 - cl -ph ) -3 - (4 -meo -ph )- 5 -p - sisting of hydrogen , halogen , C .- C . alkyl, Cz -Co cycloalkyl, US 2018 /0273906 A1 Sep . 27 , 2018

substituted or unsubstituted aromatic or heteroaromatic ring , isoxazole - 3 -carboxaldehyde , 3 -( 4 -methoxyphenyl ) cyano , nitro , and acyl; X is 0 , NH or S ; and Y is 0 , NH or isoxazole - 5 - carbaldehyde , 3 - ( 2 -methoxyphenyl ) isoxazole 5 - carbaldehyde , 5 - ( 4 -methoxyphenyl ) isoxazole , 3 - ( 4 [0251 ] In a yet further aspect, the isoxazole compound has methoxyphenyl ) isoxazole , 3 - ( 2 -methoxy - phenyl) - 4 , 5 the formula : dihydro - isoxazole -5 -carboxylic acid , 3 -methoxy - isoxazole 5 -carboxylic acid , isoxazole -5 -carboxylic acid, isoxazole -4 carboxylic acid , isoxazole - 5 - carbothioamide , isoxazole - 5 carbonyl chloride, isoxazole - 3 - carbonitrile , isoxazole - 3 N carbaldehyde, isoxazole - 4 -boronic acid , isoxazole , 5 -cyclopropyl - 4 -[ 2 - (methylsulfonyl ) -4 - ( trifluoromethyl) IKA benzoyl] isoxazole , 6 - (5 -( thiophen -2 -yl ) isoxazole- 3 -carbox Rz R2 R5R5 amido ) hexyl 5 - ( ( 3as, 4s, bar )- 2 -oxohexahydro - 1h -thieno [ 3 , 4 -djimidazol - 4 -yl ) pentanoate , isocarboxazid 5 -methyl - 3 isoxazole- carboxylic acid 2 -benzylhydrazide , 5 - isobutyl wherein R1 and R2 is each selected from C1 - C4 alkyl, isoxazole - 3 -carboxylic acid , 4 - iodo - 5 -methyl - isoxazole, phenyl, benzyl, trifluoromethyl or halogen , R3 is selected 3 , 3 ' - iminobis ( n , n - dimethylpropylamine ), 3 - ( 3 -hydroxy from hydrogen , hydroxy, C1 - C4 alkyl or alkoxy , R4 , in phenyl) - isoxazole - 5 - carboxylic acid methyl ester , 5 - ( 4 - hy position 3 or 5 , is selected from hydrogen , trifluoromethyl, droxy - phenyl) - isoxazole - 3 - carboxylic acid , 5 - ( 3 -hydroxy C1 - C4 alkoxy, C1- C4 alkyl, or C1 -C4 hydroxyalkyl, R5 is phenyl ) - isoxazole - 3 - carboxylic acid , 5 - hydroxymethyl) - 3 selected from hydrogen or C4 - C4 alkyl or R4 and R5 methylisoxazole , 3 -hydroxy - 5 -methylisoxazole , 5 - ( 1 together form a tetramethylene group , Z at position 3 or 5 on hydroxyethyl) - 3 - (4 -trifluoromethylphenyl ) isoxazole , 3a, 4 , the heterocycle is selected from : — N (R6 ) - CO — , - CO 5 , 6 , 7 ,7a - hexahydro -benzo [ djisoxazole - 3 - carboxylic acid , N ( R6) -, _ N ( R6 ) CO _ NRG ) - , CH( RF ) - NHCO … , 5 - ( 2 - furyl) isoxazole - 3 - carbaldehyde, 5 - furan - 2 - yl- isox or - NH CO CH (RO ) , in which R6 is selected from azole - 3 - carboxylic acid , 6 - fluoro - 3 -( 4 -piperidinyl ) ben hydrogen or C1- C4 alkyl. zisoxazole , 5 - ( 4 - fluorophenyl) isoxazole - 3 - methanol, 3 - ( 2 [0252 ] In a yet further aspect, the isoxazole compound or fluoro -phenyl ) - isoxazole - 5 -carboxylic acid , 5 - ( 4 a derivative thereof is selected from the group : 5 - ( trifluo fluorophenyl) isoxazole - 3 - carboxaldehyde, 3 - ( 4 romethyl) - 3 - ( 4 -methoxyphenyl ) isoxazole - 4 - carboxylic fluorophenyl) isoxazole - 5 -carbaldehyde , 3 -( 3 - fluorophenyl) acid , 5 - (trifluoromethyl ) - 3 - ( 4 - fluorophenyl) isoxazole - 4 isoxazole - 5 -carbaldehyde , 3 - ( 2 - fluorophenyl) isoxazole - 5 carboxylic acid , 5 - ( thiophen - 2 - yl) isoxazole - 3 - carboxalde carbaldehyde, 5 -( 4 - fluorophenyl) isoxazole , 3 -( 4 hyde , 5 , 6 , 7 , 8 - tetrahydro -4h - cyclohepta [ d ]isoxazole - 3 - car fluorophenyl) isoxazole , 5 - ( 3 - fluoro - 4 -methoxy -phenyl ) boxylic acid , 4 ,5 ,6 , 7 - tetrahydro -benzo [d ] isoxazole - 3 isoxazole -3 - carboxylic acid , ethyl 5 -( trifluoromethyl) - 3 -( 4 carboxylic acid , 3 - amino - 5 -methylisoxazole , 4 - amino - n - ( 5 methoxyphenyl) isoxazole -4 -carboxylate , ethyl- 5 methyl- 3 - isoxazolyl) benzenesulfonamide , 3 -phenyl (tributylstannyl ) isoxazole- 3 - carboxylate , ethyl 5 - ( thiophen isoxazole - 5 - boronic acid pinacol ester , 5 - phenylisoxazole , 2 - yl) isoxazole - 3 - carboxylate, 5 - ethyl- isoxazole - 4 1 - phenyl- 1 -cyclopentanecarboxylic acid , 3 -phenyl - benzo [ c ] carboxylic acid , 5 - ethyl- isoxazole - 3 - carboxylic acid , ethyl isoxazole -5 -carboxylic acid , 5 -methyl - 3 - phenylisoxazole - 4 5 -( 4 - fluorophenyl) isoxazole - 4 - carboxylate , ethyl 5 - ( 4 - fluo carboxylic acid , 3a ,4 , 5 ,6 , 7 ,8 , 9 , 9a - octahydro - cycloocta [ d ] rophenyl) isoxazole - 3 - carboxylate , ethyl 5 - ( 2 , 3 - dihyd isoxazole - 3 - carboxylic acid , 5 - ( 3 - nitrophenyl) isoxazole , robenzo [ b ] [ 1 , 4 ] dioxin - 7 -yl ) isoxazole - 3 - carboxylate , ethyl 3 -( 4 -nitrophenyl ) isoxazole , 3 -hydroxy -5 - aminomethyl 3 -( 4 - chlorophenyl) - 5 -( trifluoromethyl) isoxazole -4 - car isoxazole , 5 - (morpholinomethyl ) isoxazole - 3 - carboxylic boxylate , ethyl 5 - ( 4 -chlorophenyl ) isoxazole - 3 - carboxylate , acid hydrochloride, 5 - (morpholinomethyl ) isoxazole - 3 - carb ethyl 3 - ( 4 -bromophenyl ) - 5 - ( trifluoromethyl) isoxazole - 4 aldehyde, 3 -methyl - 5 - (trifluoromethyl ) isoxazole - 4 - carbox carboxylate, ethyl 5 - ( 4 -bromophenyl ) isoxazole - 3 - carboxy ylic acid , methyl 5 - (thiophen - 2 - yl ) isoxazole - 3 -carboxylate , late , ethyl 5 -amino - 4 - ( 4 -chlorophenyl ) isoxazole - 3 -carboxy 3 - (methylsulfonyl ) - 5 - ( 2 - thienyl) isoxazole - 4 -carbonitrile , late , ethyl 5 - amino - 4 - ( 4 - bromophenyl) isoxazole - 3 5 -methyl - 3 -( 2 -pyrrolidinyl ) isoxazole , 3 -methyl - 5 -( 2 - pyrro carboxylate , ethyl 6b -acetyl - 2 - ( acetyloxy ) - 4a ,6a - dimethyl lidinyl) isoxazole , 3 - ( 1 -methyl - 1h - pyrazol- 4 -yl ) - isoxazole 2 , 3 , 4 , 4a ,4b , 5 , 6 ,6a , 6b ,9a , 10 , 10a ,106 , 11 - tetradecahydro - 1 5 - carboxylic acid , 3 - ( 1 -methyl - 1h -pyrazol - 4 -yl ) - 4 , 5 - di h -naphtho [ 2 ', 1 ": 4 , 5 ] indeno [ 2 , 1 - d ] isoxazole - 9 - carboxylate , hydro - isoxazole - 5 - carboxylic acid , 5 - ( 4 -methylphenyl ) 3 , 5 - dimethyl - 4 - (tributylstannyl ) isoxazole , 5 - ( 1 , 5 - dimethyl isoxazole - 3 - carboxylic acid , 5 -methyl - 3 -phenylisoxazole - 4 1h -pyrazol - 4 -yl ) - isoxazole - 3 -carboxylic acid , 5 -( 1 , 3 -dim carboxylic acid , 5 -( 4 -methylphenyl ) isoxazole - 3 ethyl - 1h - pyrazol - 4 - yl) - isoxazole - 3 - carboxylic acid , 5 - ( 1 , 5 carboxaldehyde , 5 -methyl - 3 - (4 -phenoxyphenyl ) isoxazole dimethyl -1h -pyrazol - 4 -yl ) - isoxazole , 3, 5 4 - carboxylic acid , 3 -methyl - 5 - ( 4 -methyl - 1 , 2 , 3 -thiadiazol - 5 dimethylisoxazole - 4 - boronic acid pinacol ester, 3 , 5 yl) isoxazole -4 - carboxylic acid , 3 -methyl - 5 -( 5 dimethylisoxazole , 3 -( dimethylamino )- 1 -( 2 -pyridyl ) - 2 methylisoxazol- 3 -yl ) isoxazole - 4 -carboxylic acid , methyl propen - 1 - one , 5 - ( 3 , 5 - difluorophenyl) isoxazole, [ 2 ,6 5 - ( 4 -methoxyphenyl ) isoxazole- 4 - carboxylate , methyl 5 - ( 4 dichloro - 4 -( trifluoromethyl) phenyl] hydrazine , 5 -( 2 ,5 methoxyphenyl) isoxazole -3 - carboxylate , 5 -methylisox dichlorophenyl) isoxazole- 3 - carboxylic acid , danazol, 3 - ( 4 azole , methyl 5 - ( 4 - fluorophenyl) isoxazole - 4 -carboxylate , chlorophenyl) -5 -( trifluoromethyl ) isoxazole - 4 - carboxylic methyl 5 - ( 4 - fluorophenyl) isoxazole - 3 - carboxylate , methyl acid , 5 - ( 4 - chlorophenyl) isoxazole - 3 - propionic acid , 5 - ( 4 5 - ( 4 - chlorophenyl) isoxazole - 4 - carboxylate, methyl 5 - ( 4 chlorophenyl) isoxazole - 4 -carboxylic acid , 5 - ( 4 - chlorophe bromophenyl) isoxazole - 4 - carboxylate , 5 - ( 4 -methoxyphe nyl ) isoxazole - 3 - carboxylic acid , 3 - ( 4 - chlorophenyl) isox nyl) isoxazole - 3 -carboxylic acid , 5 - ( 3 -methoxy -phenyl ) azole - 5 -carboxylic acid , 3 - (3 - chlorophenyl) isoxazole - 5 isoxazole - 3 - carboxylic acid , 3 - ( 2 -methoxyphenyl ) carboxylic acid , 5 - ( 4 -chlorophenyl ) isoxazole - 3 isoxazole -5 -carboxylic acid , 5 -( 4 -methoxyphenyl ) carboxaldehyde, 3 - ( 4 - chlorophenyl) isoxazole -5 US 2018 /0273906 A1 Sep . 27 , 2018 carbaldehyde, 3 - ( 3 - chlorophenyl ) isoxazole - 5 - carbaldehyde , methoxyphenyl) - 2 -phenyl - 3 - (3 -pyridinyl ) dihydro - 2h 3 - ( 2 - chlorophenyl) isoxazole - 5 - carbaldehyde, 5 - ( 4 -chloro pyrrolo [ 3 ,4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) - dione, 5 - ( 4 -methoxy phenyl) isoxazole , 3 - ( 4 - chlorophenyl ) isoxazole , 5 - ( chlorom ph ) - 2 , 3 - diphenyldihydro - 2h - pyrrolo ( 3 ,4 - d )isoxazole - 4 , 6 ethyl) isoxazole - 4 - carboxylic acid , 3 - (chloromethyl ) - 5 - ( 2 ( 3h , 5h )- dione, 5 - ( 4 - fluorophenyl) - 3 - ( 4 -methoxyphenyl ) - 2 furyl) isoxazole , 4 - chloromethyl- 3 , 5 -dimethylisoxazole , phenyldihydro - 2h -pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h , 5h ) 5 -( chloromethyl ) -3 - ( 4 -chlorophenyl ) isoxazole , 5 - (3 -chloro dione , 5 - (4 - fluorophenyl) - 2 -( 2 -methylphenyl ) - 3 - ( 4 4 -methoxy - phenyl) - isoxazole - 3 - carboxylic acid , 3 - chloro pyridinyl) dihydro - 2h -pyrrolo [ 3 ,4 -d ]isoxazole -4 ,6 ( 3h ,5h ) 4 - fluorobenzaldehyde , 3 - 5 - chloro - 2 , 4 -dimethoxy -phenyl ) dione, 5 - ( 4 - ethoxyphenyl) - 3 - ( 4 - nitrophenyl) - 2 4 , 5 - dihydro - isoxazole - 5 - carboxylic acid , 5 - tert - butyl - 4 , 5 , 6 , phenyldihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) 7 -tetrahydro -benzo [ d ]isoxazole - 3 -carboxylic acid , 3 - (4 dione , 5 - ( 4 - ethoxyphenyl) - 3 - ( 4 - fluorophenyl) - 2 bromophenyl) -5 - (trifluoromethyl ) isoxazole -4 -carboxylic phenyldihydro - 2h - pyrrolo [ 3 ,4 - d ] isoxazole -4 ,6 ( 3h ,5h ) acid , 5 - ( 4 - bromophenyl ) isoxazole - 3 - propionic acid , 5 - ( 4 dione , 5 - (4 -ethoxyphenyl ) - 2 -methyl - 3 - (4 -nitrophenyl ) bromophenyl) isoxazole - 3 - carboxylic acid hydrazide, 5 - ( 4 dihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) -dione , 5 - ( 4 bromophenyl) isoxazole- 4 -carboxylic acid , 5 - ( 4 -bromophe ethoxy -ph ) - 2 - ph - 3 - thiophen - 2 - yl- 4h -pyrrolo ( 3 , 4 - d ) nyl) isoxazole - 3 -carboxylic acid , 3 - ( 4 -bromophenyl ) isoxazole - 4 ,6 - dione , 5 - ( 4 - cl- ph ) - 3 - ( 3 -nitro - ph ) - 2 -phenyl isoxazole - 5 -carboxylic acid , 3 - ( 4 - bromophenyl) isoxazole tetrahydro -pyrrolo ( 3 ,4 -d ) isoxazole - 4 ,6 -dione , 5 - ( 4 5 - carboxaldehyde, 5 - ( 4 -bromophenyl ) isoxazole , 5 - ( 3 bromophenyl) - 3 - (4 -methoxyphenyl ) - 2 -phenyldihydro - 2h bromophenyl ) isoxazole , 3 - ( 4 - bromophenyl) isoxazole , pyrrolo [ 3 ,4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) - dione, 5 - (4 5 - (bromomethyl ) - 3 - ( 4 -methoxyphenyl ) isoxazole, 4 - (bro bromophenyl) - 3 - ( 2 - furyl) - 2 - ( 2 -methylphenyl ) dihydro - 2h momethyl) isoxazole , 5 - bromomethyl) - 3 - ( 4 - fluorophenyl) pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 (3h ,5h ) -dione , 5 - ( 4 isoxazole , 5 - (bromomethyl ) - 3 - ( 4 -chlorophenyl ) isoxazole , bromophenyl) - 2 -phenyl - 3 -( 2 -pyridinyl ) dihydro -2h -pyrrolo 5 - (bromomethyl ) - 3 - ( 4 -bromophenyl ) isoxazole , 6 -bromo - 3 [ 3 , 4 - d ] isoxazole- 4 , 6 ( 3h ,5h ) - dione, 5 - ( 4 -br - ph ) 2 -ph - 3 - ( 2 -ph methylbenzo [ d ] isoxazole , 5 - bromo- 3 -methylbenzo [ d ] isox vinyl )dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) dione , azole , 4 - bromo - 5 - ( 4 -methoxyphenyl ) isoxazole , 3 -bromo 5 - ( 2 - cl - ph ) - 3 - ( 4 - dimethylamino -ph ) 2 - 0 - tolyl- 4h -pyrrolo ( 3 , isoxazole , 3 -bromo - 5 - (2 -hydroxyethyl ) isoxazole , 4 -bromo 4 -d ) isoxazole - 4 ,6 -dione , 5 -( 2 -chlorophenyl ) -3 -[ 4 -( dimeth 5 - (4 -fluorophenyl ) isoxazole , 3 -bromo - 5 -( 4 - fluorophenyl) ylamino )phenyl ) -2 -phenyldihydro - 2h -pyrrolo [3 ,4 -d ] isox isoxazole , 4 - bromo - 5 - ( 4 - chlorophenyl) isoxazole , 4 -bromo azole - 4 , 6 ( 3h ,5h ) -dione , 5 - ( 2 - chlorophenyl) - 3 - ( 4 5 - ( 4 - bromophenyl) isoxazole , 6 - bromo- benzo [ d ] isoxazole methoxyphenyl) - 2 -phenyldihydro - 2h -pyrrolo [ 3 , 4 - d ] 3 - carboxylic acid , benzo [ d ] isoxazole - 3 - carboxylic acid , isoxazole - 4 , 6 ( 3h ,5h ) - dione , 4 - ( 3 -( 2 - cl - ph )- 5 -methyl 3 -amino - 5 -methylisoxazole , 5 -amino -3 -( 4 -methoxyphenyl ) isoxazole- 4 -carbonyl ) - amino )- benzoic acid ethyl ester, 4 , 5 , isoxazole , 3 - aminoisoxazole , 3 - amino - 5 - ( 4 - fluorophenyl) 6 ,6a - tetrahydro - 3ah - cyclopenta [ d ] isoxazole - 3 - carboxylic isoxazole , 5 - amino - 3 - ( 4 - chlorophenyl) isoxazole , 5 -amino acid , 3 -phenyl - 3a ,ba - dihydrothieno [ 2 , 3 - d ] isoxazole 4 , 4 - di 4 -( 4 -bromophenyl ) isoxazole , 3 -amino -5 - (4 -bromophenyl ) oxide , 3 -methyl -5 - ( 3 -phenylpropyl ) isoxazole, 3 -methyl - 4 isoxazole , 5 - acetyl- 3 - ( 4 - fluorophenyl) isoxazole , 5 - acetyl- 3 nitro - 5 -[ ( e )- 2 -phenylethenyl ] isoxazole , 3 -methyl - 4, 5 ,8 , 9 ( 3 - fluorophenyl) isoxazole , 3 - methyl- 5 - [ (25 ) - 1 -methyl - 2 tetrahydrocycloocta ( d ) isoxazole , 3 -methyl - 4 , 5 ,5a ,ba , 7 , 8 pyrrolidinyl ] isoxazole hydrochloride , 7 -methoxy - 5 -methyl hexahydrooxireno ( 2 , 3 ': 5 , 6 ) cycloocta ( 1 , 2 - d ) isoxazole , 4 ,5 -dihydronaphtho [2 , 1- d ] isoxazole , 5 -methyl - 3 -phenyl 3 -methyl - 3a ,4 ,5 ,8 ,9 ,9a -hexahydrocycloocta ( d )isoxazole , isoxazole - 4 - carboxylic acid methylamide , 5 -methyl - 3 3 - furan - 2 -yl - 2 -phenyl - 5 -p -tolyl - tetrahydro -pyrrolo (3 ,4 - d ) phenylisoxazole - 4 - carbothioic acid methylamide , isoxazole -4 , 6 -dione , 3 -chloro - 4 , 5 -dihydro ( 1 ) -benzothiepino 5 -methyl - 3 -phenyl - 4 -( 1h -pyrazol - 5 -yl ) isoxazole , 5 -benzyl ( 5 ,4 -c ) isoxazole , 3 - [ 4 -( dimethylamino )phenyl ] - 5 -( 4 3 - furan - 2 - yl- 2 - phenyl- tetrahydro -pyrrolo ( 3 , 4 - d ) isoxazole methoxyphenyl) - 2 - ( 2 -methylphenyl ) dihydro - 2h -pyrrolo [ 3 , 4 ,6 -dione , 5 -benzyl - 3 - [4 - (dimethylamino )phenyl ] - 2 -phe 4 -djisoxazole - 4 , 6 ( 3h ,5h ) - dione , 3 -( 5 -br - 2 -ho -phenyl ) - 2 , 5 nyldihydro - 2h - pyrrolo [ 3 , 4 - d ]isoxazole - 4 , 6 ( 3h , 5h )- dione , diphenyl- tetrahydro - pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 - dione , 5 -benzyl - 3 - ( 5 - br- 2 -ho - phenyl) - 2 -ph - tetrahydro - pyrrolo ( 3 , 4 3 - ( 5 - br- 2 -ho - ph ) - 5 - ( 2 - cl - ph ) - 2 - ph - tetrahydro -pyrrolo ( 3 , 4 - d ) d ) isoxazole -4 , 6 -dione , 5 -benzyl - 3 - (4 -nitro -ph )- 2 -ph -di isoxazole - 4 , 6 - dione , 3 - ( 4 -meo - phenyl) - 5 - phenyl- 2 - 0 - tolyl hydro -2h -pyrrolo (3 , 4 - d )isoxazole - 4 ,6 (3h , 5h )- dione, 5 -ben tetrahydro - pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 -dione , 3 - ( 4 - fluoro zyl- 3 - ( 4 -methoxy - phenyl) - 