COMPOSITION FOR REPROGRAMMING SOMATIC CELLS TO GENERATE INDUCED PLURIPOTENT STEM CELLS, COMPRISING Oct4 IN COMBINATION WITH Bmi1 OR ITS UPSTREAM REGULATOR, AND METHOD FOR GENERATING INDUCED PLURIPOTENT STEM CELLS USING THE SAME

Abstract

Disclosed is a composition for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising: a) a Bmi1 (B cell-specific Moloney murine leukemia virus integration site 1) protein or a nucleic acid molecule coding for Bmi1; and b) an Oct4 protein or a nucleic acid molecule coding for Oct4. Also, a method is provided for reprogramming somatic cells to generate embryonic stem cell-like cells using the composition. In addition to reducing the number of the genetic factors conventionally needed, the composition and method allow the generation of pluripotent embryonic stem cell-like cells which have high potential in the cell therapy of various diseases.

1 . A composition for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising:
 a) a Bmi1 (B cell-specific Moloney murine leukemia virus integration site 1) protein or a nucleic acid molecule coding for Bmi1; and   b) an Oct4 protein or a nucleic acid molecule coding for Oct4.   
     
     
         2 . The composition according to  claim 1 , wherein the Bmi1 protein is expressed by an expression vector carrying the nucleic acid molecule coding therefore and the Oct4 protein is expressed by an expression vector carrying the nucleic acid molecule coding therefore. 
     
     
         3 . The composition according to  claim 1 , wherein the Bmi1 protein is expressed by a virus anchoring the nucleic acid molecule coding therefore and the Oct4 protein is expressed by a virus anchoring the nucleic acid molecule coding therefore. 
     
     
         4 . A method for reprogramming somatic cells to generate embryonic stem cell-like cells by introducing a Bmi1 (B cell-specific Moloney murine leukemia virus integration site 1) gene and an Oct4 gene into the somatic cells. 
     
     
         5 . The method according to  claim 4 , comprising:
 (i) culturing fibroblasts in a medium   (ii) infecting the fibroblasts with a packaging cell into which respective vectors carrying a Bmi1 gene and an Oct4 gene are transfected; and   (iii) culturing the infected fibroblasts under the conditions used to culture embryonic stem cells.   
     
     
         6 . The method according to  claim 5 , wherein the vector carrying a Bmi1 gene is constructed by inserting a nucleic acid molecule coding for a Bmi1 protein into a pBabe puro vector and the vector carrying an Oct4 gene is constructed by inserting a nucleic acid molecule coding for an Oct4 protein into a pBabe neo vector. 
     
     
         7 . The method according to  claim 5 , wherein the packaging cell is a PT67 packaging cell. 
     
     
         8 . An embryonic stem cell-like cell, prepared according to the method of one of  claim 4 . 
     
     
         9 . The embryonic stem cell-like cell according to  claim 8 , showing positive responses to alkaline phosphatase and antibodies against SSEA-1, Oct-4, and Sox2. 
     
     
         10 . A method for reprogramming somatic cells to generate neural stem cells, comprising:
 introducing a Bmi1 (B cell-specific Moloney murine leukemia virus integration site 1) gene into the somatic cells; and   culturing the somatic cells in a condition used to culture neural stem cells.   
     
     
         11 . A composition for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising:
 a) a reprogramming factor selected from a group consisting of a Shh (Sonic hedgehog signaling) protein, a nucleic acid molecule coding for a Shh protein, oxysterol, purmorphamine and a combination thereof; and   b) an Oct4 protein or a nucleic acid coding for an Oct4 protein.   
     
     
         12 . The composition according to  claim 11 , wherein the Shh protein is expressed by an expression vector carrying the nucleic acid molecule coding therefore and the Oct4 protein is expressed by an expression vector carrying the nucleic acid molecule coding therefore. 
     
     
         13 . The composition according to  claim 11 , wherein the reprogramming factor is selected from the group consisting of a Shh protein, oxysterol, purmorphamine and a combination thereof, and the Oct4 protein is expressed by a virus anchoring a nucleic acid molecule coding therefore. 
     
     
         14 . A method for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising:
 treating the somatic cells with a reprogramming factor selected from a group consisting of a Shh (Sonic hedgehog), a Shh-encoding nucleic acid molecule, oxysterol, purmorphamine and a combination thereof; and   introducing an Oct4 gene into the somatic cells.   
     
     
         15 . The method according to  claim 14 , comprising:
 (i) culturing fibroblasts in a medium;   (ii) infecting the fibroblasts with a packaging cell into which a vector carrying an Oct4 gene are transfected while treating the fibroblasts with the reprogramming factor selected from the group consisting of a Shh protein, a Shh-encoding nucleic acid, oxysterol, purmorphamine and a combination thereof; and   (iii) culturing the infected fibroblasts under the conditions used to culture embryonic stem cells.   
     
     
         16 . The method according to  claim 15 , wherein step (ii) is conducted by transducing the Oct4 gene into the fibroblasts while treating them with a reprogramming factor selected from the group consisting of a Shh protein, oxysterol and purmorphamine in a condition used to culture neural stem cells. 
     
     
         17 . An embryonic stem cell-like cell, prepared according to the method of one of  claim 14 .