Reprogramming cardiomyocytes with one transcription factor

Abstract

Compositions and methods are described herein for chemically inducing cells that express a single pluripotency transcription factor to change their differentiation state and become cardiac cells, cardiac progenitor cells, cardiomyocytes, or a combination thereof.

What is claimed is: 
     
         1 . A composition comprising one or more of the following agents: a WNT agonist, a GSK3 inhibitor, a TGF-beta inhibitor, an epigenetic modifier, an adenylyl cyclase agonist, an agent that induces Oct polypeptide expression, and any combination thereof. 
     
     
         2 . The composition of  claim 1 , containing at least two of the agents, or at least three of the agents, or at least four of the agents, or at least five of the agents, or at least six of the agents. 
     
     
         3 . The composition of  claim 1 , wherein the WNT agonist is one or more of WNT-3a, a GSK-inhibitor, WNT5, WNT-6a, Norrin, or another WNT family protein. 
     
     
         4 . The composition of  claim 1 , wherein the GSK3 inhibitor is one or more of CHIR99021 (6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile); 1-azakenpaullone (9-Bromo-7,12-dihydro-pyrido[3′,2′:2,3]azepino[4,5-b]indol-6(5H)-one), BIO ((2′Z,3′E)-6-Bromoindirubin-3′-oxime); AR-A014418 (N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea); Indirubin-3′-monoxime; 5-Iodo-indirubin-3′-monoxime; kenpaullone (9-Bromo-7,12-dihydroindolo-[3,2-d] [1]benzazepin-6(5H)-one); SB-415286 (3-[4(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitro-phenyl)-1H-pyrrole-2,5-dione); SB-216763 (3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione); Maybridge SEW00923SC (2-anilino-5-phenyl-1,3,4-oxadiazole); (Z)-5-(2,3-Methylenedioxyphenyl)-imidazolidine-2,4-dione; TWS119 (3-(6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy)phenol); CHIR98014 (N2-(2-(4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-ylamino)ethyl)-5-nitropyridine-2,6-diamine); SB415286 (3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione); Tideglusib (2-(1-naphthalenyl)-4-(phenylmethyl)); LY2090314 (3-imidazo[1,2-a]pyridin-3-yl-4-[1,2,3,4-tetrahydro-2-(1-piperidinylcarbonyl)-pyrrolo[3,2,1-jk][1,4]benzodiazepin-7-yl]); lithium salt; or any combination thereof. 
     
     
         5 . The composition of  claim 1 , wherein the GSK3 inhibitor is CHIR99021. 
     
     
         6 . The composition of  claim 1 , wherein the TGFβ inhibitor is one or more of 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB 431542); 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01); 2-(3-(6-Methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (SJN 2511); 4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D 4476); 4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-quinoline (LY 364947); 2-(4-(benzo[d][1,3]dioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl)-6-methylpyridine (SB505124); 6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB 525334); 2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine (SD 208); 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, or any combination thereof. 
     
     
         7 . The composition of  claim 1 , wherein the TGFβ inhibitor is SB431542. 
     
     
         8 . The composition of  claim 1 , wherein the epigenetic modifier is a methylation modifying agent, an acetylation modifying agent, a monoamine oxidase (MAO) inhibitor, a lysine-specific demethylase 1 inhibitor, or a combination thereof. 
     
     
         9 . The composition of  claim 1 , wherein the epigenetic modifier is parnate. 
     
     
         10 . The composition of  claim 1 , wherein the adenylyl cyclase agonist is Forskolin, CGP 12177 (4-[3-[(1,1-Dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride), or a combination thereof. 
     
     
         11 . The composition of  claim 1 , wherein the adenylyl cyclase agonist is Forskolin. 
     
     
         12 . The composition of  claim 1 , comprising one or more of the following agents: SB431542 (an ALK4/5/7 inhibitor), CHIR99021 (a GSK3 inhibitor), parnate (an LSD1/KDM1 inhibitor), forskolin (an adenylyl cyclase activator), or a combination thereof. 
     
     
         13 . The composition of  claim 1 , further comprising a cell culture media. 
     
