Improved methods for reprograming non-pluripotent cells into pluripotent stem cells

Abstract

Provided are chemical inducers of pluripotency (CIP) which include glycogen synthase kinase inhibitors, TGFβ receptor inhibitors, cyclic AMP agonists and S-adenosylhomocysteine hydrolase (SAH) inhibitors or histone acetylators. A method of inducing pluripotency in a partially or completely differentiated cell by using such chemical inducers of pluripotency is also provided. The method includes: (i) contacting a cell with the CIPs for a sufficient period of time to result in reprogramming the cell into a pluripotent stem cell having ESC-like characteristics (CiPSC). Isolated chemically induced pluripotent stem cells (CiPSCs) and their progeny, produced by inducing differentiation of the CiPSCs, can be used in a number of applications, including but not limited to cell therapy and tissue engineering.

1 . A kit or cell culture media composition for inducing pluripotency in non-pluripotent eukaryotic cells, the composition comprising chemical inducers of pluripotency (CIPs) from each of the following groups
 (1) glycogen synthase kinase (GSK) inhibitors,   (2) TGFβ receptor inhibitors,   (3) cyclic AMP (cAMP) agonists,   (4) S-adenosylhomocysteine hydrolase (SAH) inhibitors,   (5) histone acetylators,   (6) DOT1L methyltransferase inhibitors,   (7) Retinoic acid receptor (RAR) agonist,   (8) epigenetic modulator, and   (9) inhibitors of histone demethylation in amounts effective to induce reprogramming of XEN-like cells into pluripotent cells.   
     
     
         2 . The kit or composition of  claim 1 , wherein the GSK inhibitor is [6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile] (“C”); the TGFβ receptor inhibitor is [2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine] (“6”); the cAMP agonist is Forskolin (“F”), the SAH inhibitor is 3-deazaneplanocin A (“Z”), the DOT1L methyltransferase inhibitor is SGC 0946 (“S”) (1-[3-[[[(2R,3S,4R,5R)-5-(4-Amino-5-bromo-7H-pyrrolo[2,3-d] pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl](isopropyl)amino]propyl]-3-[4-(2,2-dimethylethyl)phenyl]urea) or EPZ004777, “1-(3-((((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea (“E”); RAR agonists include AM 580 (“A”) (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid); the epigenetic modulator is 5-azacytidine (“D”) and the inhibitor of histone demethylation is tranylcypromine (“T”) and optionally, wherein the composition comprises 2i-medium, the 2i medium optionally further comprising N2B27. 
     
     
         3 . The kit or composition of  claim 2  comprising VC6TFZASD. 
     
     
         4 . (canceled) 
     
     
         5 . The kit or composition of  claim 1 , further comprising a least one small molecule selected from the group consisting of: (i) small molecules that facilitate late reprogramming; (ii) epigenetic modulators selected from the group consisting of sodium butyrate and RG108 [N-Phthalyl-L-tryptophan] (iii) small molecules that improve/boost chemical reprogramming efficiency selected from the group consisting of SF1670 [N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide]; DY131 [N-(4-(Diethylaminobenzylidenyl)-N-(4-hydroxybenzoyl)-hydrazine]; UNC0638 [2-Cyclohexyl-6-methoxy-N-[1-(1-methylethyl)-4-piperidinyl]-7-[3-(1-pyrrolidinyl)propoxy]-4-quinazolinamine]; SRT1720 [N-(2-(3-(piperazin-1-ylmethyl)imidazo[2,1-b]thiazol-6-yl)phenyl)quinoxaline-2-carboxamide hydrochloride]; 2-Me-5HT (2-methyl-5-hydroxytryptamine); IBMX [3,7-Dihydro-1-methyl-3-(2-methylpropyl)-1H-purine-2,6-dioneand]; (4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide) and TTNPB [4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and (iv) protein growth factors. 
     
     
         6 . The composition of  claim 5  wherein the late reprogramming facilitator is selected from the group consisting of prostaglandin E2 (PGE2) and rolipram. 
     
