Treatment of pluripotent cells

Abstract

The present invention provides a method for treating human pluripotent cells. In particular, the methods of the invention are directed to the treatment of human pluripotent cells, whereby the human pluripotent cells can be efficiently expanded in culture and differentiated by treating the pluripotent cells with an inhibitor of glycogen synthase kinase 3β (GSK-3B) enzyme activity.

What is claimed is: 
     
         1 . A method to expand and differentiate human pluripotent cells, comprising the steps of culturing pluripotent cells, and treating the pluripotent cells with an inhibitor of GSK 3B enzyme activity having the Formula III: 
       
         
           
           
               
               
           
         
         wherein A and E are each independently selected from a hydrogen substituted carbon atom and a nitrogen atom: 
       
       
         
           
           
               
               
           
         
         wherein N is 1H-indole, 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine or 1H-indazole; 
         Z is O or dihydro; 
         wherein each hydrogen atom is attached by a single bond; 
         R 4  and R 5  are independently selected from C 1-8 alkyl, C 2-8 alkenyl and C 2-8 alkynyl optionally substituted with oxo; 
         R 2  is —C 1-8 alkyl-, —C 2-8 alkenyl-, —C 2-8 alkynyl-, —O—(C 1-8 )alkyl-O—, —O—(C 2-8 )alkenyl-O—, —O—(C 2-8 )alkynyl-O—, —C(O)—(C 1-8 )alkyl-C(O)—, halogen, (halo) 1-3 (C 1-8 )alkyl, (halo) 1-3 (C 1-8 )alkoxy, hydroxy, hydroxy(C 1-8 )alkyl, oxo, cycloalkyl, heterocyclyl, aryl, heteroaryl; and 
         R 1  and R 3  are independently selected from hydrogen, C 1-8 alkyl, C 2-8 alkenyl, and C 2-8 alkynyl, amino(C 1-8 )alkyl; 
         wherein each alkyl, alkenyl and alkynyl linking group is optionally substituted with one to four substituents independently selected from heterocyclyl, aryl, heteroaryl, spirocycloalkyl, spiroheterocyclyl, heterocyclyl(C 1-8 )alkyl, aryl(C 1-8 )alkyl, heteroaryl(C 1-8 )alkyl, C 1-8 alkyl, C 1-8 alkoxy, C 1-8 alkoxy(C 1-8 )alkyl, carboxyl, carboxyl(C 1-8 )alkyl, —C(O)O—(C 1-8 )alkyl, —C 1-8 alkyl-C(O)O—(C 1-8 )alkyl, amino, amino(C 1-8 )alkyl, cyano, halogen, hydroxy and nitro; 
         wherein each amino group is optionally substituted with hydrogen and C 1-4 alkyl wherein each cycloalkyl, heterocyclyl, aryl and heteroaryl group is optionally substituted with one to four substituents independently selected from the group consisting of C 1-8 alkyl, C 1-8 alkoxy, C 1-8 alkoxy(C 1-8 )alkyl, carboxyl, carboxyl(C 1-8 )alkyl, amino, and amino(C 1-8 )alkyl, cyano, halogen, hydroxy and nitro; 
         wherein each heterocyclyl is optionally substituted with halogen, cyano, hydroxyl, nitro (halo) 1-3 (C 1-8 )alkyl, (halo) 1-3 (C 1-8 )alkoxy, hydroxy, hydroxy(C 1-8 )alkyl), oxo, —(O—(CH 2 ) 1-6 ) 0-5 —O—, —O—(CH 2 ) 1-6 —O—(CH 2 ) 1-6 —O—, —O—(CH 2 ) 1-6 —O—(CH 2 ) 1-6 —O—(CH 2 ) 1-6 —O—, —(O—(CH 2 ) 1-6 ) 0-5 —NR 6 —, —O—(CH 2 ) 16 —NR 6 —(CH 2 ) 1-6 —O—, —O—(CH 2 ) 1-6 —O—(CH 2 ) 1-6 NR 6 —, —(O—(CH 2 ) 1- ) 0-5 —S—, —O—(CH 2 ) 1-6 —S—(CH 2 ) 1-6 —O—, —O—(CH 2 ) 1-6 —O—(CH 2 ) 1-6 —S—, —NR 6 —, —NR 6 —NR 7 —, —NR 6 —(CH 2 ) 1-6 —NR 6 —, —NR 6 —(CH 2 ) 1-6 —NR 7 —(CH 2 ) 1-6 —NR 8 —, —NR 6 —C(O)—, —C(O)—NR 6 —, —C(O)—(CH 2 ) 0-6 —NR 6 —(CH 2 ) 0-6 —C(O)—, —NR 6 —(CH 2 ) 0-6 —C(O)—(CH 2 ) 1-6 —C(O)—(CH 2 ) 0-5 —NR 7 —, —NR 6 —C(O)—NR 7 —, —NR 6 —C(NR 7 )—NR 8 —, —O—(CH 2 ) 1-6 —NR 6 —(CH 2 ) 1-6 —S—, —S—(CH 2 ) 1-6 —NR 6 —(CH 2 ) 1-6 —O—, —NR 9 —C(O)—, —C(O)—NR 9 —, —S—(CH 2 ) 1-6 —NR 6 —(CH 2 ) 1-6 —S—, —NR 6 —(CH 2 ) 1-6 —S—(CH 2 ) 1-6 —NR 7 — and —SO 2 —, wherein R 6 , R 7  and R 8  are each independently selected from hydrogen, C 1-8 alkyl, C 1-8 alkoxy(C 1-8 )alkyl, carboxyl(C 1-8 )alkyl, amino(C 1-8 )alkyl, hydroxy(C 1-8 )alkyl, heterocyclyl(C 1-8 )alkyl, aryl(C 1-8 )alkyl and heteroaryl(C 1-8 )alkyl; and wherein R 9  is selected from the group consisting of C 1-8 alkyl, C 1-8 alkoxy(C 1-8 )alkyl, carboxyl(C 1-8 )alkyl, amino(C 1-8 )alkyl; 
         with the proviso that, if A and E are a hydrogen substituted carbon atom, then R 2  is —C 2-8 alkynyl-, —O—(C 1-8 )alkyl-O—, —O—(C 2-8 )alkenyl-O—, —O—(C 2-8 )alkynyl-O—, or —C(O)—(C 1-8 )alkyl-C(O)—. 
       