2 -ph - tetrahydro - pyrrolo ( 3 , 4 - d ) phenyl) - 5 -( 4 - nitrophenyl) -2 -phenyldihydro - 2h -pyrrolo [3 ,4 isoxazole -4 ,6 -dione , 5 -benzyl - 3 -( 4 - fluorophenyl) - 2 -( 2 d ]isoxazole -4 ,6 ( 3h ,5h )- dione , 3 -( 4 - fluorophenyl) - 5 -( 4 methylphenyl) dihydro - 2h -pyrrolo [ 3 ,4 - d ] isoxazole -4 , 6 (3h , methylphenyl) - 2 -phenyldihydro - 2h -pyrrolo [3 , 4 -d ] 5h ) - dione , 5 -benzyl - 3 - ( 3 -nitro -ph ) - 2 -phenyl - tetrahydro isoxazole -4 ,6 (3h ,5h )- dione , 3 -( 4 - dimethylamino -ph )- 5 - ph pyrrolo ( 3 ,4 -d )isoxazole -4 ,6 -dione , 5 -benzyl - 2 - ph - 3 -( 2 2 - 0 - tolyl- 4h - pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 - dione , 3 - ( 4 pyridinyl) dihydro -2h -pyrrolo (3 ,4 -d ) isoxazole - 4 ,6 (3h , 5h ) fluorophenyl) - 5 - ( 4 -methoxyphenyl ) - 2 -phenyldihydro - 2h dione , 5 -benzyl - 2 - ph - 3 -( 2 - ph - vinyl) dihydro - 2h - pyrrolo ( 3 , pyrrolo [ 3, 4 -d ]isoxazole -4 ,6 ( 3h ,5h )- dione , 3 - ( 4 -br - ph )- 2 -ph 4 -d ) isoxazole - 4 ,6 (3h ,5h )- dione , 5 -benzyl - 2 - (4 5 - ( 2 - trifluoromethyl- ph ) -4h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 chlorophenyl) - 3 - ( 2 - thienyl) dihydro - 2h -pyrrolo [ 3 , 4 - d ] dione, 3 -( 3 -nitro -phenyl ) - 2 ,5 -diphenyl - tetrahydro -pyrrolo isoxazole - 4 , 6 ( 34 , 5h ) - dione , 5 - benzyl- 2 , 3 - diphenyldihydro ( 3 ,4 - d ) isoxazole - 4 , 6 - dione , 3 - ( 3 -br -phenyl ) - 2 , 5 2h - pyrrolo ( 3 , 4 - d ) isoxazole- 4 , 6 (3h ,5h ) -dione , 5 -benzyl - 2 ( 4 diphenyldihydro - 2h - pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 ( 3h ,5h ) cl- ph ) 3 - ( 2 - furyl) dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 dione , 3 - ( 3 - br - ph ) - 5 - ( 2 -meo - ph )- 2 - 0 - tolyl - tetrahydro ( 3h , 5h )dione , 5 - benzyl- 2 (4 - cl - ph ) - 3 - ( 4 - f -ph ) dihydro - 2h pyrrolo ( 3 , 4 - d ) isoxazole- 4 ,6 - dione , 3 - ( 2 - furyl ) - 5 - [ 4 - (4 pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 (3h , 5h ) dione , 5 - (p - tolyl) morpholinyl) phenyl] - 2 -phenyldihydro - 2h -pyrrolo [ 3 , 4 - d ] isoxazole , 5 - ( 4 -methylphenyl ) - 3 - ( 4 - nitrophenyl) - 2 isoxazole - 4 , 6 ( 3h , 5h ) - dione, 3 - ( 2 - furyl) - 2 - ( 2 -me - ph ) - 5 -ph phenyldihydro -2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h , 5h ) dihydro -2h -pyrrolo ( 3 ,4 - d ) isoxazole - 4 ,6 (3h , 5h )- dione, 3 -( 2 dione, 5 - ( 4 -methoxyphenyl ) - 2 - phenyl- 3 - ( 4 -pyridinyl ) furyl ) - 2 , 5 - diphenyldihydro - 2h - pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 dihydro - 2h -pyrrolo [ 3 ,4 - d ] isoxazole- 4 ,6 ( 3h ,5h )- dione , 5- ( 4 ( 3h , 5h )- dione, 3 - ( 2 -cl - phenyl) - 5 -methyl - isoxazole - 4 US 2018 /0273906 A1 Sep . 27 , 2018 30 carboxylic acid ( 2, 5 -dichloro -phenyl ) - amide , 3 -( 2 -cl - ph )- 5 subject in need thereof. Methods to determine the effective me- isoxazole - 4 -carboxylic acid ( 4, 5 -dihydro -thiazol - 2 -yl ) ness of the therapy are known in the art , some of which are amide, 3 -( 2 -chloro -phenyl ) - 5 -methyl - isoxazole - 4 described herein . carboxylic acid cyanomethyl - amide, 3 - ( 2 , 4 [ 0256 ] In another aspect , the populations are prepared by dichlorophenyl) - 5 - ( 4 -methoxyphenyl ) - 2 - phenyldihydro - 2h culturing the cells in the presence of serum - free medium and pyrrolo [ 3 , 4 - d ] isoxazole - 4 , 6 ( 3h , 5h ) - dione , 3 - ( 2 , 4 - di- cl - ph ) a TGF -beta type - I receptor inhibitor, non - limiting examples 2 ,5 -diphenyldihydro -2h -pyrrolo ( 3 ,4 - d ) isoxazole -4 ,6 ( 3h , of such TGF -beta type - I receptor inhibitor include 5h )- dione , 3 - ( 2 , 2 - dichloro - vinyl ) - 5 - phenylisoxazole , 3 , 5 SB431542 and A8301, which are commercially available diphenyl - isoxazole , 3, 5 - dimethyl- 4 - (1 -pyrrolidinylsulfonyl ) from Cayman Chemical and Sigma Aldrich ; LY2157299 , isoxazole , 3 ( 4 -dimethylamino -ph ) -5 -( 4 -eto -ph ) 2 -o - tolyl- 4h which can be purchased from AdooQ Bioscience (Catalog No. A11017 ) ; and LY2109761, which can be purchased pyrrolo (3 ,4 - d ) isoxazole - 4 ,6 -dione , 2 -( 4 -cl -ph ) 5 -ph -3 - ( 2 from AdooQ Bioscience and Selleckchem (Catalog No . thienyl) dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 ,6 ( 3h , 5h ) S2704 ) . The cell populations can be heterogeneous , homo dione , 2 - ( 4 - cl - ph ) - 5 - ( 3 -meo - ph ) - 3 - ( 3 - nitro - ph ) -4h -pyrrolo geneous , substantially homogeneous or highly purified . In ( 3 , 4 - d ) isoxazole - 4 ,6 - dione , 2 - ( 4 - cl- ph ) - 3 - ( 4 -meo - ph ) - 5 - p one aspect, the populations comprise at least 80 % , or tolyl- tetrahydro -pyrrolo ( 3 ,4 - d ) isoxazole - 4 , 6 - dione, 2 - (4 alternatively 85 % , or alternatively 90 % , or alternatively chlorophenyl) - 5 -( 4 -methylphenyl )- 3 -( 2 -thienyl )dihydro - 2h 95 % , or alternatively 97 % or alternatively 99 % , or alterna pyrrolo [ 3 ,4 -d ] isoxazole- 4 ,6 (3h , 5h )- dione , 2 - ( 4 tively 100 % , of the cells express the exosomes or microve chlorophenyl) - 3 - [ 4 - ( dimethylamino ) phenyl] - 5 sicles . Methods to isolate the populations are known in the phenyldihydro -2h - pyrrolo [ 3 , 4 - d ] isoxazole - 4 ,6 ( 3h , 5h ) art , some of which are described herein . dione, 2 - ( 4 - chlorophenyl) - 3 - ( 4 - fluorophenyl) - 5 - ( 4 [0257 ] These cells and exosomes or microvesicles are nitrophenyl) dihydro - 2h - pyrrolo [ 3 , 4 - d ] isoxazole- 4 , 6 (3h , useful for regenerating cardiac muscle by administering to a 5h )- dione , 2 - ( 4 - chlorophenyl) - 3 - ( 2 - thienyl) - 5 - [ 3 subject in need thereof an effective amount of the population ( trifluoromethyl ) phenyl] dihydro - 2h - pyrrolo [ 3 , 4 - d ] or exosomes or microvesicles. They also are useful for isoxazole- 4 ,6 ( 3h ,5h ) - dione, 2 - ( 4 - chlorophenyl) - 3 - ( 2 , 4 treating cardiac dysfunction associated with Duchenne Mus dichlorophenyl) - 5 -phenyldihydro - 2h -pyrrolo [ 3, 4 - d ] cular Dystrophy (DMD ) , by administering to a subject in isoxazole - 4 , 6 ( 3h ,5h ) -dione , 2 , 3 -di - ph - 5 - ( 3 - ( tri- f -me ) ph ) need thereof an effective amount of the population of cells dihydro - 2h -pyrrolo ( 3 , 4 - d ) isoxazole - 4 , 6 ( 3h , 5h ) - dione, or the exosomes or microvesicles. Methods to determine the danazol, and n - cyclopropyl- 5 - ( thiophen - 2 - yl) isoxazole - 3 effectiveness of the therapy are known in the art, some of carboxamide. which are described herein . [ 0253] Also provided are cell populations prepared by the [ 0258 ] In a further aspect , the population of cells overex above methods as well as exosomes or microvesicles iso press at least skeletal myogenic genes selected from the lated and / or purified from these cell populations. The popu group of: Meox1 , Meox2 , Tcf15 , Pax3 , Pax7 , MyoDi, lations can be homogenous , heterogeneous, purified , highly dystrophin , Myf5 , or DESMIN . In one aspect , the popula purified , homogenous or substantially homogeneous . In one tions overexpress , at least one , or at least two, or at least aspect , at least 80 % , or alternatively 85 % , or alternatively three , or at least four , or at least five , or at least at least six , 90 % , or alternatively 95 % , or alternatively 97 % or alterna or at least seven , or at least eight, or at all nine eighteen tively 99 % , or alternatively 100 % , of the cells express the skeletal myogenic genes. The cells are prepared by culturing protein or proteins . or contacting an iPSC in the presence of an effective amount VMS. of Givinostat (GIV ) , and optionally in serum free media . The [ 0254 ] Further provided are cell populations and exo effective amount of GIV is from about 100 nM to about 30 somes or microvesicles isolated from these populations , nM per 1x10 cells . In a further aspect , the cells are also wherein the populations are cardiac or skeletal myogenic cultured in the presence of or contacted in the presence of an progenitors prepared from a population of human induced effective amount of ROCK inhibitors , non - limited examples pluripotent stem cells (hiPSCs ) by culturing the hiPSCs with of such include Thiazovivin , Y27632 , SR3677 , or an effective amount of Givinostat (GIV ) for an effective GSK429286 . In a yet further aspect, the cells are cultured (or amount of time. In one aspect the effective amount of GIV contacted ) in the presence of a TGF -beta type I inhibitor. comprises from about 100 nM to about 30 nM per 1x106 Non - limiting examples of such include SB431542 , A8301 , cells , or from about 100 nM to about 50 nM per 1x10 cells , LY2157299 , or LY2109761. The cell populations can be or from about 90 nM to about 30 nM per 1x100 cells , or from heterogeneous , homogeneous , substantially homogeneous about 30 nM to about 75 nM per 1x100 cells , or from about or highly purified . In one aspect, the populations comprise at 70 nM to about 50 nM per 1x100 cells . In a further aspect, least 80 % , or alternatively 85 % , or alternatively 90 % , or the cells and exosome or microvesicle populations are alternatively 95 % , or alternatively 97 % or alternatively prepared by methods that further comprise culturing the 99 % , or alternatively 100 % , of the cells express the genes . cells in the presence of serum - free medium and Rho [0259 ] The cells and /or exosomes or microvesicles iso associated kinase (ROCK inhibitors . Non - limiting lated from the cells are useful in methods for regenerating examples of the Rho - associated kinase (ROCK ) inhibitors skeletal muscle or for treating Duchenne Muscular Dystro are Thiazovivin or Y27632 , which are commercially avail phy (DMD ) by administering to a subject in need thereof an able from R & D Systems, Sigma Aldrich and Stemgent. effective amount of the population of cells and / or exosomes Methods to isolate the compositions are known in the art , or microvesicles isolated from the cells . Methods to deter some of which are described herein . mine the effectiveness of the therapy are known in the art , [0255 ] The cells and exosomes or microvesicles are useful some of which are described herein . in regenerating skeletal muscle or treating DMD by admin - (0260 ) In a further aspect, also provided is a population of istering to cells and /or exosomes or microvesicles to a cells that overexpress xESI myogenic genes selected from US 2018 /0273906 A1 Sep . 27 , 2018 31 the group of: Pitx2, ISL1, Nkx2 . 5 , Hand1 , GATA4 , Tbx5 , the compositions also contain a preservative and /or a cryo TnnT2 , My17 , MLC2v , Myf2c , Cdh4 , or Lhx2 as well as protectant. The compositions can be freeze - dried or exosomes or microvesicles isolated from the cells . In one lyophilized and provided in one or more dosage formula aspect, the populations overexpress , at least one , or at least tions. In one embodiment, the composition is intended for two , or at least three , or at least four, or at least five , or at therapeutic use and therefore , an effective amount of the least at least six , or at least seven , or at least eight, or at least modified cell , exosomes or microvesicles , miRNA , and nine , or at least ten , or at least eleven , or all twelve xESI populations are provided , alone or in combination with the myogenic genes . The cell or exosome or microvesicle popu isoxazole or isoxazole similar compound , in the composi lations can be heterogeneous , homogeneous, substantially tion . homogeneous or highly purified . In one aspect , the popula Methods for Preparing Certain Cell Populations, Exosomes tions comprise at least 80 % , or alternatively 85 % , or alter or Microvesicles and miRNA Populations , and Uses Thereof natively 90 % , or alternatively 95 % , or alternatively 97 % or [ 0265 ] Also provided herein aremethods for preparing an alternatively 99 % , or alternatively 100 % , of the cells over isolated population of exosomes or microvesicles that over express the genes. Methods to isolate cells overexpressing express a microRNA miRNA ) selected from the group of the one or more genes , and exosomes or microvesicles mir - 373 , mir -210 mir - 377 , mir - 367 , mir - 520 , mir -548ah , isolated from these populations are known n the art some of mir - 335 , mir - 30c , mir - 214 , or mir - 548q and /or one or more which are described herein . protein selected from Tsg101 , CD9, Hsp 70 , Flotillin - 1 , or [ 0261] The populations and exosomes or microvesicles GAPDH . In one aspect, at least 70 % , or 80 % , or 85 % , or are useful for regenerating cardiac muscle and / or for cardiac 90 % or 95 % , or 98 % of the cells express the stated mir dysfunction associated with Duchenne Muscular Dystrophy and / or protein . (DMD ) by administering to a subject in need thereof an [0266 ] In one aspect, the following method is provided . effective amount of the population of cells and / or exosomes Briefly , more than 50 mice with MI, and 3 time points of or microvesicles isolated from the cells . Methods to deter cardiac function assessment during one month observation mine the effectiveness of the therapies are known in the art , period were assessed for the Applicant' s exosome data . some of which are described herein . miRNAs secreted by exosomes derived from specific car [0262 ] Also provided is a composition for the repair or diac progenitor cells exerted a strong therapeutic effect on regeneration of damaged or diseased cardiac tissue or for the myocardial infarction . Paracrine effects of cardiac exosomes treatment of one or more of Hoyeraal -Hreidarsson syn derived from progenitors on cell survival, migration and drome, dyskeratosis congenita , pulmonary fibrosis , aplastic differentiation play an important role in cardiac regenera anemia , liver fibrosis , dyskeratosis congenita , bone marrow tion . miRNA signatures are unique in exosomes amongst failure , lung disease , endocrine diseases , polycystic ovary different parent cells . Therefore , Applicant isolated and syndrome (PCOS ) , Cushing ' s syndrome, and acromegaly , characterized exosomes from iPSC and isoxazole induced Cerebrovascular Disease (Strokes ), High Blood Pressure cardiac progenitor cells (Exo - CPCSO ) and determined their Hypertension , Parkinson ' s Disease, Dementia , Alzheimer ' s effects on cardiac cells in vitro and on mice hearts following Disease , Age - related hearing loss , Celiac disease (CD ) , myocardial infarction (MI ) . Applicant characterized the exo COPD , bipolar