GENERATION OF INDUCED PLURIPOTENT STEM (iPS) CELLS

Abstract

The present invention relates to a method of generating an induced pluripotent stem (iPS) cell comprising the step of introducing into a target cell one or two coding sequences each giving rise upon transcription to a factor that contributes to the reprogramming of said target cell into an induced pluripotent stem cell and selected from Oct3/4 or a factor belonging to the Myc, Klf and Sox families of factors, wherein the target cell endogenously expresses at least the factors that are not encoded by the coding sequences to be introduced and selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, and wherein the cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox. Furthermore, the present invention relates to an induced pluripotent stem cell generated by the method of the invention and a method of identifying a compound that contributes to the reprogramming of a target cell into an induced pluripotent stem cell. Also, a method of generating a transgenic non-human animal and a composition comprising an iPS cell generated by the method of the present invention for gene therapy, regenerative medicine, cell therapy or drug screening are envisaged.

1 . A method of generating an induced pluripotent stem (iPS) cell comprising the step of introducing into a target cell one or two coding sequences each giving rise upon transcription to a factor that contributes to the reprogramming of said target cell into an induced pluripotent stem cell and selected from Oct3/4 or a factor belonging to the Myc, Klf and Sox families of factors, wherein the target cell endogenously expresses at least one of the factors that are not encoded by the coding sequences to be introduced and selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, and wherein the cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox. 
     
     
         2 . The method of  claim 1 , wherein the factors belonging to the factor families of Myc, Klf and Sox and endogenously expressed by or encoded by the coding sequences to be introduced into the target cell are selected from the group consisting of 1-Myc, n-Myc, c-Myc, Klf1, Klf2, Klf4, Klf15, Sox1, Sox2, Sox3, Sox15 and Sox18. 
     
     
         3 . The method of  claim 1 , wherein the target cell does not endogenously express said at least one of the factors encoded by the one or two coding sequences to be introduced into said target cell. 
     
     
         4 . The method of  claim 1 , wherein the target cell is a multipotent stem cell. 
     
     
         5 . The method of  claim 4 , wherein the multipotent stem cell is an ectodermal cell. 
     
     
         6 . The method of  claim 1 , wherein the target cell is a neural stem cell (NSC). 
     
     
         7 . The method of  claim 6 , wherein the coding sequence to be introduced encodes the factor Oct3/4. 
     
     
         8 . The method of  claim 6 , wherein the two coding sequences to be introduced encode factors Oct3/4 and c-Myc or Oct3/4 and Klf4. 
     
     
         9 . The method of  claim 7 , wherein the target cell endogenously expresses the factors c-Myc, Klf4 and Sox2. 
     
     
         10 . The method of  claim 9 , wherein the target cell endogenously expresses the factors c-Myc, Klf4 and Sox2 at levels at least 10-fold lower or at most 10-fold higher as compared to the corresponding expression levels in embryonic stem cells of the same genus as the target cell. 
     
     
         11 . The method of  claim 7 , wherein the target cell is a murine neural stem cell. 
     
     
         12 . An induced pluripotent stem cell generated by the method of  claim 1 . 
     
     
         13 . A method of identifying a compound that contributes to the reprogramming of a target cell into an induced pluripotent stem cell comprising the steps of:
 (a) reprogramming a target cell according to the method of  claim 1 , wherein one coding sequence to be introduced is replaced by the compound to be tested; and   (b) assessing whether iPS cells are formed in the presence and absence of the compound to be tested,
 wherein the formation of iPS cells from target cells in which the compound to be tested has been introduced is indicative of the compound contributing to the reprogramming of a target cell into an induced pluripotent stem cell. 
   
     
     
         14 . A method of generating a transgenic non-human animal comprising the steps of:
 (a) introducing the induced pluripotent stem cell generated by the method of  claim 1  into a non-human preimplantation embryo;   (b) transferring the embryo of step (a) into the uterus of a female non-human animal; and   (c) allowing the embryo to develop and to be born.   
     
     
         15 . A transgenic non-human animal generated by the method of  claim 14 . 
     
     
         16 . A composition comprising an iPS cell generated by the method of  claim 1  for gene therapy, regenerative medicine, cell therapy or drug screening.