     
     
         2 . The method of  claim 1 , wherein the pluripotent cells are embryonic stem cells. 
     
     
         3 . The method of  claim 1 , wherein the pluripotent cells are cells expressing pluripotency markers derived from embryonic stem cells. 
     
     
         4 . The method of  claim 3 , wherein the cells expressing pluripotency markers express at least one of the following pluripotency markers selected from the group consisting of: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, and Tra1-81. 
     
     
         5 . The method of  claim 1 , wherein the pluripotent cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage. 
     
     
         6 . The method of  claim 1 , wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about one to about 72 hours. 
     
     
         7 . The method of  claim 1 , wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 48 hours. 
     
     
         8 . The method of  claim 1 , wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about 48 hours. 
     
     
         9 . The method of  claim 1 , wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 100 nM to about 100 μM. 
     
     
         10 . The method of  claim 1 , wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 1 μM to about 10 μM. 
     
     
         11 . The method of  claim 1 , wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 10 μM. 
     
     
         12 . The method of  claim 1 , wherein the compound of the Formula (III) is:
 6,7,9,10,12,13,15,16-octahydro-23H-5,26:17,22-dimetheno-5H-dipyrido[2,3-k:3′,2′-q]pyrrolo[3,4-n][1,4,7,10,19]trioxadiazacyclohenicosine-23,25(24H)-dione;   10,11,13,14,16,17,19,20,22,23-decahydro-9,4:24,29-dimetheno-1H-dipyrido[2,3-n:3′,2′-t]pyrrolo[3,4-q][11,4,7,10,13,22]tetraoxadiazacyclotetracosine-1,3(2H)-dione;   10,11,13,14,16,17,19,20,22,23,25,26-dodecahydro-9,4:27,32-dimetheno-1H-dipyrido[2,3-q: 3′,2′-w]pyrrolo[3,4-t][1,4,7,10,13,16,25] pentaoxadiazacycloheptacosine-1,3(2H)-dione;   6,7,9,10,12,13-hexahydro-20H-5,23:14,19-dimetheno-5H-dibenzo[h,n]pyrrolo[3,4-k][1,4,7,16]dioxadiazacyclooctadecine-20,22(21H)-dione;   6,7,9,10,12,13,15,16-octahydro-23H-5,26:17,22-dimetheno-5H-dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]trioxadiazacycloheneicosine-23,25(24H)-dione;   10,11,13,14,16,17,19,20,22,23-decahydro-9,4:24,29-dimetheno-1H-dibenzo[n,t]pyrrolo[3,4-q][1,4,7,10,13,22]tetraoxadiazacyclotetracosine-1,3(2H)-dione;   3-[1-[3-[(2-hydroxyethyl)methylamino]propyl]-1H-indazol-3-yl]-4-[1-(3-pyridinyl)-1H-indol-3-yl]-1H-pyrrole-2,5-dione;   3,5-dichloro-N-[3-chloro-4-[(3,4,12,12a-tetrahydro-1H-[1,4]thiazino[3,4-c][1,4]benzodiazepin-11(6H)-yl)carbonyl]phenyl]-benzamide;   3-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-2,5-dione;   3-(2-methoxy-phenyl)-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-2,5-dione;   6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile;   3-(5-chloro-1-methyl-1H-indol-3-yl)-4-[1-(3-imidazol-1-yl-propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione;   3-(5-chloro-1-methyl-1H-indol-3-yl)-4-[1-(3-[1,2,3]triazol-1-yl-propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione;   3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(1-methyl-1H-pyrazol-3-yl)-pyrrole-2,5-dione;   3-[1-(3-hydroxy-3-methyl-butyl)-1H-indazol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-2,5-dione;   3-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-4-(1-pyrimidin-5-yl-1H-indol-3-yl)-pyrrole-2,5-dione;   3-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-4-(1-pyrimidin-5-yl-1H-indol-3-yl)-pyrrole-2,5-dione;   (11Z)-8,9,10,13,14,15-hexahydro-2,6:17,21-di(metheno)pyrrolo[3,4-h][1,15,7]dioxazacyclotricosine-22,24(1H,23H)-dione;   3-(5-chloro-1-pyridin-3-yl-1H-indol-3-yl)-4-[1-(3-hydroxy-propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione;   3-(2-methoxy-phenyl)-4-[1-(3-methoxy-propyl)-1H-pyrrolo[3,2-c]pynidin-3-yl]-pyrrole-2,5-dione;   3-[1-(3-hydroxy-propyl)-1H-indazol-3-yl]-4-[1-(tetrahydro-pyran-4-yl)-1H-indol-3-yl]-pyrrole-2,5-dione;   