disorder, hydroxyurea , sickle cell diseases , somes isolated from iPSC and CPCs. Transmission electron hypertension , atherosclerosis , arthritis , osteoporosis , microscopy ( TEM ) of exosomes isolated from iPSC and osteoarthritis , vasculardementia or macular degeneration , CPC SX -9 showed typical morphology . Western blot further cancer, type 2 diabetes , or diseases with telomerase dys confirmed that exosomes from iPSC and CPC SX - 9 were function dealing with a shortened telomere length , the enriched in Exosome ( Exo ) - specific markers Tsg101 . Other composition comprising , or alternatively consisting essen common exosome proteins including CD9, Hsp70 and Flo tially of, or yet further consisting of, a synthetic microRNA tillin - 1 . Calnexin was absent in exosomes . No significant 146a and a pharmaceutically acceptable carrier , and option difference in average size of exosomes from iPSC and ally administration of an effective amount of Danazol. These CPC /SX -9 was observed . compositions are useful for regenerating tissue in a subject [0267 ] Applicant also investigated the miRNA cargo con in need thereof , by administering one or more microRNA tents in the exosomes from CPCs. We found miR - 373 , fragments , or derivatives thereof to the individual, wherein miR - 367 , miR -520 and miR -548 were significantly overex after administration of the one or more microRNA frag pressed in Exo -CPC · So compared with exosomes from iPSC , ments, the one or more microRNA fragments alter gene commercial CPCs and embryoid body (EB ) and these expression in the damaged tissue , improve the viability of miRNA microarray data were confirmed with real- time said damaged tissue, and facilitate the formation of new PCR . Go enrichment analysis based on miRNA - targeted tissue in the subject .Methods to determine the effectiveness genes showed that biological process included organelle , of the therapy are known in the art , some of which are molecular function , response to stress and mitotic cell cycle . described herein . Applicant further studied the effects of Exo -CPCSO on [0263 ] For the purposes of this disclosure , the exosomes cultured fibroblasts . PKH26 labeled exosomes from or microvesicles have an average diameter of from about 20 CPCISA - 9 were observed inside the fibroblasts Calcein AM ) , nm to about 90 nm . mostly located at the perinuclear region . Interestingly , Compositions expression of fibrotic genes (CTCF , FN1, TIMP1, TIMP2 , MMP2 and MMP9) in cultured fibroblasts stimulated with [ 0264 ] This invention also provides compositions contain TGF - B was significantly downregulated after treatment with ing the cells , exosomes or microvesicles , miRNA , and Exo -CPCiso . Loss -of - function analysis using miR - 373 populations containing the same, as described above , in inhibitor confirmed that enrichment of miR -373 was the combination with a carrier, such as a biocompatible scaffold major contributor to reversion of TGF - B stimulation in or a pharmaceutically acceptable carrier. In a further aspect fibroblasts . Applicant also tested the therapeutic effects in US 2018 /0273906 A1 Sep . 27 , 2018 32 murine MI model . Applicant found intramyocardial injec [ 0272 ] The populations are prepared by culturing a popu tion of Exo - CPC ' exerted favorable cardioprotective effects lation of stem cells or progenitor cells cultured in the on cardiomyocyte proliferation , angiogenesis and preserva presence of an effective amount of an isoxazole compound , tion of cardiac function in mice one month post- MI com a derivative thereof or an equivalent thereof, and for an pared with PBS and Exo - iPSC treated mice . ki67 positive effective amount of time . Exemplary isoxazole and deriva CM (cTnT positive ) in Exo - CPCSX -9 treated mouse 30 days tives there of are provided herein . In one aspect , the effective after MI and quantitative analysis of proliferating CMS amount comprises an amount that results in overexpression revealed that Exo - CPCSX -9 significantly increased prolifera of the miRNA , such as for example from about 5 uM to tion of CMs in peri - infarct region 30 days after MI as about 25 uM or ranges in between as described above . determined by Ki67 (data not shown) . Quantitate analysis of Non -limiting exemplary stem cell or progenitor cells are arterioles density showed Exo - CPCSX- 9 significantly selected from the group of an iPSC -derived CPC , a cardio increased arterioles density after MI. Furthermore , func - myocyte , a skeletal muscle cell , an endothelial cell, a tional measurement demonstrated Exo - CPC /SX - 9 treatment myocyte , a smooth muscle cell , or an iPSC -derived significantly improved EF and FS ( data not shown ). Quan embryoid body. titative analysis showed smaller fibrosis area from Exo [0273 ] After the cells have been cultured under the appro CPC SA -9 treated mice after MI ( data not shown ) . Hence, a priate conditions , the exosomes ormicrovesicles are isolated Micro -RNA 373 mimic would overexpress Micro -RNA 373 from the cells or cell culture supernatant using methods which would reduce scar tissue in fibrotic diseases. described herein and methods known in the art . Depending [ 0268] The exosome or microvesicle populations can be on the purification methods, the compositions can be het heterogeneous , homogeneous , substantially homogeneous erogeneous, purified , highly purified , homogenous, or sub or highly purified . In one aspect , the populations comprise at stantially homogeneous with the desired level of purity as least 80 % , or alternatively 85 % , or alternatively 90 % , or described herein . The exosomes or microvesicles and cells alternatively 95 % , or alternatively 97 % or alternatively can be detectably labeled by adding to the cells and / or exosomes or microvesicles a detectable label by conjugating 99 % , or alternatively 100 % , of the exosomes or microve to them the label using methods described herein or known sicles contain express the miRNA . in the art . [ 0269 ] Also provided are methods to prepare and isolate 0274 ] Depending on the purification methods, the cell population of one or more of: a cardiac progenitor cell, a compositions can be purified , highly purified , homogenous, cardiomyocyte , a myocyte , an endothelial cell , a smooth or substantially homogeneous and having the desired per muscle cell , a skeletal muscle cell, each generated from a centage purity . The cells can be detectably labeled by adding cell selected from the group of: an iPSC , an embryonic stem to the cells a detectable label by conjugating to them the cell or a stem cell that was contacted with an isoxazole label using methods described herein or known in the art. compound , derivative, or an equivalent of each thereof , [0275 ] The cells can be admixed or combined with a wherein the cells or the population containing same over carrier wherein the carrier is optionally a non -naturally express one or more ofmir - 373 , mir - 210 mir - 377 , mir - 367 , occurring carrier . mir - 520 , mir -548ah , mir - 335 , mir - 30c, mir -214 , or mir [0276 ] A preservative or cryoprotectant can be combined 548q ; and / or a muscle gene selected from the group of: or admixed with the cells or compositions containing them . paZ3 , PAX7, MYF5 ,MYOD ,MYOG , or dystrophin . In one These compositions can be lyophilized using methods aspect , at least 70 % , or 80 % , or 85 % , or 90 % or 95 % , or known in the art and / or formulated into appropriate dosage 98 % of the cells express the stated mir and /or muscle gene. forms for ease of use . The exosomes or microvesicles can be isolated for any one [0277 ] The cells and / or exosomes or microvesicles can be of the following: a cardiac progenitor cell, a cardiomyocyte , admixed or combined with a carrier wherein the carrier is a skeletal muscle , an endothelial cell, a myocyte , or a optionally a non -naturally occurring carrier . In one aspect , smooth muscle cell . the population of cells is cultured in serum - free media . [0270 ] Also provided herein are methods for preparing f0278 ] A preservative or cryoprotectant can be combined cells that overexpress one or more of mir - 373 , mir - 210 or admixed with the cells , exosomes or microvesicles or mir - 377 , mir - 367 , mir - 520 , mir - 548ah , mir - 335 , mir- 30c , compositions containing them . These compositions can be mir - 214 , or mir- 548q ; and/ or a muscle gene selected from lyophilized using methods known in the art and / or formu the group of: paX3 , PAX7 , MYF5 , MYOD , MYOG , or lated into appropriate dosage forms for ease of use . dystrophin . In one aspect, at least 70 % , or 80 % , or 85 % , or [0279 ] The cells and compositions prepared by these 90 % or 95 % , or 98 % of the cells overexpress the stated mir methods and comprising a microRNA (miRNA ) selected and / or muscle gene . Expression levels are determined using from the group of mir - 373, mir -210 , mir - 377 , mir - 367 , methods described herein and known in the art , e . g . , high mir - 520 , mir - 548ah , mir - 335 , mir- 21, mir - 30c , mir - 214 or throughput assays , ELISA and hybridization techniques . mir- 548q; and / or one or more of a protein selected from : One ormore of the mir and /or genes , as described above can Tsg101, CD9, Hsp70 , Flotillin - 1 or GAPDH , or alternatively be overexpressed . cells or a population of cells that overexpress one or more of [0271 ] Also provided are methods for preparing compo mir -373 , mir -210 mir - 377 , mir - 367, mir -520 , mir -548ah , sitions containing the exosomes or microvesicles and/ or mir- 335 , mir -30c , mir - 214 , or mir - 548q ; and /or a muscle cells . The compositions can further comprise a protein that gene selected from the group of: paX3, PAX7 , MYF5 , facilitates regeneration and / or improved function of a tissue MYOD , MYOG , or dystrophin are useful for one or more of or a nucleic acid that encodes the protein and / or an agent that providing in a subject in need thereof regenerating damaged inhibits the expression of an inflammatory protein , that are tissue such as muscle and /or cardiac tissue ; improving the optionally a cytokine . Examples of such are described viability of damaged tissue , such as muscle and /or cardiac herein . tissue; facilitating the formation of new tissue, such as US 2018 /0273906 A1 Sep . 27 , 2018 33 cardiac tissue, muscle tissue, skeletal muscle tissue, a blood compositions are delivered locally or systemically to the vessel, a capillary , or a myocyte ; promoting cardiac regen tissue of the subject or by an intramyocardial or an intrac eration ; promoting cardiac regeneration in a subject suffer oronary route . ing from an acute cardiac event; promoting cardiac regen [0282 ] Also provided are methods of preparing a popula eration in a subject suffering from a myocardial infarction ; tion of cardiac or skeletal myogenic progenitors from a promoting cardiac regeneration in subject suffering from population of human induced pluripotent stem cells (hip Duchenne Muscular Dystrophy (DMD ) or Duchenne Mus SCs ) by contacting the hiPSCs with an effective amount of cular Dystrophy (“ DMD ” )- associated cardiomyopathy , pro Givinostat (GIV ) or culturing the cells in the presence of an moting cardiac regeneration in a subject suffering from effective amount of Givinostat (GIV ) for an effective age -related diseases, such as for example , chronic obstruc amount of time. In one aspect, the effective amount of GIV tive pulmonary disease (“ COPD " ) , arthritis , osteoporosis , comprises from about 100 nM to about 30 nM per 1x106 cells and ranges in between as described herein . In a further osteoarthritis , diabetes , vascular dementia , or macular aspect, the cells are cultured in the presence of serum - free degeneration ; promoting tissue regeneration in tissue dam medium and an effective amount of a Rho - associated kinase aged from one or more of stroke , arthritis , Alzheimer' s , (ROCK ) inhibitors for an effective amount of time. Non memory loss discorders , cystic fibrosis , inflammatory dis limiting examples of such are described above and incor orders and cancer ; decreasing cardiac wall thickness in a porated herein by reference . In a further aspect, an effective tissue damaged from a cardiac infarction ; altering gene expression of one or more of protein kinase C , iL -6 , mmp , amount of a TGF - beta type - 1 receptor inhibitor is added to PDGF ; reducing or inhibiting the expression of an inflam the culture medium , non - limiting examples of the TGF -beta matory protein that is optionally a chemokine, macrophage type - 1 receptor inhibitor are described above , and incorpo or a cytokine ; directly or indirectly stimulating angiogen rated herein by reference . esis , promoting cardiac regeneration in a subject suffering [0283 ] After the cells have been cultured under the appro from a disease selected from the group of coronary artery priate conditions, the cells are isolated from the cell culture disease , myocardial infarction , heart failure , hypoplasic left supernatant using methods described herein and methods heart syndrome, peripheral artery disease (PAD ), cardiac known in the art . mypertrophy, valvular heart disease ( aortic stenosis ) , myo [ 0284 ] Use of the markers can be used to isolated and cardial hypertrophy, hypertrophy fibrosis ; and /or directly or purify the compositions , Depending on the purification indirectly inhibit cellular replication . The methods are methods, the cell compositions can be purified , highly accomplished by administering an effective amount of the purified , homogenous , or substantially homogeneous and /or exosomes or microvesicles , cells, populations or composi having the desired percentage purity as described above . The tions containing the same to a subject in need thereof. The cells can be detectably labeled by adding to the cells a methods can further comprise administering an effective detectable label by conjugating to them the label using amount of non -embryonic stem cell or a progenitor cell to methods described herein or known in the art. In a further the subject that is optionally the same or different type of aspect , exosome or microvesicle are isolated from the cells tissue that is in need of repair . In one aspect, the non using methods described herein