     
         14 . The composition of  claim 1 , further comprising one or more cells that comprise Oct RNA, Oct polypeptide, or a combination thereof. 
     
     
         15 . The composition of  claim 1 , further comprising one or more cells that comprise an introduced RNA. 
     
     
         16 . The composition of  claim 1 , further comprising one or more cells that comprise an introduced RNA comprising an open reading frame (ORF) for the Oct polypeptide flanked by a 5′ untranslated region (UTR) containing a translational initiation signal. 
     
     
         17 . The composition of  claim 1 , furthering comprising one or more cells, where the cells can express an Oct polypeptide. 
     
     
         18 . The composition of  claim 1 , furthering comprising one or more selected cells, wherein the one or more selected cells can express an Oct RNA from a heterologous promoter. 
     
     
         19 . The composition of  claim 1 , furthering comprising one or more selected cells, wherein the one or more selected cells can express a micro-RNA-320 from a heterologous promoter. 
     
     
         20 . The composition of  claim 1 , furthering comprising one or more non-cardiac, differentiated, adult, multipotent, unipotent, or progenitor cells. 
     
     
         21 . The composition of  claim 1 , furthering comprising one or more newborn cord blood cells, or newborn stem cells. 
     
     
         22 . The composition of  claim 1 , furthering comprising one or more allogenic or autologous cells. 
     
     
         23 . The composition of  claim 1 , further comprising a heterogeneous or homogeneous mixture of cells. 
     
     
         24 . The composition of  claim 1 , wherein each of the agents or compounds is present in an amount sufficient to reprogram one or more cells into a cardiac, cardiac progenitor or cardiomyocyte cell type. 
     
     
         25 . The composition of  claim 1 , wherein the agent(s) or compound(s) is present in an amount sufficient to induce a cell to express one or more of Myh6, Tnnt2, Ryr2, Gata4, Nkx2-5, α-Actinin, Mlc2v, Mlc2a, MY20, cMHC, MEF2c, ISL1, cTNT, cTNI, or any combination thereof. 
     
     
         26 . The composition of  claim 1 , wherein the agent(s) or compound(s) is present in an amount sufficient to induce a cell to contract or beat rhythmically; and/or to induce action potentials and calcium transients characteristic of cardiac ventricular cells. 
     
     
         27 . A cell culture media comprising the composition of  claim 1 . 
     
     
         28 . A method of generating a cardiac progenitor cell, a cardiomyocyte, or a cardiac cell comprising contacting one or more selected cells with the cell culture media of  claim 27 , to thereby generate one or more cardiac progenitor cells, cardiomyocytes, cardiac cells, or a combination thereof. 
     
     
         29 . The method of  claim 28 , wherein the one or more selected cells comprise Oct RNA molecules, Oct polypeptides, or a combination thereof, or can express Oct RNA molecules, Oct polypeptides, or a combination thereof. 
     
     
         30 . The method of  claim 28 , further comprising one or more cells that comprise an introduced RNA. 
     
     
         31 . The method of  claim 28 , further comprising one or more cells that comprise an introduced RNA comprising an open reading frame (ORF) for the Oct polypeptide flanked by a 5′ untranslated region (UTR) containing a translational initiation signal. 
     
     
         32 . The method of  claim 28 , wherein one or more of the selected cells expresses an Oct polypeptide from a heterologous promoter. 
     
     
         33 . The method of  claim 28 , wherein one or more of the selected cells transiently expresses an Oct polypeptide while contacted with the culture media. 
     
     
         34 . The method of  claim 28 , wherein one or more of the selected cells can express an Oct polypeptide but does not have a heterologous Oct nucleic acid integrated into the cell's genome. 
     
     
         35 . The method of  claim 28 , wherein one or more of the selected cells can express an Oct polypeptide from a replication-defective expression vector. 
     
     
         36 . The method of  claim 28 , wherein one or more of the selected cells are allogenic or autologous differentiated cells, non-cardiac cells, somatic cells, adult cells, or a combination thereof. 
     
     
         37 . The method of  claim 28 , wherein one or more of the selected cells is a newborn cord blood cell, or a newborn stem cell. 
     
