Compositions and Methods for Establishing and Maintaining Stem Cells in an Undiffferentiated State

Abstract

The present invention embraces compositions and methods for establishing and maintaining stem cells and inhibiting stem cell differentiation using a selective Protein Kinase C (PKC) inhibitor.

1 . A method for maintaining a stem cell in an undifferentiated state comprising contacting an isolated stem cell with a selective Protein Kinase C inhibitor thereby maintaining the stem cell in an undifferentiated state. 
     
     
         2 . The method of  claim 1 , wherein the inhibitor inhibits at least Protein Kinase C zeta. 
     
     
         3 . The method of  claim 2 , wherein the inhibitor further inhibits Protein Kinase C alpha and delta. 
     
     
         4 . The method of  claim 1 , wherein the inhibitor concentration is in the range of 100 nm to 5 μM. 
     
     
         5 . The method of  claim 1 , wherein the undifferentiated state is maintained in the absence of a feeder cell or leukemia inhibitory factor. 
     
     
         6 . The method of  claim 1 , wherein the stem cell is isolated from a mouse. 
     
     
         7 . The method of  claim 1 , wherein the stem cell is isolated from a rat. 
     
     
         8 . The method of  claim 1 , wherein the stem cell is isolated from a human. 
     
     
         9 . The method of  claim 1 , wherein the stem cell is an embryonic stem cell, adult stem cell, induced pluripotent stem cell, or cancer stem cell. 
     
     
         10 . The method of  claim 1 , wherein the PKC inhibitor is a PKC inhibitor of Formula I, II, III, IV, or V, 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein
 each of R 1  and R 2  is independently selected from —H, halogen, haloalkoxy, —CN, —NO 2 , —OR 3 , —N(R 3 )R 3 , —S(O)O-2R 3 , —N(R 3 )C(═O)N(R 3 )R 3 , —N(R 3 )C(═O)C(═O)N(R 3 )R 3 , —SO 2 N(R 3 )R 3 ), —CO 2 R 3 —C(═O)N(R 3 )R 3 , —C(═NR 5 )N(R 3 )R 3 , —C(═NR 5 )NR 3 , —N(R 3 ) SO 2 R 3 , —N(R 3 )C(O)R 3 , —NCO 2 R 3 , —N(R 3 )C(O) 2 R 3 , —C(═O)R 3 , optionally substituted alkoxy, optionally substituted lower alkyl, optionally substituted aryl, optionally substituted lower arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, or optionally substituted lower heterocyclylalkyl; 
 Y is selected from O, or HH; 
 each of m and n is independently 1 to 5; 
 each of A and B is independently selected from a five- to ten-membered aryl of heteroaryl; 
 R 3  is selected from —H, optionally substituted lower alkyl, optionally substituted aryl, optionally substituted lower arylalkyl, optionally substituted heterocyclyl, or optionally substituted lower heterocycylalkyl; 
 two of R 3 , together with nitrogen to which they are attached, can combine to form an optionally substituted heterocycyl containing between one and three additional heteroatoms; 
 R 4  is selected from —H and optionally substituted lower alkyl; 
 each R 5  is independently selected from —H, —CN, —NO 2 , —OR 3 , —S(O)O-2R 3 , —CO 2 R 3 , optionally substituted lower alkyl, optionally substituted lower alkenyl, or optionally substituted lower alkynyl; and 
 each of L 1  and L 2  is independently selected from absent and —C(R 3 )(R 3 )—, —C(R 3 )(R 3 )C(R 3 )(R 3 )—, —C(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )—, —C(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )—. 
 
     
     
         11 . A method for inhibiting differentiation of a stem cell comprising contacting an isolated stem cell with a selective Protein Kinase C inhibitor thereby inhibiting differentiation of the stem cell. 
     
     
         12 . The method of  claim 11 , wherein the inhibitor inhibits at least Protein Kinase C zeta. 
     
     
         13 . The method of  claim 12 , wherein the inhibitor further inhibits Protein Kinase C alpha and delta. 
     
     
         14 . The method of  claim 11 , wherein the inhibitor concentration is in the range of 100 nm to 5 μM. 
     
     
         15 . The method of  claim 11 , wherein differentiation is inhibited under differentiation conditions. 
     
     
         16 . The method of  claim 11 , wherein the stem cell is isolated from a mouse. 
     
     
         17 . The method of  claim 11 , wherein the stem cell is isolated from a rat. 
     
     
         18 . The method of  claim 11 , wherein the stem cell is isolated from a human. 
     
     
         19 . The method of  claim 11 , wherein the stem cell is an embryonic stem cell, adult stem cell, induced pluripotent stem cell, or cancer stem cell. 
     