and known in the art . embryonic stem or progenitor cell is autologous to the [0285 ] The cells and / or exosomes or microvesicles can be subject. In another aspect, the non - embryonic stem or pro admixed or combined with a carrier wherein the carrier is genitor cell is allogeneic to the subject. The exosomes or optionally a non - naturally occurring carrier. microvesicles , cells , populations can be locally or systemi [0286 ] A preservative or cryoprotectant can be combined cally delivered to the subject . Subjects suitably treated by or admixed with the cells , exosomes or microvesicles or these methods include animals , mammals and human compositions containing them . These compositions can be patients . Methods to determine the effectiveness of the lyophilized using methods known in the art and /or formu therapy are known in the art, some of which are described lated into appropriate dosage forms for ease of use. herein , by administering an effective amount of the compo [0287 ] The cells , exosomes or microvesicles and compo sition of cells , exosomes or microvesicles or compositions sition containing them are useful in methods for regenerat containing the same to the subject in need thereof. As noted ing skeletal muscle , or regenerating cardiac muscle , and /or above , methods for determining the effectiveness of the treating DMD by administering to a subject in need thereof therapy are known in the art, some of which are described an effective amount of the population of cells . Any appro herein . priate method of administration can be used , e . g ., topical, by infusion or intravenously, as determined by the treating [ 0280 ] The methods can further comprise administering veterinarian or physician . It also depends on the age , health an effective amount of a non - embryonic stem cell or pro and gender of the subject being treated as well as the genitor cell to the subject, that is optionally of the same type formulation . Effective amounts can be determined empiri as the tissue in need of repair of a type different from the cally by the treating veterinarian or physician . In one aspect , type of tissue of repair. The cells can be non - embryonic stem the cells , exosomes or microvesicles or compositions are or progenitor cell is autologous or allogeneic to the subject. delivered locally or systemically to the tissue of the subject 10281 ] Any appropriate method of administration can be or by an intramyocardial or an intracoronary route . Methods used , e . g ., topical, by infusion or intravenously , as deter to determine the effectiveness of the therapy are known in mined by the treating veterinarian or physician . The appro the art , some of which are described herein . priate route of administration and dosage also depends on [0288 ] Also provided are compositions for the repair or the age , health and gender of the subject being treated as regeneration of damaged or diseased cardiac tissue , the well as the formulation . Effective amounts can be deter composition comprising , or alternatively consisting essen mined empirically by the treating veterinarian or physician . tially of, or yet consisting of synthetic microRNA - 146a In one aspect, the cells , exosomes or microvesicles or (miRNA - 146a ) or a fragment thereof, and optionally a US 2018 /0273906 A1 Sep . 27 , 2018 34 pharmaceutically acceptable carrier , and optionally wherein products /lightswitch -mirna - mimics - inhibitors /inhibitors / the composition is purified , highly purified , or substantially mir - 300 -399 (mir373 inhibitors ; switchgeargenomics .com / homogeneous. In a further aspect, the composition can be products / lightswitch -mirna -mimics - inhibitors /mir - 300 - 399 . heterogeneous . Synthetic mRNA - 146a is commercially (miR373 mimics (switchgeargenomics . com / products / syn available from vendors such as Sigma Aldrich or can be thetic -Zutr - goclone -reporters /mir -300 - 399 (miR 3' UTR manufactured using conventional techniques . In one aspect, reporter ) ) ; (addgene . org /78127 / (miR - 373 expression vec the microRNA fragments , or derivatives thereof, are syn tor ) ; Thermo - Fischer ( thermofisher. com / us / en /home / life thetically generated using techniques such as Artificial sciencelepigenetics - noncoding - rna - research /mirna - analysis / microRNA (amiRNA ) technology which exploits micro mirna -mimics - inhibitors /mirvana -mimics - inhibitors .html RNA (miRNA ) biogenesis pathway to produce artificially and thermofisher. com / us /en /home / life - science / epigenetics designed small RNAs using miRNA gene backbone vis the noncoding - rna - research /mirna -analysis /mirna -mimics - in following steps : Step 1 : predict the target miRNAs from hibitors / ambion - pre- mir - precursors .html . gene coding sequence . Step 2 : predict artificial miR * [0290 ] IDT oligos can be used and are commercially sequence from miR . Step 3 : predict primers from the sec available ( see idtdna. com / pages /decoded / decoded - articles/ ondary structure of pre -microRNA . In a further aspect , the product- spotlight/ decoded / 2012 /04 / 11/ inhibiting -mirnas microRNA fragments , or derivatives thereof are synthesized using - antisense - oligonucleotides ) . Others can be purchase with a sequence that mimics one or more endogenous from Qiagen ( qiagen . com / us/ shop / genes -and -pathways / microRNA molecules using techniques such as miRNA mirna -details . aspx ? mirnaid = 7344 ) . mimics which are small, chemically modified double [0291 ] The miRNA compositions can be detectably stranded RNAs that mimic endogenous miRNAs and enable labeled by adding to the miRNAs a detectable label by miRNA functional analysis by up - regulation of miRNA conjugating to them the label using methods described activity . miRNA inhibitors are small, chemically modified herein or known in the art . The miRNAs can be admixed or single -stranded RNA molecules designed to specifically combined with a carrier wherein the carrier is optionally a bind to and inhibit endogenous miRNA molecules and non -naturally occurring carrier. A preservative or cryopro enable miRNA functional analysis by down - regulation of tectant can be combined or admixed with the miRNA or miRNA activity . In a yet further aspect, the wherein the compositions containing them . These compositions can be microRNA fragments, or derivatives thereof are modified to lyophilized using methods known in the art and / or formu enhance their stability using techniques such as anti- miRNA lated into appropriate dosage forms for ease of use. oligonucleotides which inhibit miRNA activity to fine - tune [0292 ] In a further aspect, the methods to generate these specific signaling pathways or to block miRNA function compositions are encapsulated or conjugated to synthetic induced by disease . These anti -miRNA oligonucleotides liposomes using techniques known in the art . “ Liposomes” may be modified to enhance stability , target affinity , and are microscopic vesicles consisting of concentric lipid bilay promote cellular uptake . AntagomiRs are anti -miRNAs that ers . Structurally, liposomes range in size and shape from are modified in one or more of the following ways : ( 1 ) long tubes to spheres, with dimensions from a few hundred 2 '- O -methylation of the ribose sugar to enhance nuclease Angstroms to fractions of a millimeter . Vesicle - forming resistance and improve the binding affinity to target RNAs; lipids are selected to achieve a specified degree of fluidity or ( 2 ) phosphorothioate linkage between nucleotides to provide rigidity of the final complex providing the lipid composition stability , with the balance between phosphodiester and phos of the outer layer . These are neutral ( cholesterol) or bipolar phorothioate enhancing oligonucleotide stability against and include phospholipids, such as phosphatidylcholine nucleases; and (3 ) cholesterol conjugation at the 3 'UTR to (PC ), phosphatidylethanolamine (PE ), phosphatidylinositol facilitate cellular uptake. Locked nucleic acid (LNA - ) -modi (PI ) , and sphingomyelin (SM ) and other types of bipolar fied oligonucleotides are anti- miRNAs with a 2 ' sugar modi lipids including but not limited to dioleoylphosphatidyletha fication in which the ribose is locked in a C3 - endo confor nolamine ( DOPE ) , with a hydrocarbon chain length in the mation by a 2 - 0 , 4 - C methylene bridge that strongly range of 14 - 22 , and saturated or with one or more double increases the affinity for complementary RNA and increases C = C bonds. Examples of lipids capable of producing a the duplex melting temperature miRNA inhibition is stable liposome, alone , or in combination with other lipid achieved by anti -miRNAs , which are single - stranded oligo components are phospholipids, such as hydrogenated soy nucleotides complementary to target miRNAs. AntagomiRs phosphatidylcholine (HSPC ) , lecithin , phosphatidyletha and LNA oligonucleotides are anti -miRNAs modified to nolamine, lysolecithin , lysophosphatidylethanol- amine , improve uptake and stability . Examples of Micro -ma 195 phosphatidylserine , phosphatidylinositol, sphingomyelin , inhibitors are known and are sold by Sigma - Aldrich cephalin , cardiolipin , phosphatidic acid , cerebrosides, dis ( HSTUD0320 SIGMA MISSION® Synthetic microRNA tearoylphosphatidylethan - olamine (DSPE ) , dioleoylphos Inhibitor, Human hsa- miR - 195 -5p ) and abmgood . com ( e. g ., phatidylcholine (DOPC ), dipalmitoylphosphatidylcholine inhibitory hsa- miR - 195 - 5p miRNA /microRNA Lentivec (DPPC ), palmitoyloteoylphosphatidylcholine (POPC ) , tor ). Hence , a Micro - RNA 373 mimic would overexpress palmitoyloleoylphosphatidylethanolamine (POPE ) and dio micro - RNA 373 which would reduce scar tissue in fibrotic leoylphosphatidylethanolamine 4 - ( N -maleimido - triethyl ) diseases . cyclohexane - 1 -carboxylate (DOPE -mal ) . Additional non [ 0289] Examples ofmicro -ma 373 mimic ' s are known and phosphorous containing lipids that can become incorporated sold by Sigma - Aldrich under MISSION® microRNA into liposomes include stearylamine , dodecylamine , hexa Mimic hsa -miR - 373 * ( sigmaaldrich . com / catalog /product / decylamine , isopropyl myristate , triethanolamine - lauryl sul sigma/ hmi0531 ? lang = en & region = US) . Additional Micro fate , alkyl- aryl sulfate, acetyl palmitate , glycerol ricinoleate , ma 373 mimic ' s , mir- 373 inhibitors , mir - 373 oglio ' s , mir hexadecyl stereate , amphoteric acrylic polymers , polyethyl 373 expression vector 's , mir - 373 precursers are known and oxylated fatty acid amides, and the cationic lipids mentioned sold by : Switchgear genomics ( switchgeargenomics. com / above ( DDAB , DODAC , DMRIE , DMTAP, DOGS, US 2018 /0273906 A1 Sep . 27 , 2018 35

DOTAP (DOTMA ) , DOSPA , DPTAP, DSTAP, DC -Chol ) . and / or an effective amount of the chemically modified cell Negatively charged lipids include phosphatidic acid (PA ) , or population of chemically modified cells described above . dipalmitoylphosphatidylglycerol (DPPG ), dioteoylphospha In a further aspect, the isolated cell, e . g . , an iPS cell or other tidylglycerol and (DOPG ) , dicetylphosphate that are able to progenitor or stem cell is locally administered within an form vesicles . Typically , liposomes can be divided into three effective amount of isoxazole or isoxazole similar com categories based on their overall size and the nature of the pound . In one aspect, the treated iPS cells were differentiated lamellar structure . The three classifications, as developed by into myocytes forming myofibers in the scar tissue of the the New York Academy Sciences Meeting , “ Liposomes and heart . The cells can be autologous or allogeneic to the host Their Use in Biology and Medicine , ” December 1977, are or patient. The subject and cells can be any species as multi - lamellar vesicles (MLVs ) , small uni- lamellar vesicles described herein . (SUVs ) and large uni- lamellar vesicles (LUVs ) . The bio [0298 ] Yet another embodiment of the invention is a logical active agents can be encapsulated in such for admin method for regenerating cardiac muscle tissue in a suitable istration in accordance with the methods described herein host by administering to the host an effective amount of the and described for example in U . S . Pat. Nos. 5 ,512 , 295 ; chemically modified cell or population of chemically modi 7 ,401 ,707 , and 9 ,554 , 999 . These compositions are further fied cells as described above . The cells can be autologous or provided herein . allogeneic to the host or patient. The subject and cells can be [0293 ] The synthetic miRNA compositions are useful to any species as described above . regenerate tissue in a subject in need thereof, by adminis [0299 ] In another aspect , the method comprises, or alter tering one or more microRNA fragments , or derivatives natively consists essentially of, or yet further consists of thereof to the subject wherein after administration of the one administering to a patient in need thereof an effective or more microRNA fragments , the one or more microRNA amount of an iPS cell and an effective amount an isoxazole fragments alter gene expression in the damaged tissue, or isoxazole similar compound . Administration can be local improve the viability of said damaged tissue, and facilitate to the site of damage , and can include direct injection of the the formation of new tissue in the subject . cells and isoxazole or isoxazole similar compound into the [ 0294 ] Any appropriate method of administration can be heart of the patient. The method also includes combination used , e . g ., topical, by infusion or intravenously , as deter therapy as described herein . For example , the method can be mined by the treating veterinarian or physician . It also combined with an effective amount of electrical stimulation , depends on the age , health and gender of the subject being before, after or concurrent to this therapy . The method can treated as well as the formulation . Effective amounts can be also be combined with an effective amount of other cardiac determined empirically by the treating veterinarian or phy inducing small molecules including : sician . Methods to determine if the methods are effective are [ 0300 ] 1 ) Wnt/ beta - catenin inhibitors , e . g ., IWR - 1 , IWP known in the art some of which are described herein . 