     
         38 . The method of  claim 28 , wherein one or more of the selected cells is contacted with the composition for a time and/or with an amount of each agent sufficient to induce the selected cell to express Myh6, Tnnt2, Ryr2, Gata4, Nkx2-5, α-Actinin, Mlc2v, Mlc2a, MY20, cMHC, MEF2c, ISL1, cTNT, cTNI, or any combination thereof. 
     
     
         39 . The method of  claim 28 , furthering comprising administering one or more of the cardiac progenitor cells, the cardiomyocytes, the cardiac cells, or a combination thereof to a subject. 
     
     
         40 . The method of  claim 39 , furthering comprising administering at least about 100 of the cardiac progenitor cells, the cardiomyocytes, the cardiac cells, or a combination thereof to a subject. 
     
     
         41 . The method of  claim 40 , wherein the subject suffers or is suspected of suffering from a heart condition or disease. 
     
     
         42 . The method of  claim 40 , wherein the subject's heart is abnormally enlarged, thickened and/or stiffened. 
     
     
         43 . The method of  claim 40 , wherein the subject suffers from, or is suspected of suffering from, congestive heart failure, myocardial infarction, cardiomyopathy, cardiac ischemia, myocarditis, arrhythmia, Duchenne muscular dystrophy, Emery Dreiffuss dilated cardiomyopathy, or any combination thereof. 
     
     
         44 . A method comprising administrating the composition of  claim 1  to a subject. 
     
     
         45 . The method of  claim 44 , wherein the composition contains one or more cardiac cells, cardiac progenitor cells, mature cardiomyocytes, or a combination thereof. 
     
     
         46 . The method of  claim 44 , wherein the composition contains one or more allogenic or autologous cells. 
     
     
         47 . The method of  claim 44 , wherein the composition contains one or more cells that express Myh6, Tnnt2, Ryr2, Gata4, or a combination thereof. 
     
     
         48 . The method of  claim 44 , wherein the subject is in need of administration of the composition. 
     
     
         49 . The method of  claim 44 , wherein the subject is in need of one or more cardiac cells, cardiac progenitor cells, mature cardiomyocytes, or a combination thereof. 
     
     
         50 . The method of  claim 44 , wherein the composition and/or one or more cardiac cells, cardiac progenitor cells, mature cardiomyocytes, or a combination thereof, are administered for a time and/or in an amount of each agent per cell sufficient to reduce the symptoms of a heart condition or disease. 
     
     
         51 . A kit comprising the composition of  claim 1 , and instructions for using the composition. 
     
     
         52 . The kit of  claim 51 , further comprising components for in vitro cell culture of a selected cell. 
     
     
         53 . The kit of  claim 51 , further comprising one or more selected cells. 
     
     
         54 . The kit of  claim 51 , further comprising one or more selected cells, wherein the one or more selected cells comprise Oct RNA molecules, Oct polypeptides, or a combination thereof, or can express Oct RNA molecules, Oct polypeptides, or a combination thereof. 
     
     
         55 . The kit of  claim 51 , further comprising a cell culture medium, one or more sterile cell collection devices, or a supplementary factor. 
     
     
         56 . The kit of  claim 55 , wherein the supplementary factor comprises at least one bone morphogenic protein, brain derived neurotrophic factor, ciliary neurotrophic factor, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β, β endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor, growth related protein, heparin binding epidermal growth factor, hepatocyte growth factor, insulin-like growth factor, keratinocyte growth factor, leukemia inhibitory factor, neurotrophin, placenta growth factor, platelet-derived endothelial cell growth factor, platelet derived growth factor, pre-B cell growth stimulating factor, stem cell factor, transforming growth factor, latent transforming growth factor, transforming growth factor β binding protein, vascular endothelial growth factor or any combination thereof. 
     
     
         57 . The kit of  claim 51 , further comprising a population of cardiac progenitor cells, mature cardiomyocytes, or a combination thereof generated by contacting the cells with the composition. 
     
     
         58 . The kit of  claim 51 , further comprising a diluent, a pharmaceutically acceptable carrier, a syringe, a catheter, or a device for delivery of cells or of the composition.