     
         20 . The method of  claim 11 , wherein the PKC inhibitor is a PKC inhibitor of Formula I, II, III, IV, or V, 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein
 each of R 1  and R 2  is independently selected from —H, halogen, haloalkoxy, —CN, —NO 2 , —OR 3 , —N(R 3 )R 3 , —S(O)O-2R 3 , —N(R 3 )C(═O)N(R 3 )R 3 , —N(R 3 )C(═O)C(═O)N(R 3 )R 3 , —SO 2 N(R 3 )R 3 ), —CO 2 R 3  (═O)N(R 3 )R 3 , —C(═NR 5 )N(R 3 )R 3 , —C(═NR 5 )NR 3 , —N(R 3 )SO 2 R 3 , —N(R 3 )C(O)R 3 , —NCO 2 R 3 , —N(R 3 )C(O)2R 3 , —C(═O)R 3 , optionally substituted alkoxy, optionally substituted lower alkyl, optionally substituted aryl, optionally substituted lower arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, or optionally substituted lower heterocyclylalkyl; 
 Y is selected from 0, or HH; 
 each of m and n is independently 1 to 5; 
 each of A and B is independently selected from a five- to ten-membered aryl of heteroaryl; 
 R 3  is selected from —H, optionally substituted lower alkyl, optionally substituted aryl, optionally substituted lower arylalkyl, optionally substituted heterocyclyl, or optionally substituted lower heterocycylalkyl; 
 two of R 3 , together with nitrogen to which they are attached, can combine to form an optionally substituted heterocycyl containing between one and three additional heteroatoms; 
 R 4  is selected from —H and optionally substituted lower alkyl; 
 each R 5  is independently selected from —H, —CN, —NO 2 , —OR 3 , —S(O)O-2R 3 , —CO 2 R 3 , optionally substituted lower alkyl, optionally substituted lower alkenyl, or optionally substituted lower alkynyl; and 
 each of L 1  and L 2  is independently selected from absent and —C(R 3 )(R 3 )—, —C(R 3 )(R 3 )C(R 3 )(R 3 )—, —C(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )—, —(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )—. 
 
     
     
         21 . A method for establishing a pluripotent stem cell line comprising culturing a cell with a selective Protein Kinase C inhibitor thereby establishing a pluripotent stem cell line. 
     
     
         22 . The method of  claim 21 , wherein the inhibitor inhibits as least Protein Kinase C zeta. 
     
     
         23 . The method of  claim 21 , wherein the inhibitor further inhibits Protein Kinase C alpha and delta. 
     
     
         24 . The method of  claim 21 , wherein the inhibitor concentration is in the range of 100 nm to 5 μM. 
     
     
         25 . The method of  claim 21 , wherein the cell is isolated from a mouse. 
     
     
         26 . The method of  claim 21 , wherein the cell is isolated from a rat. 
     
     
         27 . The method of  claim 21 , wherein the cell is isolated from a human. 
     
     
         28 . The method of  claim 21 , wherein the cell is a blastocyte or fibroblast. 
     
     
         29 . The method of  claim 21 , wherein the PKC inhibitor is a PKC inhibitor of Formula I, II, III, IV, or V, 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein
 each of R 1  and R 2  is independently selected from —H, halogen, haloalkoxy, —CN, —NO 2 , —OR 3 , —N(R 3 )R 3 , —S(O)O-2R 3 , —N(R 3 )C(═O)N(R 3 )R 3 , —N(R 3 )C (═O)C(═O)N(R 3 )R 3 , —SO 2 N(R 3 )R 3 ), —CO 2 R 3 , —C(═O)N(R 3 )R 3 , —C(═NR 5 )N(R 3 )R 3 , —C(═NR 5 )NR 3 , —N(R 3 )SO 2 R 3 , —N(R 3 )C(O)R 3 , —NCO 2 R 3 , —N(R 3 )C(O)2R 3 , —C(═O)R 3 , optionally substituted alkoxy, optionally substituted lower alkyl, optionally substituted aryl, optionally substituted lower arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, or optionally substituted lower heterocyclylalkyl; 
 Y is selected from 0, or HH; 
 each of m and n is independently 1 to 5; 
 each of A and B is independently selected from a five- to ten-membered aryl of heteroaryl; 
 R 3  is selected from —H, optionally substituted lower alkyl, optionally substituted aryl, optionally substituted lower arylalkyl, optionally substituted heterocyclyl, or optionally substituted lower heterocycylalkyl; 
 two of R 3 , together with nitrogen to which they are attached, can combine to form an optionally substituted heterocycyl containing between one and three additional heteroatoms; 
 R 4  is selected from —H and optionally substituted lower alkyl; 
 each R 5  is independently selected from —H, —CN, —NO 2 , —OR 3 , —S(O)O-2R 3 , —CO 2 R 3 , optionally substituted lower alkyl, optionally substituted lower alkenyl, or optionally substituted lower alkynyl; and 
 each of L 1  and L 2  is independently selected from absent and —C(R 3 )(R 3 )—, —C(R 3 )(R 3 )C(R 3 )(R 3 )—, —C(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )—, —C(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )C(R 3 )(R 3 )—.