1 . Include an FDA approved drug called Pyrvinium ( com [ 0295 ] Also provided are methods to isolate a population mon brand name is Vanquin ) . Another novel small molecule of exosomes or microvesicles , comprising culturing a popu includes ICG - 001 (Chemical Name: (65 , 9aS ) -Hexahydro lation of non - embryonic human regenerative cells in the 6 -[ (4 -hydroxyphenyl ) methyl ] - 8 -( 1 -naphthalenylmethyl ) - 4 , presence of a hydrolase enzyme to induce the cells to secrete 7 -dioxo - N - (phenylmethyl ) - 2H - pyrazino [ 1 , 2 - a ]pyrimidine - 1 exosomes or microvesicles , thereby generating exosomes or (6H ) - carboxamide ) that targets the Wnt/ B - catenin pathway . microvesicles . In a further aspect, the secreted exosomes or [0301 ] 2 ) TGF - B inhibitors , such as the small molecule microvesicles are isolated or purified from the culture media . ITD - 1 defined as Chemical Name: 4 - [ 1 , 1 ' - Biphenyl] - 4 - yl In one aspect , the hydrolase comprises a member of the 1 ,4 , 5 ,6 , 7 ,8 -hexahydro - 2 , 7 ,7 -trimethyl - 5 - oxo - 3 - quinolin DNAse I superfamily of enzymes , non - limiting examples of ecarboxylic acid ethyl ester ), such include a sphingomyelinase , selected from the group of [0302 ] 3 ) Prostaglandins and COX - 2 . Activation of lysosomal acid sphingomyelinase, secreted zinc - dependent cyclooxygenase 2 ( COX - 2 ) and subsequent production of acid sphingomyelinase , neutral sphingomyelinase , and alka prostaglandin E2 ( PGE2 ) induced by MI ( PGE2 is an line sphingomyelinase . In another aspect, the neutral sphin FDA -approved treatment for induction of labor under the gomyelinase comprises one or more of magnesium - depen brand name Dinoprostone. Another example includes small dent neutral sphingomyelinase and magnesium - independent molecule ONO - 1301 — a small molecule agonist of PGI2 neutral sphingomyelinase . Non - limiting examples of such with a synymom of 7 , 8 - Dihydro - 5 -[ ] ( E )- [[ c -( 3 pyridyl) include neutral sphingomyelinase type I , neutral sphingo benzylideneJaminooxylethyl] - 1 -naphthyloxy ] acetic acid myelinase type 2 , and neutral sphingomyelinase type 3 . and supplied by Sigma - Aldrich , [0303 ] 4 ) DPP - IV inhibitors in combination with granu Methods and Uses of Cell Populations locyte colony stimulating factor or G - CSF . ( This approach [ 0296 ] Yet another embodiment of the invention is a combines two molecules : a small molecule inhibitor of method for restoring cardiac function in a tissue or host in dipeptidylpeptidase IV (DPP - IV ) , an enzyme that degrades need thereof. This and other therapeutic , diagnostic , and SDF - 1a , and granulocyte colony -stimulating factor research uses are described herein . ( G -CSF ) , a biological molecule that enhances the release of [ 0297] In one embodiment, the invention provides meth stem cells from the bone marrow through matrix metallo ods for one or more of: regenerating cardiac muscle tissue proteinase 2 , that in one aspect , is scar tissue in the damaged or diseased [0304 ] 5 ) Angiotensin ( 1 - 7 ) and Mas receptor ( formula : heart ; improving cardiac function or for treating a cardiac C41H62N 2011 and supplied by Tochris ) . disease or condition in a patient in need thereof. The f0305 ] The combination therapy can be sequential or methods comprise contacting the tissue to be regenerated concurrent, as determined by the condition being treated , the with an effective amount of isoxazole or isoxazole similar health of the patient and the selection of the particular compound or by administering to a subject in need thereof, combination therapy. US 2018 /0273906 A1 Sep . 27 , 2018 36

[0306 ] In one aspect, the stem cells or iPS cells are therapy and the subject being treated . Single or multiple administered in the form of a cardiac patch derived from administrations can be carried out with the dose level and stem cells ( e . g . , adult or bone marrow derived progenitors pattern being selected by the treating physician . Suitable cells , iPS cells , iPS cells derived cardiac lineage cells , small dosage formulations and methods of administering the cell juvenile stem cells and/ or engineered tissue cardiac patch or agents are known in the art . In a further aspect , the cells transplantation for the heart repair in heart diseases ). Thus, and composition of the invention can be administered in in one aspect , the stem cells comprise very small juvenile combination with other treatments . stem cells that are present in the bone marrow ( that are [0311 ] The cells and populations of cell are administered comprised within bone marrow stem cells ) and / or peripheral to the host using methods known in the art and described , for blood cells ( that are comprised within circulating blood and example , in U . S . Pat. No. 6 ,638 , 369. This administration of heart - derived stem cells ) . These cells are with significantly the cells or compositions of the invention can be done to high proliferative and differentiation potentials and can also treat disease as noted herein , and to produce an animal be easily reprogrammed and/ or converted to cardiac pro model of the desired disease , disorder , or condition for genitors with isoxazole or isoxazole similar compounds or experimental and screening assays . Wnt inhibitors . Thus, this method would not require the derivation of iPS cells for safe cell transplantation . This disclosure also provides a method for promoting stem cell Screening Assays survival and differentiation by applying cardiac patches with [0312 ] The present invention provides methods for screen derivatives of iPS cells treated with isoxazole or isoxazole ing various agents that modulate the therapeutic functions as similar compounds and Wnt inhibitors administered over described herein as well as gene expression , cell differen scar area allowing better penetration , migration and integra tiation , exosome or microvesicle expression . For the pur tion of patch cells (myocytes , endothelial cells , smooth poses of this invention , an “ agent" is intended to include , but musclem cells . (FIGS . 14A - 14H ) with host heart. not be limited to a biological or chemical compound such as [ 0307] This disclosure also provides a method for promot a simple or complex organic or inorganic molecule , a ing stem cell survival and differentiation in vitro , comprising peptide, a protein ( e . g . , antibody ) , a polynucleotide ( e . g ., applying an effective amount of electrical stimulation to the anti -sense ) or a ribozyme. A vast array of compounds can be stem cell . In one aspect, the effective amount comprises synthesized , for example polymers , such as polypeptides wherein the electrical stimulation comprises from about and polynucleotides , and synthetic organic compounds 1. 0V / 1 . 5 cm to about 2 . 0V / 2 . 0 cm for about 1 to about 5 based on various core structures , and these are also included hours . in the term “ agent. ” In addition , various natural sources can [ 0308 ] This disclosure also provides a method for gener provide compounds for screening , such as plant or animal ating iPSC derived muscle progenitor cells (MPC ) by isox extracts , and the like . It should be understood , although not azole and isoxazole like compounds and their precondition always explicitly stated that the agent is used alone or in ing or treatment of skeletal muscle that will induce combination with another agent, having the same or different functional CXCR4 expression in MPC to facilitate mobili biological activity as the agents identified by the inventive zation and engraftment of progenitor cells in dystrophic screen . skeletal muscles. See FIGS . 15A - 15B . For the MPC to be a [0313 ] To practice the screening method in vitro , suitable viable therapeutic option for DMD , dystrophin gene expres cell cultures or tissue cultures containing the modified cell ( s ) sion must be sufficient to restore muscle contractility . Hence are first provided . When the agent is a composition other MPC from the disclosed methods provide an abundant than a DNA or RNA , such as a small molecule as described source of cells for transplantation and provide an effective above, the agent can be directly added to the cell culture or and safe therapy for regeneration of dystrophic muscle . added to culture medium for addition . As is apparent to those Transplantation of iPSC - derived progenitors coupled with skilled in the art , an “ effective ” a mount must be added methods to optimize the host muscle microenvironment, which can be empirically determined . When agent is a such as treatment with an effective amount of a steroid will polynucleotide, it can be directly added by use of a gene gun more effectively ameliorate dystrophic pathology and or electroporation . Alternatively , it can be inserted into the improve the quality of life for patients with DMD . cell using a gene delivery vehicle or other method as [ 0309 ] Patients suitably treated by this method include described above . Positive and negative controls can be those suffering from a disease or disorder associated with cardiac malfunction including , but not limited to , congestive assayed to confirm the purported activity of the drug or other heart failure , isolated diastolic heart failure , myocardial agent. infarction , and cardiac arrhythmia . There are several forms [0314 ] Also provided herein is a method for selecting a of cardiac arrhythmia that can be treated including, but not cell or cell population for reprogramming , comprising deter limited to , sick sinus syndrome, bradyarrhythmia , abnormal mining the expression level of miR - 195 in a sample wherein sinus node function , atrioventricular block , and atrial and low expression of miR - 195 selects the cell or population and ventricular tachyarrhythmia . lack of low expression of miR - 195 does not select the cell [0310 ] Local or systemic administration , by use of a or population . In a further aspect, the method further com catheter or cardiac patch with isoxazole or similar com prises determining the expression level of one or more of pound treated iPS cells , of the cells or compositions can be marker selected from miR - 29b , miR - 205, miR - 378 , and effected in one dose , continuously or intermittently through miR - 542 - 3p , wherein low expression of the one or more out the course of treatment. Methods of determining the marker selects the cell or population and lack of low most effective means and dosage of administration are expression ofmiR -195 does not select the cell or population . known to those of skill in the art and will vary with the [0315 ] The following methods are useful in executing the composition and /or cells used for therapy, the purpose of the inventions as described herein . US 2018 /0273906 A1 Sep . 27 , 2018 37

Experiment No. 1 Amicon Ultra- 4 10 KDa centrifugal filter units to a final [ 0316 ] In Vitro Generation of Muscle Progenitor Cells volume of < 100 ul. The purified exosomes or microvesicles (MPCs ) from hiPSC were stored at - 80° C . and subsequently characterized by [ 0317 ] Human Induced Pluripotent Stem (iPS ) Cells nanotracking analysis , protein and ultrastructure analysis . (ATCC® ACS - 1021TM ) induced from human cardiac fibro Particle Size and Concentration Distribution Measurement blasts were cultured with mTeSRTM1 (STEMCELL Tech with Tunable Resistive Pulse Sensing nologies Inc. ) on Vitronectin XF (STEMCELL Technologies [0323 ] Particle size and concentration distribution of exo Inc . ) coated 6 -well plates . iPS Cells were passaged every 4 somes or microvesicles isolated analysis were performed to 6 days with ReLeSRTM (STEMCELL Technologies Inc. ). using tunable resistive pulse sensing method with a qNano [0318 ] For differentiation of iPS Cells into MPCs iPS instrument ( Izon Science ) . Briefly , the number of particle Cells were dissociated into single cells with ACCUTASETM was counted at least 600 to 1000 events using 20 mbar ( STEMCELL Technologies Inc. ) into single cells and seeded pressure and NP200 nanopore membranes stretched at 1x10 cells / cm2with mTeSRTM1 supplemented with 5 uM between 46 . 5 -47 . 5 mm . Calibration was performed using RHO /ROCK pathway inhibitor ( Y - 27632 , STEMCELL known concentration of beads CPC200 ( diameter : 210 nm ) . Technologies Inc. ). After 24 hr, the medium was changed to Data were processed using Izon Control Suite software . fresh mTeSRTM1. mTeSRTM1 was refreshed daily during first 3 days . After 3 days , culture medium was changed to Transmission Electron Microscopy mTeSRTM1 supplemented with 20 uM ISX - 9 (MedChem [0324 ] Exosomes or microvesicles pellet were fixed with Express ) . The medium was refreshed every other day . After 4 % paraformaldehyde ( PFA ). Following a total of 8 washes 6 days, the medium was switched to RPMI 1640 Medium using distilled water, grids were contrasted with a uranyl ( Thermo Fisher Scientific ) supplemented with N - 2 Supple oxalate solution for 5 minutes , and transferred to methyl ment ( Thermo Fisher Scientific ) and 20 uM ISX - 9 and cellulose -uranyl acetate for 10 minutes on ice according to refreshed every other day for another 3 to 6 days. the description in previous study . Samples were examined Methods for Exosome or Microvesicle and Cpc Generation on a JEOL JEM - 1220 transmission electron microscope Include Cell Culture , hiPSC Maintenance . ( TEM ) ( JEOL USA , Inc . ) [0319 ] Human iPSC cells (ACS - 1021 , ATCC , USA ) were maintained in mTeSR1 media (Stem Cell Technology ) on Selected Exemplary Embodiments vitronectin coated six - well plates with daily medium changes. Cells were passaged with ReLeSRTM reagent every [0325 ] Applicant has discovered that iPSc can be chemi 4 - 7 days according to the manufacturer' s protocol (Stem cally induced to DNA hypomethylation causing upregula Cell Technology ) . tion of cardiac genes and allowing successful propagation in the diseased heart with no or limited chances for tumor CPC Generation . genecity . [ 0326 ] Skeletal myoblasts ( SMS) purified from young [0320 ] Briefly , hiPSCs maintained on vitronectin coated male Oct3 / 4 -GFP + transgenic mouse were treated with DNA six -well plate in m TeSR1 media ( Stem Cell Technology ) methyltransferase inhibitor 500 UM RG - 108 in 0 . 