2-{3-[4-(5-chloro-1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-indazol-1-yl}-N-(2-hydroxy-ethyl)-acetamide;   4-(3-chloro-phenyl)-6-(3-dimethylamino-propyl)-5,6-dihydro-4H-2,4,6-triaza-cyclopenta[c]fluorine-1,3-dione;   14-ethyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-dimethenodibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione;   14-benzyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26;17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione;   3-(1-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethyl}-1H-indol-3-yl)-4-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-pyrrole-2,5-dione;   6,7,8,9,10,11,12,13-octahydro-8,11-dimethyl-5,23:14,19-dimetheno-20H-dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10]tetraazacyclooctadecine-20,22(21H)-dione;   7,8,9,10,12,13,16,17,18,19-decahydro-8,17-dimethyl-15H,26H-5,29:20,25-dimetheno-6H-dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10, 19,22]dioxatetraazacyclotetracosine-26,28(27H)-dione;   14-(2-furylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione;   14-(2-thienylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][11,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione;   14-(1-naphthylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione;   14-(pyridin-4-ylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione;   3-[1-(2-{2-[2-(1,2,3,4-tetrahydro-naphthalen-1-ylamino)-ethoxy]-ethoxy}-ethyl)-1H-indol-3-yl]-4-{1-[2-(1,2,3,4-tetrahydro-naphthalen-1-ylamino)-ethyl]-1H-indol-3-yl}-pyrrole-2,5-dione;   3-[1-(3-dimethylamino-phenyl)-1H-indol-3-yl]-4-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-pyrrole-2,5-dione;   3-[5-chloro-1-(6-dimethylamino-pyridin-3-yl)-1H-indol-3-yl]-4-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-pyrrole-2,5-dione; or   5-(5-chloro-3-{4-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl}-indol-1-yl)-nicotinic acid methyl ester.   
     
     
         13 . A method to expand and differentiate human pluripotent cells comprising:
 culturing the pluripotent cells; and   treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity wherein the inhibitor is selected from the group consisting of:   3-[1-(3-hydroxy-3-methylbutyl)-1H-indazol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;   3-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-2,5-dione;   6-[(2-{[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-yl]amino}ethyl)amino]pyridine-3 carbonitrile;   3-[1-{3-[(2-hydroxyethyl)methyl)amino]propyl}-1H-indazol-3-yl)-4-(1-pyridin-3-yl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;   14-ethyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-di(metheno-dizenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione;   14-(thiophen-2-ylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione; and   14-(napthalen-1-ylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione.   
     
     
         14 . The method of  claim 13 , wherein the pluripotent cells are cells expressing pluripotency markers of embryonic stem cells. 
     
     
         15 . The method of  claim 13 , wherein the cells expressing pluripotency markers express at least one of the markers selected from the group consisting of: ABCG1, crypto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, HTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81. 
     
     
         16 . The method of  claim 13 , wherein the pluripotent cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage. 
     
     
         17 . The method of  claim 13 , wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about one to about 72 hours. 
     
     
         18 . The method of  claim 13 , wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 48 hours. 
     
     
         19 . The method of  claim 13 , wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 100 nM to about 100 μM. 
     
     
         20 . The method of  claim 13 , wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 10 μM.