Methods for creating integration-free, virus-free, exogenous oncogene-free ips cells and compositions for use in such methods

Abstract

Methods are disclosed for reprogramming a somatic cell, including an adherent cell and a cell in suspension, into an induced pluripotent stem comprising expressing exogenous Sox-2, exogenous Klf-4, exogenous Oct3/4 from DNA that has not integrated into the genome of the somatic cell, suppressing p53 activity within the somatic cell, and exposing the somatic cell to reprogramming-assistance factors comprising an exogenous Alk-5 inhibitor, an exogenous histone deacetylase inhibitor, and an exogenous activator of glycolysis. Compositions and kits for use in such methods are also disclosed as are cells made by such a method.

What is claimed is: 
     
         1 . A method for reprogramming a somatic cell comprising:
 culturing a somatic cell comprising one or more non-integrating vectors encoding Sox-2, Klf-4, and Oct3/4 in a reprogramming medium comprising an exogenous Alk-5 inhibitor, an exogenous histone deacetylase inhibitor, an exogenous activator of glycolysis, and exogenous ascorbic acid; and   obtaining an integration-free, virus-free, exogenous oncogene-free induced pluripotent stem (iPS) cell.   
     
     
         2 . The method for reprogramming a somatic cell according to  claim 1 , wherein the one or more non-integrating vectors also encode EBNA-1. 
     
     
         3 . The method for reprogramming a somatic cell according to  claim 1 , further comprising maintaining the obtained iPS cell in a dedifferentiation maintenance medium comprising basic fibroblast growth factor and transforming growth factor beta after being cultured in the reprogramming medium. 
     
     
         4 . The method for reprogramming a somatic cell according to  claim 1 , wherein the culturing does not require feeder cells. 
     
     
         5 . The method for reprogramming a somatic cell according to  claim 1 , wherein
 the Alk-5 inhibitor comprises 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide, 4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide, or 6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline;   the histone deacetylase inhibitor comprises sodium butyrate or valproic acid; and   the activator of glycolysis comprises 5-(4-Chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48); α,α,-Dimethyl-4-[2-methyl-8-[2-(3-pyridinyl)ethynyl]-1H-imidazo[4,5-c]quinolin-1-yl]-benzeneacetonitrile (BAG956); N-[3-[[5-Iodo-4-[[3-[(2-thienylcarbonyl)amino]propyl]amino]-2-pyrimidinyl]amino]phenyl]-1-pyrrolidinecarboxamide (BX795); (3S,6R)-1-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide (GSK 2334470); 2-Amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]acetamide (OSU03012); or 4-Dodecyl-N-1,3,4-thiadiazol-2-yl-benzenesulfonamide (PHT427).   
     
     
         6 . The method for reprogramming a somatic cell according to  claim 5 , wherein the Alk-5 inhibitor comprises A83-01, the histone deacetylase inhibitor comprises sodium butyrate, and the activator of glycolysis comprises PS48. 
     
     
         7 . The method for reprogramming a somatic cell according to  claim 1 , wherein the somatic cell is a human cell and reprogramming efficiency exceeds 0.0006%. 
     
     
         8 . A composition comprising a somatic cell and a cell culture medium, the somatic cell comprising episomal polynucleotides encoding Sox-2, Klf-4, and Oct3/4, and the medium comprising an exogenous Alk-5 inhibitor, an exogenous histone deacetylase inhibitor, an exogenous activator of glycolysis, and exogenous ascorbic acid. 
     
     
         9 . The composition according to  claim 8 , wherein the somatic cell further comprises an episomal polynucleotide encoding EBNA-1. 
     
     
         10 . The composition according to  claim 8 , wherein the somatic cell is a human cell. 
     
     
         11 . The composition according to  claim 8 , wherein the medium or the somatic cell further comprises an agent for inhibiting p53 activity. 
     
     
         12 . The composition according to  claim 11 , wherein the agent for inhibiting p53 activity comprises a compound selected from pifithrin-α, cyclic pifithrin-α, pifithrin-μ, RITA, SJ 172550, nutlin-3, a pharmaceutically acceptable salt of one of the aforementioned compounds, or an antisense oligonucleotide. 
     
     
         13 . The composition according to  claim 8 , wherein
 the Alk-5 inhibitor comprises 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide, 4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide, or 6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline;   the histone deacetylase inhibitor comprises sodium butyrate or valproic acid; and   the activator of glycolysis comprises 5-(4-Chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48); α,α,-Dimethyl-4-[2-methyl-8-[2-(3-pridinyl)ethynyl]-1H-imidazo[4,5-c]quinolin-1-yl]-benzeneacetonitrile (BAG956); N-[3-[[5-Iodo-4-[[3-[(2-thienylcarbonyl)amino]propyl]amino]-2-pyrimidinyl]amino]phenyl]-1-pyrrolidinecarboxamide (BX795); (3S,6R)-1-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide (GSK 2334470); 2-Amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]acetamide (OSU03012); or 4-Dodecyl-N-1,3,4-thiadiazol-2-yl-benzenesulfonamide (PHT427).   
     
     
         14 . The composition according to  claim 13 , wherein the Alk-5 inhibitor comprises A83-01, the histone deacytylase inhibitor comprises sodium butyrate, and the activator of glycolysis comprises PS48. 
     
     
         15 . A kit comprising:
 (a) one or more non-integrating vectors encoding Sox-2, Klf-4, and Oct3/4;   (b) an Alk-5 inhibitor,   (c) a histone deacetylase inhibitor,   (d) an activator of glycolysis, and   (e) ascorbic acid.   
     
     
         16 . The kit according to  claim 15 , wherein the one or more non-integrating vectors also encode EBNA-1. 
     
     
         17 . The kit according to  claim 15 , which further comprises an agent for inhibiting p53 activity. 
     
     
         18 . The kit according to  claim 17 , wherein the agent for inhibiting p53 activity comprises a compound selected from pifithrin-α, cyclic pifithrin-α, pifithrin-μ, RITA, SJ 172550, nutlin-3, a pharmaceutically acceptable salt of one of the aforementioned compounds, or an antisense oligonucleotide. 
     
     
         19 . The kit according to  claim 15 , wherein
 the Alk-5 inhibitor comprises 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide, 4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide, or 6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline;   the histone deacetylase inhibitor comprises sodium butyrate or valproic acid; and   the activator of glycolysis comprises 5-(4-Chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48); α,α,-Dimethyl-4-[2-methyl-8-[2-(3-pridinyl)ethynyl]-1H-imidazo[4,5-c]quinolin-1-yl]-benzeneacetonitrile (BAG956); N-[3-[[5-Iodo-4-[[3-[(2-thienylcarbonyl)amino]propyl]amino]-2-pyrimidinyl]amino]phenyl]-1-pyrrolidinecarboxamide (BX795); (3S,6R)-1-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide (GSK 2334470); 2-Amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]acetamide (OSU03012); or 4-Dodecyl-N-1,3,4-thiadiazol-2-yl-benzenesulfonamide (PHT427).   
     
     
         20 . The kit according to  claim 19 , wherein the Alk-5 inhibitor comprises A83-01, the histone deacetylase inhibitor comprises sodium butyrate, and the activator of glycolysis comprises PS48.