5 % DMSO were dissociated into single cells using Accutase solution in knock out DMEM for 5 - days . Two weeks later, GFP + ( Invitrogen ) at 37° C . for 10 min and then were seeded on colonies of SM derived iPS cells ( SiPS ) expressing GFP and to a vitronectin - coated six -well plate at 1x106 cell/ well in morphological features of mouse embryonic stem cells were m TeSR1 supplemented with 5 UM ROCK inhibitor isolated and propagated in vitro . SiPS were positive for ( Y - 27632 , Stem Cell Technology ) for 24 h . The second day , alkaline phosphatase activity , expressed SSEA1 and dis cells were cultured in m TesR1 with daily change for 3 days . played a panel of pluripotency markers ( FIG . 1 ) similar to Afterwards the medium was switched to RPMI/B27 minus ES cells and developed teratomas in nude mice . Although insulin supplemented with ISX - 9 (20 uM , dissolved in the research described herein was conducted in a murine DMSO , Stem Cell Technology ) for 7 days . model, the use of the same markers and agents will provide similar if not identical results in human cells and tissue . EB Generation 103271 In order to direct SiPS towards cardiac lineage [ 0321] Applicant generated EBs using hanging drop cells , they were treated with a small molecule ( Isoxazole , 20 method in RPMI/B27 minus insulin medium . uM , Sigma -Aldrich or Isox 9 , StemCell Technologies, Inc Generation and Isolation of Exosomes or Microvesicles Vancouver, BC ) for five days and analyzed for DNA meth from Cardiac Progenitor Cells , EB or hiPSCs yltransferase (Dnmt ) activity , cell proliferation , and cardiac [ 0322] Exosomes or microvesicles were generated from gene expression . DNMT activity was completely abolished Human iPSC cell line ACS - 1021 (ATCC , USA ), Embryoid with 95 % reduction in global DNA methylation in small bodies (EB ) and CPCs induced by ISX - 9 . CPCs were molecule treated SiPS ( FIG . 2 ) . These SiPS showed generated as described in Experiment No . 10 . Conditioned increased proliferative activity ( p < 0 .01 vs . non treated Sips ) media was collected from hiPSC and CPCs . The conditioned evaluated by cell proliferation assay and also become tol media was centrifuged at 3000 rpm for 30 min to remove erant to apoptosis ( FIGS . 3A - 3B ) an important consideration cells and debris, followed by filtration through a 0 . 22 um for preventing cell death in the ischemic environment. Small filter to remove the remaining debris . Then the medium was molecule treated IPS cells show a significant decrease in further concentrated to 500 ul using Amicon Ultra - 15 100 cytochrome c translocation to cytoplasm as compared to the kDa centrifugal filter units (Millipore ) . Isolation of exo untreated IPS cells ( FIG . 4 ) . somes or microvesicles in the concentrated medium was [ 0328 ] RT PCR analysis showed cardiomyocyte - like gene used qEV size exclusion columns ( Izon science ) . Exosome expression profile with significant upregulation of Nkx - 2 .5 or microvesicle fractions were collected and concentrated by ( p < 0 .01 vs non treated IPS ; see FIGS . 5A - 5B ) . Approxi US 2018 /0273906 A1 Sep . 27 , 2018 38 mately 60 % treated iPS cells were positive for Nkx - 2 .5 . J . L . et al . ( 2012 ) ACS Chem Biol. 7 ( 6 ) : 1067 - 1076 ) . Small Given that posttranscriptional regulation is crucial for gene molecule induced iPS cells were engrafted in post ischemic expression and cell survival molecular phenotypic analysis model and improved global cardiac function compared to was performed , Affymetrix array - based gene expression non - treated iPS cells . Recovery of cardiac function was profiling further confirmed 2 - 3 folds downregulation of dependent on the survival of the iPS cell -derived progenitors Dnmt1 , Dnmt3b and Max gene associated protein which cells . Small molecules are innovative in inducing the tissue were associated with global DNA hypomethylation and myc specific gene expression in iPS cell towards tissue differen dependent cell transformation (see Table 1 ) . Additionally , tiation , as well as determining the temporal and spatial there was a 2 - 3 fold concomitant upregulation in the CCL7 , patterns of development. CXCR2 , CXCR5 , integral membrane protein 2A , and ephrin [0334 ] Applicant has discovered that the induced pluripo A3. ( FIG . 6 ) . These were associated with DNA synthesis , tent stem cells , if treated with the appropriate small mol cell proliferation , cell matrix interaction and chemoattrac ecule , can pharmacologically inhibit the DNA methylation , tion . miR microarray analysis showed upregulation of car hence a critical player in the regulation of cardiac develop diac specific miR - 133 , 762 and down regulation of pluripo mental genes . Without being bound by theory , the effect of tency associated miR - 290 cluster, miR - 574 -5p and let - 7 small molecule on epherin family may manipulate the family (see FIGS . 7A - 7D ) . Western blot analysis showed process of hematopoietic progenitors generation and could significant upregulation ofGai protein levels as compared to be beneficial for clinical hematopoietic malignancies . untreated IPS cells ( see FIG . 7E ) . [0329 ] The Sips treated with the small molecule were [0335 ] Applicant also discovered that isoxazoles upregu stained with PKH 26 for locating them in the heart and lated cardiac specific miR - 133 , miR - 762 and down regula transplanted in the myocardium after 30 minutes of coronary tion of pluripotency associated miR -290 cluster and let- 7 artery ligation for 6 weeks. Before harvesting the hearts for family in iPS cells as compared to nontreated iPS cells . visualizing the fate of transplanted Sips, cardiac function [0336 ] Eric Oslon in U .S . Pat. No . 8, 318 , 951 (951 patent) was monitored with echocardiography . The treated Sips previously reported the use of these compounds in neuro were differentiated into myocytes forming myofibers in the genesis , epicardial progenitors cells . Regardless of these scar tissue . The scar area in the left ventricle was muscu studies , the findings reported herein are unique and innova larized with the treatment with isox treated SiPS ( FIGS . 8B , tive in that the findings address the small molecule induced 8C , 8D ) compared to untreated /control hearts (FIG . 8A ) epigenetic changes that can be manipulated for rendering the These are significant findings and have been reported pre iPS cells for propagation in the infarcted heart. This work viously in an initial patent application as only new pictures emphasizes the iPS cells which can be made from a patient are graphically shown in figures ( see FIGS. 8A - 8F ) . cell in this case skeletal muscle ) and reintroduced into the [0330 ] Cardiac function : The temporal changes in global same patient after pretreatment with small molecule . These heart function including left ventricle ejection fraction findings are distinct from the work of Oslon since iPS cells (LVEF ) and left ventricle fractional shortening (LVFS ) were of the present disclosure were therapeutically predesigned measured in control infarcted and treated infarcted hearts . ( or pretreated ) for propagation in the scar tissue of the mice The pathological remodeling of left ventricle chamber heart after coronary artery ligation or heart attack . There was dimensions during systole ( LVDs ) and diastole (LVDd ) was significant regeneration in the scar tissue by iPS cells . On the also significantly reduced in CPs treated hearts ( 2 . 97 mm other hand , Olson injected the drug into live mice to act on and 3 . 99 mm ) as compared with DMEM treated hearts ( 4 .77 heart progenitors present in the heart . It is Applicant' s belief mm and 4 .78 ). See FIG . 8E . Transplantation of CPs signifi that the drug worked on different cells of the heart . Here , the cantly improved LVEF and LVFS ( 56 .8 + - 1 .32 % ; 25 . 5 + - 0 . composition and methods predesigned the iPS cells into 4 % ) in comparison with the DMEM injected infarcted hearts cardiac progenitors in the dish and then reintroduced into ( n = 4 ; 32. 42 + - 1 . 03 % and 13. 0 + - 0 . 4 % respectively ). FIG . damaged hearts . 8F . [0337 ] Applicant also found that IPS pretreatment with [0331 ] Applicant also discovered that isoxazoles induced small molecule reduced the apoptosis which is also novel DNA hypomethylation and myc dependent cell transforma finding. For example , less apoptosis was observed in small tion in the iPS cells and were associated with DNA synthe molecule induced iPS cells as compared to non - induced iPS sis , cell proliferation , cell matrix interaction and chemoteris . cells . The results reported in the ' 951 patent were generated The isoxazoles compound upregulated the CXC chemokine only using the Trypan blue exclusion assay for cell viability receptors and integral membrane proteins, Epherin family not cell apoptosis which is critical in cell survival under and related receptors that specifically involved in the devel ischemic condition . opment of erythropoiesis . (0338 ] Applicant also found that DNA methyltransferase [ 0332 ] Applicant further discovered that isoxazoles ( DNMT) activity was completely abolished in the chemi upregulated cardiac specific miR -133 , miR - 762 and down cally modified IPS cells . There was 2 - 3 folds downregula regulation of pluripotency associated miR - 290 cluster and tion of Dnmt1, Dnmt3b and Max gene associated protein in let - 7 family in iPS cells as compared to untreated iPS cells . small molecule modified iPS cells which are associated with 10333 ) Thus, the above reports that small molecule -medi DNA hypomethylation and cell transformation . There was ated modification of iPS cells can grow in the infarcted heart 2 - 3 folds upregulation of CCL7 , CXCR2, CXCR5 , integral and replace the scar tissue with working myocytes coupled membrane protein 2A , and EphrinA3 which are associated together with electrical connections ( gap junctions ) . The with cell mobilization , chemotaxis , cancer and erythropoi sulfonyl- hydrazone family of small molecules can induce esis was also observed . Applicant further observed upregu cardiac genes in iPS cells derived from myoblasts . These lation of cardiogenic specific miR - 133 , miR - 762 and down small molecules are known to induce muscle differentiation regulation of pluripotency markers and pluripotency asso in Notch activated epicardium derived progenitors ( Russell, ciated miR - 290 - 295 cluster and let - 7 family which confirms US 2018 /0273906 A1 Sep . 27 , 2018 39 the induction of the cardiac regulatory genes by Micro USA ), 0 .2 mM L - glutamine ( Invitrogen , USA ), 0 . 1 mM RNAs by down regulating the pluripotency genes . B -mercaptoethanol ( Invitrogen , CA , USA ) and 1000 U /ml [ 0339 ] In addition , the ' 951 patent didn 't observe Ga LIF (Millipore ) 0 . 5 % penicillin and streptomycin . The colo protein level , as compared to the work reported here, which nies thus generated were detached regularly at an interval of did observe the Ga protein level upregulation which was 3 - 4 days with 0 . 2 % collagenase - 1V ( Invitrogen , CA , USA ) blocked when the chemically modified iPS cells were treated dissociated into single cell suspension with 0 .025 % trypsin with GPCR blocker and abolished the all in vitro effect in (Sigma Aldrich , MO , USA ) and re -plated onto MEFs for small molecule induced iPS cells . Oslon previously has propagation . reported the use of these compounds in neurogenesis , epi cardial progenitors cells . Regardless of the previous studies, Cell Proliferation Assay it is Applicant' s belief that the current findings are unique [0344 ] The cell proliferation assay was performed with the and innovative that address the small molecule induced use of the MTS [ 3 - ( 4 , 5 -dimethylthiazol - 2 - yl) - 5 - ( 3 - car epigenetic changes that can be manipulated for rendering the boxymethoxyphenyl) - 2 - ( 4 -sulfophenyl ) - 2H - tetrazolium ] iPS cells suitable for propagation in the infarcted heart . assay according to the manufacturer ' s recommendations Another novelty is shown here that initially iPS cells primed ( Promega ) . The plates were read at 490 nm using an auto with isoxazoles and similar small molecules can be con verted into vascular ( endothelial) progenitors and smooth mated ELISA plate - reader for the quantity of formazan muscle cells as well besides myocytes . product which was directly proportional to the number of [ 0340] To the best of Applicant' s knowledge and the living cells in culture. results reported in Example 2 , below , that isoxazoles and DNA Methyltransferase (DNMT ) Activity Assay other similar small molecules work on other stem cells from the body. For example , stem cells derived from the bone [0345 ] Nuclear extracts were isolated using the NE - PER marrow and the heart can be directly reprogrammed into Nuclear and Cytoplasmic Extraction Kit ( Thermo Scientific , myoctyes or endothelial cells and smooth muscles with IL USA ) . Total DNMT activity was determined using an small molecule drugs like isoxazoles . Thus, isoxazoles and EpiQuik DNA methyltransferase activity assay kit (Epigen similar small molecules can be directly administered , e . g ., tek , Brooklyn , N . Y . ) per manufacturer ' s protocol. Enzyme by injection into the hearts of patients, e . g . , heart attack activity for samples and controls was measured on a patients , for converting inflammatory cells and stem cells microplate reader (Hidex Chameleon , Finland ) at 450 nm mobilized to the ischemic sites into cardiac cells in order to and DNMT activity (OD / h /ml ) was calculated according to replace the scar tissue . The net effect is to assist with the the formula : (Sample OD -blank OD )/ ( sample volume) regrowth of the damaged heart after the heart attack . The 1000 . disclosed methods have the advantage of being non - viral based , using a previously approved compound , that simpli RT- PCR and Quantitative RT- PCR fies clinical adoptions. Moreover , it is Applicant' s belief that [ 0346 ] Total RNA from small molecule treated and non the disclosed methods will dramatically increase cardiac treated SiPS cells was isolated using RNeasy mini kit progenitors in one step treatment of iPSC in mono layer (Qiagen , Maryland , USA ) and Omniscript Reverse Tran without coverting them into embryoid bodies within a few scription kit ( Qiagen , Maryland , USA ) was used for the short weeks after treating with isoxazoles or other similar respective cDNA synthesis per manufacturer ' s instructions . small molecules like cardionogin ; CDNG1/ vuc230 , For PCR amplification , 1 ug of the cDNA from the reverse CDNG2/ vuc198 , and CDNG3/ vuc247 . transcription reaction was added to PCR mix containing the [ 0341] Current approaches in using iPS cells include limi suggested quantity of the PCR buffer, Q solution , dNTP mix , tations such as genetic mutations and or tumor ( i. e ., cancer ) reverse and forward primers , Taq DNA polymerase and growth versus Applicant' s methods , approach and tech distilled water. PCR conditions included initial denaturation nique . The methods are beneficial in that genetic mutations at 95° C . for 4 minutes, 32 cycles of denaturation at 95° C . are not necessary , improving safety for clinical use. for 1 minute , annealing at 55° C . for 1 minute , extension at [ 0342 ] It is Applicant' s belief and to the best of his 72° C . for 1 minute and final extension at 72° C . for 7 knowledge , the current reported study provides the first minutes . The PCR products were separated on 1 . 