     
         7 - 10 . (canceled) 
     
     
         11 . The kit of  claim 2  in a kit, wherein the small molecular weight compounds are present in relative amounts to put into cell culture media for differentiated cells to induce expression of XEN markers selected from the group consisting of SALL4, GATA4 and SOX1. 
     
     
         12 . A method of inducing pluripotency in partially or completely differentiated cells, the method comprising:
 (a) contacting the cell to be reprogrammed with a first cocktail of CIPs (XEN-Cocktail) for a sufficient period of time to bias the cells into a XEN-like cell population;   (b) contacting the population of XEN-like cells for a sufficient period of time to reprogram the cell into a chemically induced pluripotent stem cell (CiPSC) with a second cocktail of CIPS (herein, XEN-CiPSC cocktail); and (c) culturing the cells in 2i-medium.   
     
     
         13 . The method of  claim 12 , wherein the differentiated cells are selected from the group consisting of multipotent stem cells, cells of hematological origin, cells of embryonic origin, skin derived cells, fibroblasts, adipose cells, epithelial cells, endothelial cells, mesenchymal cells, parenchymal cells, neurological cells, and connective tissue cells. 
     
     
         14 . The method of  claim 14 , wherein the cells to be induced are selected from the group consisting of fibroblasts, adipose-derived cells, neural derived cells and intestinal epithelial cells, preferably not expressing Oct4 and optionally, wherein the cells are not transfected to express any of Oct4, KLF4, SOX2, C-Myc or NANOG. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 12 , wherein the cells to be induced are cultured in reprogramming medium comprising the CIPs for a period between 26-30 days. 
     
     
         17 . The method of  claim 12  wherein the cell culture medium comprises VC6TFA the entire time before the use of 2i-medium. 
     
     
         18 . The method of  claim 12  wherein the cells are replated at a density between 50,000 to 100,000 per well in a 6-well plate between day 6-10. 
     
     
         19 . The method of  claim 12  wherein the XEN-cocktail comprises VC6TFAE and/or the XEN-CiPSC cocktail comprises VC6TFZASD. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 12  wherein the medium is changed into 2i-medium between day 26 to and day 30 and cultured in 2i-medium for about 10-14 days and optionally, wherein the 2i-medium further comprises N2B27. 
     
     
         22 - 23 . (canceled) 
     
     
         24 . The method of  claim 12  wherein the cell culture medium further comprising small molecules that facilitate late reprogramming selected from the group consisting of cAMP agonists, epigenetic modulators and small molecules that improve/boost chemical reprogramming efficiency. 
     
     
         25 . The method of  claim 12  wherein the XEN-cocktail does not comprise SGC 0946. 
     
     
         26 . The method of  claim 12 , wherein the media comprising the small molecule that boosts reprogramming efficiency, TTNPB. 
     
     
         27 . The method of  claim 12 , comprising identifying the pluripotent cells based on morphology, doubling time, the ability of the cell to differentiate into tissues of the three embryonic germ layers, expression of ESC markers and combinations thereof wherein the ESC markers are selected from the group consisting of alkaline phosphatase (AP); nanog; Rex1; Sox2; Dax1; Sall4; undifferentiated embryonic cell transcription factor (Utf1); stage specific embryonic antigen-4 (SSEA-4) and combinations thereof; and optionally, isolating the pluripotent cells. 
     
     
         28 - 29 . (canceled) 
     
     
         30 . Pluripotent cells obtained by the method of  claim 12 . 
     
     
         31 . A therapeutic composition comprising the pluripotent cells of  claim 30 , formulated for administration to an individual by injection, implantation of a prosthetic device or tissue engineering matrix. 
     
     
         32 . The pluripotent cells of  claim 31 , wherein the the ESC markers are selected from the group consisting of alkaline phosphatase (AP); nanog; Rex1; Sox2; Dax1; Sall4; undifferentiated embryonic cell transcription factor (Utf1); stage specific embryonic antigen-4 (SSEA-4) and combinations thereof; and optionally, isolating the pluripotent cells.