5 % agarose evidence that iPS cells can be therapeutically rendered safe gel , stained with ethidium bromide and visualized and for use by altering their chromatin configuration for upregu photographed on a UV transluminator (Bio - Rad , USA ) . lation of cardiac genes . These so called cardiac progenitors can propagate and regenerate the dying myocardium with Myocardial Infarction Model limited cell death . Chemical reprogramming iPS cells to [0347 ] The animals were anesthetized with (ketamine / desired cardiac lineage would be smart strategy in cardiac xylazine 0 .05 ml intra -peritonealy ) . A midline cervical skin therapeutics . incision was performed for intubation . The animals were mechanically ventilated with room air supplemented with Experimental Methods oxygen ( 1 . 5 L /min ) using a rodent ventilator (Model 683 , Harvard Apparatus , MA , USA ). Body temperature was Maintenance of Mouse SiPS carefully monitored with a probe (Cole -Parmer Instrument, [ 0343] SiPS were maintained on mitomycin C - treated IL , USA ) and was maintained at 37° C . throughout the mouse embryonic fibroblasts (MEFs ) dishes in Knock out surgical procedure. The heart was exposed by left - sided Dulbecco ' s Modified Eagle ' s Medium (knock out- DMEM , limited thoracotomy and the left anterior descending (LAD ) Invitrogen , CA , USA ) supplemented with 20 % Knockout coronary artery was ligated with a prolene # 9 - 0 suture . Serum Replacement (KSR ; Invitrogen , USA ) , 0 . 1 mM Myocardial ischemia was confirmed by color change of the MEM Non -Essential Amino Acids solution (Invitrogen , CA , left ventricular wall . The animals were grouped ( n = 12 per US 2018 /0273906 A1 Sep . 27 , 2018 40 group ) for intramyocardial injection of 10 uL of basal following relations LVFS = ( LVEDd2LVESD ) /LVEDd6100 DMEM without cells ( group -1 ) or containing 3 .6x10 ^ 5 SiPS and LVEF = [ (LVEDd32LVESd3 ) /LVEDd3 ] 6100 and (group - 2 ) or 3 .6x10 ^ 5 SiPS -CPs (group - 3 ) . The cells were expressed as percentages. injected 10 minutes after coronary artery ligation at multiple Immunocytochemistry sites ( 3 - 4 sites per heart ) around the periphery of ischemic area of the free wall of the left ventricle under direct vision . [0349 ] For immunocytochemistry, differentiated colonies For post - engraftment tracking of the transplanted cells and of SiPS were immunostained with respective specific pri determination of their fate, the cells were labeled with mary antibodies ( anti -Oct3 / 4 , anti -Sox2 , anti Nanog anti PKH26 (Sigma , Product # PKH26 -GL ) according to manu bodies, all at 1: 100 concentration ; Cell Signaling , Danvers , USA ) . Small molecule treated SIPs were seeded on 0 . 1 % facturer ' s instructions. The chest was closed and the animals gelatin coated chambered slides for immunostaining. The were allowed to recover. To alleviate pain , Buprinex ( 0 .05 cells were fixed with PBS containing 4 % paraformaldehyde ml) was injected subcutaneously in first 24 hours of surgery . for 10 minutes at room temperature . After washing with The animals were euthanized on 7 days 4 weeks and 6 - 8 PBS , the cells were blocked for 45 minutes at room tem weeks after transthoracic echocardiography for the heart perature by CAS block ( Invitrogen , CA , USA ) and were function evaluation . The hearts were frozen or fixed with immunostained with antigen specific primary antibodies 10 % formalin solution and processed for embedding in Nkx - 2 . 5 , Gata 4 , a Sarcomeric actin . ( Santa Cruz , Calif . , paraffin for immunohistological studies. USA ) . The primary antibody -antigen reaction was detected with fluorescently conjugated specific secondary antibodies. Transthoracic Echocardiography Nuclei were stained with 5 ug /ml 4 '6 - diamidino - 2 - phenyl indole (DAPI ; Invitrogen , CA , USA ) staining . Fluorescence [ 0348 ] The animals (n = 8 per group ) were anesthetized and signals were observed and photographed using fluorescence lightly secured in the supine position on a warm pad . After microscopy (Olympus ; Tokyo , Japan ). the chest was shaven , Acoustic gel was applied and transt Gene Expression Profiling horacic echocardiography was performed using HDI- 5000 [0350 ) Affymetrix array -based gene expression profiling SONOS - CT (HP ) ultrasound machine with a 7 -MHz trans further confirmed 2 - 3 folds downregulation of Dnmt1 , ducer. The heart was imaged in the two - dimensional mode Dnmt3b and Max gene associated protein which were asso in the parasternal long -axis and /or parasternal short - axis ciated with global DNA hypomethylation and myc depen views which were subsequently used to position the dent cell transformation . In addition , there was 2 - 3 folds M -mode cursor perpendicular to the ventricular septum and concomitant upregulation of CCL7 , CXCR2 , CXCR5 , inte left ventricle posterior wall , after which M -mode images gral membrane protein 2A , and EphrinA3. TABLE 1 Fold change

IPS + small IPS molecule Regulation Genes description Genes symble 1 .7296195 3 . 3164034 up Chemokine ( C - C motif ) ligand 7 Cd7 1 .4497509 2 . 7316089 up Chemokine ( C — X — C motif ) ligandCxd2 1 . 0954261 2 . 136762 up Chemokine (C - X - C motif) ligandCxd5 1 . 1732435 2 .2551816 up integral membrane protein 2A Itm2a 1 . 1245799 2 . 1803806 up integralmembrane 2B Itm2b 1 . 0622039 2 .088119 up ephrin A3 Efna3 1 .5880175 3 . 0063593 up COX16 cytochrome c oxidase Cox16 assembly homolog 1 .3481197 2 . 5458012 up cytochrome c oxidase , subunit Cox6b1 Vlb polypeptide 1 / Electron Transport Chain 1 .2005587 2 . 2982864 up ubiquinol- cytochrome c Uqcr10 reductase , complex III subunit X 3 .056267 - 1 .6117706 down DNA methyltransferase Dnmt1 2 .431835 - 1 . 2820454 down DNA methyltransferase 3B Dnmt3b 2 . 1499553 - 1 . 1043067 down MAX gene associated protein Mga were obtained . For each animal, measurements were Experiment No . 2 obtained from 4 - 5 consecutive heart cycles. Measurements of ventricular septal thickness (VST ) , left ventricle internal [0351 ) Applicant has identified a bone marrow stem cell dimension (LVID ), and left ventricle posterior wall thick population named small juvenile stem cells (SJSCs ) from ness (LVPW ) were made from two - dimensionally directed aged mouse which express pluripotency and cardiac mark M -mode images of the left ventricle in both systole and ers . Applicant expects when these cells are treated with diastole . The average value from all measurements in an isoxazole compound , would be more appropriate for cardiac animal were used to determine the indices of left ventricle differentiation and can be used as an alternate to IPS cells contractile function , i . e . , left ventricle fractional shortening (Igura , K . et al. ( 2013 ) Am J Physiol Heart Circ Physiol. ( LVFS ) and left ventricle ejection fraction (LVEF ) using the 305 ( 9 ) :H1354 - H1362 ) . US 2018 /0273906 A1 Sep . 27 , 2018

Experiment No. 3 hr followed by Eles ( ElesCSCs) using a culture cell pacer [0352 ] This experiment discloses an alternate method of system (IonOptix ). Cells were subjected to EleS for 0 , 1, and generating cardiac progenitors by preconditioning with elec 3 hr at 1. 5 V / 1 .8 cm with biphasic square pulse ( 5 ms ) at 5 trical stimulation supplemented with cardiogenic small mol Hz frequency . Cells without Eles (Non -ElesCSC ) were used ecules. Applicant identified another cell population (Kim , S . as baseline controls . The cells were later harvested and used W . et al . (2013 ) Cardiovasc Res . 100 ( 2 ) :241 - 251) that for various molecular and cellular studies . expresses hematopoietic progenitor marker ( c -kit ), pluripo tency markers ( Oct- 4 , Sox2 , Nanog ) , a stem cell side popu Experiment No. 4 lation marker (Bcrpl ) , early cardiac lineage markers (Nkx [0355 ] Isolation of Old Mesenchymal Stem Cells (OM 2 .5 , GATAL , MEF2C ), and a vascular progenitor marker SCs) and Young Mesenchymal Stem Cells (YMSCs ) from ( Fiki ) . Pre - conditioning (PC ) of stem cells either through a Bone Marrow brief period of ischaemia /anoxia or treatment with alterna [0356 ] This study followed the Guide for the Care and Use tive mimetic improves their post - engraftment survival and of Laboratory Animals published by the US National Insti differentiation characteristics . However there is no known tutes of Health (NIH Publication # 85 - 23 , revised 1985 ) and report regarding the role of PC with electric stimulation protocol approved by the Institutional Animal Care and Use ( EleS ) in stem cell survival , adhesion and cardiac differen Committee , University of Cincinnati . BMSCs were isolated tiation . The heart generates a constant electrical field , the from bone marrow of C57BL /6 mice (young ; 2 months , old ; effect of which has not been explored in stem cells prior to 24 months, respectively ) as described previously in Igura K , transplantation . This study demonstrated that EleS provided et al . ( 2013 ) Am J Physiol. Heart Circ . Physiol 305 :H1354 PC effects on the survival of cardiac stem cells (Sca - 1 + H1362 . Briefly , BMSCs were cultured in 100 mm dishes CSCs) through an increase in cell adhesion via focal adhe with low glucose Dulbecco ' s modified Eagle ' s medium sion kinase (FAK ) activation , and releasing connective tis (DMEM ) (HyClone Laboratories , Logan , Utah , http :/ /www . sue growth factor (CTGF ) by miR -378 down - regulation . It hyclone . com ) supplemented with 10 % fetal bovine serum was found that connective tissue growth factor (Ctgf ) was (FBS ) for 6 - 10 days. The nonadherent hematopoietic bone responsible for EleS - induced CSC (Eles CSCS ) survival and marrow cells were discarded during the routine fresh adhesion . Importantly , knockdown of Ctgf abolished EleS medium replacement. YMSCs were isolated from old bone induced cytoprotective effects and recovery of cardiac func marrow by sieving through wells with 3 -um pores ( cell tion . Furthermore, miR - 378 was identified as a potential culture insert; BD Bioscience, San Diego , Calif . , http :/ / Ctgf regulator in Ele - CSCs . This is another stem cell type www .bdbiosciences . com ) . The homogenicity of YMSC which expresses both pluripotency and cardiac genes , and population was confirmed with surface marker expression these cells can be further exploited with isoxazoles for by fluorescence activated cell sorting analysis as described cardiac progenitors . previously in Igura , K . et al . (2013 ) Am J Physiol Heart Circ Physiol 305 :H1354 -H1362 . Isolation of Sca - 1 + CSCs [0353 ] C57BL6 mice ( Harlan ) were used for isolation of Quantitative Fluorescence In Situ Hybridization for CSCs. 12 weeks old C57BL6 mice were anesthetized by Telomere Length Measurement intraperitoneal injection of ketamine /xylazine ( 87 - 100 mg [0357 ) Telomere was detected by fluorescence in situ and 13 - 15 mg/ kg , respectively ) . The depth of anesthesia was hybridization (FISH ) with PNA Telomere Cy3 - labeled probe monitored by positive toe pinch and muscle relaxation . (F1002 ; TelC - Cy3 , PNA Bio ) according to manufacturer ' s Hearts were extracted and washed with ice -cold PBS to instructions. Briefly , cells were fixed with 4 % paraformal remove blood cells . After removal of aorta , pulmonary dehyde for 1 hour. After washing with phosphate buffer artery , and pericardium , the whole hearts were minced and solution (PBS ), cells were incubated with RNase 100 ng / 1 L digested for 20 min at 37° C . with 0 . 1 % type - II collagenase for 20 minutes and 0 .005 % pepsin for 5 minutes at 37° C . ( Invitrogen ) and 0 .01 % DNase I (Worthington Biochemical After dehydration with 70 % , 85 % , and 100 % of cold etha Corporation ) . The cells obtained were passed through 40 um nol, cells were incubated with 200 nM PNA Telomere probe filter to remove the debris , fractionated with 70 % Percoll for 10 minutes at 85° C . and incubated for 2 hours at room (Fluka ) and cultured in maintenance medium containing temperature . Then , cells were incubated with 2 3 saline serum - free DMEM /F12 ( Invitrogen ) supplemented with sodium citrate (SSC ) buffer for 10 minutes at 60° C . B27 ( Invitrogen ) , 20 ng /ml EGF ( Sigma ) , and 40 ng /ml 4 ', 6 -diamidino -2 -phenylindole (DAPI ) and Cy3 signals bFGF (basic fibroblast growth factor, Peprotech ) . One week were acquired simultaneously into separate channels using a later , the cells were transferred to new dishes with serum confocal microscope (Fluoview FV1000 , Olympus , Tokyo , free maintenancemedium with a density of 100 cells /cm² to http :/ / www .olympus - global. com ), and maximum projection initiate colony formation and each colony was mechanically from image stacks ( 10 sections at steps of 1 mm ) were picked up for individual sub - culture in 24 well dishes in generated for image quantification . Telomere length was DMEM / F12 ( Invitrogen ) supplemented with 2 % FBS , B27 analyzed by using TFL - TeloV2 - 2 free software (Vancouver , supplement, 20 ng /ml EGF, 40 ng/ ml bFGF, and 10 ng /ml Canada ) . With the program , the integrated fluorescence LIF ( Leukemia inhibitory factor , Millipore ) . Colony - derived intensity value for each telomere, which is proportional to cells were re- seeded on new dishes at 90 % confluence and the number of hybridized probes, is calculated and pre were maintained with DMEM /F12 with 2 % FB S . sented . TFL - Telo is an application program used to estimate the length of telomeres from captured images of metaphases Eles of CSCS that have been stained for quantitative FISH ( Q - FISH ) [0354 ] Twenty four hours after seeding at a cell density of analysis as described in Poon et al . ( 1999 ) Cytometry 3x10 cells / 35 mm dish , the cells were serum - starved for 15 36 : 267 -278 . US 2018 /0273906 A1 Sep . 27 , 2018

Reverse Transcription - Polymerase Chain Reaction Analysis Lentivirus -Mediated miR - 195 Inhibition in OMSCs [0363 ] Lentivirus containing miR - 195 inhibitor - express [0358 ] Total RNA was isolated from various treatment ing vector was generated by using Lenti - Pac HIV Expres groups of the cells with RNeasy Mini Kit (Qiagen , MD , sion Packaging System (GeneCopoeia ) per manufacturer 's http :/ / wwwl. qiagen . com ) , and cDNA was prepared using protocol. Briefly, 2 . 5 ul of lentiviral miR - 195 inhibitor Omniscript -RT Kit ( Qiagen ) , according to the manufactur expression plasmid or scramble , 5 . 0 mL of EndoFectin Lenti er ' s instructions . For polymerase chain reaction ( PCR ) and EndoFectin Lenti reagent were added in Opti -MEM I , amplification , 1 ug of the cDNA from the reverse transcrip and formed the DNA -EndoFectine complex . After incubat tion reaction was then added to a PCR mix containing the ing the complex at room temperature for 10 - 25 minutes, the suggested quantity of Qiagen PCR buffer , Q -Solution , dNTP DNA - EndoFectine complex was added to 293Ta cells in mix , reverse and forward primers, Taq DNA polymerase , DMEM with 10 % FBS, and then , incubated in 5 % CO2 at and distilled water. Each PCR reaction was performed with 37° C . overnight. The culture medium was replaced with specific primers . fresh DMEM with 5 % FBS and 1 /500 volume of the Isolation and Detection ofmiRNA TiterBoost reagent to the culture medium . The virus [0359 ] Extraction of miRNAs was performed by using pseudovirus - containing culture medium was collected at 48 mirVana miRNA Isolation Kit, and miR - 195 expression was hours post- transfection and concentrated after filtration . For detected by using mirVana qRT- PCR miRNA Detection Kit the transduction of OMSCs with lentivirus , 10x10 of (Ambion , Life Technologies, Austin , Tex . , http : / /www .am OMSCs was plated , and 20 ul of virus suspension was bion .com ) and QuantiTect SYBR green PCR kit (Qiagen ) as added . To enhance lentiviral transduction efficiency, cells previously described in Kim et al . (2012 ) J . Mol. Med . were placed at 4° C . for 2 hours and then incubated in a 5 % CO2 at 37° C . for 48 hours/ Senescence - Associated B -Ga 90 : 997 - 1010 . Specific miRNA primers were purchased from lactosidase (gal ) Staining Senescence -associated B - gal was Ambion . detected by Senescence Detection Kit (BioVision , CA , miRNA Microarray http : / /www .biovision .com / ) per manufacturer' s instruction . [ 0360 ] Total RNA samples obtained from OMSCs and Terminal Deoxynucleotidyl Transferase dUTP Nick End YMSCs were sent to Exiqon (Denmark , http : / /www . exiqon . Labeling Assay com /) for miRNA microarray profiling. Data were analyzed [0364 ) Apoptotic cell death was detected by terminal by Exiqon with in - house developed computer programs. deoxynucleotidyl transferase DUTP nick end labeling Intensity values were transformed into log 2 scale , and fold ( TUNEL ) according to instructions of the manufacturer changes were given in log 2 scale . At test was performed ( TMR Red ; Roche Applied Science , http :/ / www . roche - ap between OMSCs and YMSCs profiling, and statistically plied - science .com ) . For quantification , the numbers of significant difference was considered at p < 0 . 01 . TUNEL positive cells were counted in at least five randomly FISH to Detect miR - 195 selected high -power fields (magnification 3 200 ) with three [0361 ] In situ detection of miR - 195 was performed in independent samples . OMSCs and YMSCs plated on chamber slide. Samples were Western Blot Analysis fixed in 4 % para - formaldehyde for cell culture at room temperature for 20 minutes followed by two washes in PBS . [0365 ] Western blot was carried out as previously Fixed cells were then prehybridized in hybridization solu described in Kim et al. ( 2009 ) Cardiovasc Res . 100 : 241 - 251 . tion (BioChain , CA ) for 3 hours at room temperature before Briefly , cells were lysed in lysis buffer , pH 7 .4 [( in mM ) 50 hybridization . Probe ( 3 pmol ; LNA -modified and fluores HEPES , 5 EDTA , 50 NaCl] , 1 % Triton X - 100 , protease cein isothiocyanate (FITC ) - labeled oligonucleotide ; pur inhibitors [ ( 10 mg/mL aprotinin , 1 mM phenylmethylsulfo chased from Exiqon ) complementary to miR - 195 was nyl fluoride , 10 mg/ mL leupeptin ) and phosphatase inhibi hybridized to the cells for 13 - 16 hours at 22° C . lower than tors [ in mM ) 50 sodium fluoride , 1 sodium orthovanadate , predicted Tm of the probe . Subsequent to post hybridization 10 sodium pyrophosphate ) . The protein samples (40 mg) washes with SSC buffer, in situ hybridization signals were were electrophoresed using SDS - polyacrylamide gel and detected with confocal microscope ( Fluoview FV1000 , electroimmunoblotted . The specific antibodies used for the detection of Tert ( SC - 68720 ) was purchased from Santa Cruz Olympus ). ( Santa Cruz , Calif . , http : / /www . scbt . com ), p53 ( # 2524 ) , Sirt- 1 ( # 2028 ) , Phospho -Fox01 ( # 2599 ) , Bcl- 2 ( # 3498 ) , Luciferase Activity Assay cleaved caspase -3 (# 9661) , Akt ( # 2920 ), Phospho Akt [0362 ] Precursor miR - 195 expression vector was con ( # 4060 ), and Actin (# 4968) were purchased from Cell Sig structed in a feline immunodeficiency virus -based lentiviral naling (Beverly , Mass ., http : / /www .cellsignal . com ) . All anti vector system (Geneco -poeia ) . Luciferase reporter con bodies were used as diluted with 1: 1, 000 . structs containing 3 '- untranslational region (UTR ) of mouse Tert was designed to encompass mmu -miR - 195 binding Determination of Cell Proliferation Rate sites . Cells were plated into 24 -well plates in triplicate and [0366 ] Cell proliferation rate was determined by cell cotransfected with miR - 195 vector (or scramble vector ) and growth assay and colony formation assay as previously reporter construct with Lipofectamine 2000 (Invitrogen , reported in Igura , K . et al . ( 2013 ) Heart Circ . Physiol. Carlsbad , Calif. , http : / / www . invitrogen . com ) . Firefly 305 :H1354 - 1362 . luciferase activities were measured by using Dual Luciferase Reporter Assay System kit (Promega , Madison , Wis ., http : // Experimental Model of Acute Myocardial Infarction and www .promega . com ) per manufacturer' s instructions. Trans Cell Transplantation fection efficiency was normalized by Renilla luciferase [0367 ] In vivo cell transplantation using mouse acute activity. myocardial infarction ( AMI) model was performed in accor US 2018 /0273906 A1 Sep . 27 , 2018 43 dance with Applicant ' s previous report in Kim et al. ( 2013 ) OMSCs to achieve abrogation of miR - 195 (FIG . 4 ). Cardiovasc . Res. 100 :241 - 251. Briefly , C57BL / 6 mice (male mCherry signal ( red fluorescence ) in this vector system 24 months old , 30 - 35 g b . wt. , n 5 10 animals per group ) allowed Applicant to recognize the transfected OSMCs with were prepared for left anterior descending (LAD ) coronary miR - 195 inhibitor or scramble vector (FIG . 13B , upper artery ligation . Intraperitoneal anesthesia was administered panel ). Interestingly , abrogation of miR - 195 significantly with 0 . 1 % of ketamine and 0 . 02 % of xylene per body weight reduced senescence -associated B - gal expression in OSMCs ( g ) for anesthesia . After endotracheal intubation and as compared to scramble transfected OMSCs (FIG . 13B , mechanical ventilation using Rodent Ventilator (Harvard 20 .6 % 64. 9 % vs. 54 .5 % 68 . 1 % , p < 0 .01 ). More importantly , Apparatus Model 683 ) , the heart was exposed by left - sided OMSCs transfected with miR - 195 inhibitor induced signifi minimal thoracotomy and LAD coronary artery was ligated cant telomere relengthening ( 2 . 9 - fold higher as compared to with 6 - 0 silk . Immediately after ligation , 20 ul DMEM scramble transfection , FIG . 13C ). Furthermore , inhibition of without cells or containing 2x10 cells ( scrOMSCs or anti - miR - 195 reduced TUNEL -positive apoptotic cell death in 1950MSCs ) were injected into infarction and border zones . OMSCs (FIG . 13D , 19 . 7 % 6 4 . 5 % vs . 60 . 8 % 6 2 . 7 % , Following injection , the opened chests ofmice were sutured p < 0 .01 ) , suggesting that rejuvenated OMSCs by miR - 195 and all mice were allowed to recover. abrogation survive better under apoptotic condition such as miR - 195 Expression is Markedly Upregulated in OMSCs ischemia . Furthermore , expression of antiaging makers ( Tert [ 0368 ] To profile miRNA expression induced with aging, and Sirt - 1 ) and prosurvival markers ( p - Akt and Bcl- 2 ) were Applicant isolated total RNA from OMSCs and YMSCs, and significantly increased by transfection of anti -miR - 195 in performed microarray analysis in OMSCs and YMSCs) . Per OMSCs whereas expression of senescence -associated mark these results , the expression of miR -140 , miR - 146a /b , and ers (p53 ) and proapoptotic marker ( cleaved caspase - 3) was miR - 195 was significantly upregulated in OMSCs whereas markedly reduced by miR - 195 abrogation (FIG . 13E ), sup expression of miR - 29b , miR - 205 , miR - 378 , and miR - 542 porting Applicant' s hypothesis that inhibition of age- in 3p was down regulated in OMSCs. Reverse transcriptase duced miR - 195 can rejuvenate OMSCs by reactivation of PCR (RT - PCR ) and real time PCR confirmed microarray antiaging factors and suppression of senescence -associated result that miR - 195 expression was significantly upregulated markers . It is important to note that knockdown of miR - 195 in OMSCs as compared to YMSCs. FISH analysis for significantly restored the impaired cell proliferative abilities miR - 195 visualization further confirmed that miR - 195 was in OMSCs as evaluated by cell proliferation assay and highly expressed in OMSCs. This result led Applicant to colony formation assay ( FIGS . 13F and 13G ). These results hypothesize that miR - 195 was induced with aging , and its demonstrated that miR - 195 induced during stem cell aging abrogation in OMSCs may restore the regenerative capacity plays critical roles in their fate and behaviors . of OMSCs. Applicant transfected OMSCs with anti- miR 195 and confirmed that transfected OMSCs showed reduced Experiment No. 5 expression of miR - 195 while scramble did not affect it , as [0372 ] Generation of Cardiac Progenitor Cells from shown by real- time RT - PCR , indicating that miR - 195 inhibi Human iPS Cells (hiPSCs ) and Generation of Cells of tor Applicant used was specific for reduction of miR - 195 Multiple Cell Lineages Therefrom expression in stem cells . [0373 ] Cells of Human iPSC line (ACS - 1021TM ) from Tert is a Direct Target of miR - 195 in Aging Stem Cells American Type Culture Collection , Manassas, Va. , 20110 [ 0369] To investigate the biological relevance of miR - 195 USA ( ATCC ) were maintained on a vitronectin coated induction during stem cell aging and its participation in six -well plate in mTeSR1 medium and dissociated into OMSCs senescence , Applicant first carried out target pre single cells using accutase ( Invitrogen ) at 37° C . for 10 min . diction analysis by in silico search to find potential target Afterwards, the cells were seeded on to a vitronectin - coated genes of miR -195 responsible for aging and senescence . six -well plate at 1x106 cell /well in mTeSR1 supplemented [0370 ] Interestingly, computational analysis predicted that with 5 UM ROCK inhibitor ( Y - 27632 , StemCell Technolo mmu -miR - 195 directly binds to 3 ' -UTR of mouse Tert gene . gies, Vancouver , British Columbia , Canada ) (day -3 ) for 24 To experimentally validate this result , Applicant performed hours. Further, the pluripotency of those iPS cells were RT- PCR and Western blot analysis to examine whether Tert confirmed by immunostaining , for example , to confirm the expression is altered by miR - 195 knockdown in OMSCs. marker expression for OCT4, SOX2 , TRA - 1 - 60 , TRA - 1 -81 Interestingly, expression of both mRNA and protein of Tert and SSEA4 ( FIGS. 14A - 14B ) . was significantly increased by miR - 195 abrogation in [0374 ] The cells then were then cultured in mTesR1 OMSCs (FIGS . 12B and 12C ), indicating that Tert is a medium , which was changed daily according to an exem putative target ofmiR - 195 . Tert as a target gene of miR - 195 plary schematic outline as shown in FIG . 14C . At day O , the was confirmed by luciferase activity assay which showed medium was changed to RPMI/ B27 medium minus insulin that cotransfection of a miR - 195 expression vector (PEZX supplemented with 20 uM ISX - 9 (StemCell Technologies ) miR - 195 ) with the vector containing 3 '- UTR of Tert gene for 7 days . At the end of day 7 treatment, the expression of significantly reduced luciferase activity in comparison to CPC markers including Nkx2. 5 and GATA4 were observed cotransfection with miR - scramble vector ( pEZX -miR - Sc ) by RT- PCR and Immunostaining (FIG . 14D ). ( FIG . 12D ) . These results demonstrated that Tert is a direct 0375 ) For cardio myocyte differentiation , after 7 days target of miR - 195 in stem cell aging . ISX - 9 treatment, the medium was switched to RPMI/B27 Abrogation of miR - 195 Rejuvenates OMSCs Through Telo medium with insulin for another 7 - 10 days. At the end of 20 mere Relengthening and Antiaging Markers Reactivation days , all the cells began spontaneous beating as shown in [0371 ] To examine the mechanistic participation ofmiR FIG . 14E . 195 in stem cell aging , Applicant knocked down miR - 195 10376 ]. For endothelial cells differentiation , 2x105 /cm2 expression by using a lentiviral miR - 195 inhibitor vector ISX -9 induced CPC were cultured in EGM -2 medium ( Lenti -anti -miR - 195 ) which could successfully transfect (Lonza , Lonza Walkersville Inc ., Walkerswille Md. 21793