Reprogramming of somatic cells

Abstract

The disclosure relates to a method of reprogramming one or more somatic cells, e.g., partially differentiated or fully/terminally differentiated somatic cells, to a less differentiated state, e.g., a pluripotent or multipotent state. In further embodiments the invention also relates to reprogrammed somatic cells produced by methods of the invention, to uses of said cells, and to methods for identifying agents useful for reprogramming somatic cells.

1 .- 175 . (canceled) 
     
     
         176 . A method of identifying an agent that reprograms somatic cells to a less differentiated state, the method comprising:
 (a) contacting somatic cells with a candidate reprogramming agent;   (b) measuring DNA methylation of a region in the somatic cells;   (c) identifying the candidate reprogramming agent as being likely to reprogram somatic cells to a less differentiated state by itself or in combination with additional agents if the DNA methylation of the region in the somatic cells is less than a reference level of DNA methylation.   
     
     
         177 . The method of  claim 176 , wherein the DNA methylation in the somatic cells is measured by measuring the DNA methylation of the gene promoter of Oct 4. 
     
     
         178 . The method of  claim 176 , wherein the DNA methylation in the somatic cells is measured by measuring the DNA methylation of the gene promoter of Nanog gene. 
     
     
         179 . The method of  claim 176 , wherein the DNA methylation in the somatic cells is measured by measuring the DNA methylation of the gene promoter of Oct 4 and the gene promoter of Nanog gene. 
     
     
         180 . The method of  claim 176 , wherein the reference level of DNA methylation is the DNA methylation level of the region in the somatic cells prior to contacting with the candidate reprogramming agent. 
     
     
         181 . The method of  claim 176 , wherein the reference level of DNA methylation is a pre-determined level of DNA methylation. 
     
     
         182 . The method of  claim 176 , wherein the somatic cells are immune cells. 
     
     
         183 . The method of  claim 182 , wherein the immune cells are B cells. 
     
     
         184 . The method of  claim 176 , wherein the method further comprises testing the effect of the candidate reprogramming agent on reprogramming somatic cells, or wherein the method further comprises testing the effect of the candidate reprogramming agent on reprogramming somatic cells in combination with one or more additional reprogramming agents. 
     
     
         185 . The method of  claim 176 , wherein the method is for identifying an agent that reprograms somatic cells to a less differentiated state in combination with one or more additional agents selected from a group consisting of Oct-4, Sox-2, C-Myc, and Klf4. 
     
     
         186 . A method of identifying an agent that reprograms somatic cells to a less differentiated state by itself or in combination with additional agents, the method comprising:
 (a) contacting somatic cells with a candidate reprogramming agent; and   (b) determining the amount of cells that are resistant to reduced DNA methylation, wherein an increased amount of cells that are resistant to reduced DNA methylation, as compared to a reference amount, is indicative of the candidate agent being a reprogramming agent.   
     
     
         187 . The method of  claim 186 , wherein the somatic cells are sensitive to reduced DNA methylation prior to contacting with the candidate reprogramming agent. 
     
     
         188 . The method of  claim 186 , wherein at least 90%, 95%, 96%, 97%, 98%, or 99% of the somatic cells are sensitive to reduced DNA methylation prior to contacting with the candidate reprogramming agent. 
     
     
         189 . The method of  claim 186 , wherein, prior to contacting with the candidate reprogramming agent, at least 90%, 95%, 96%, 97%, 98%, or 99% of the somatic cells undergo cell cycle arrest or die within a time period under reduced DNA methylation. 
     
     
         190 . The method of  claim 186 , wherein the reference amount is the amount of somatic cells that are resistant to reduced DNA methylation prior to contacting with the candidate reprogramming agent. 
     
     
         191 . The method of  claim 186 , wherein the reduced DNA methylation is achieved by culturing cells in a DNA methylation inhibitor. 
     
     
         192 . The method of  claim 186 , wherein the reduced DNA methylation is achieved by
 (a) inhibiting expression or activity of an endogenous DNA methyltransferase;   (b) expressing an agent that inhibits expression or activity of an endogenous DNA methyltransferase;   (c) inhibiting expression or activity of an endogenous protein other than a DNA methyltransferase, wherein the endogenous protein is needed for any step of a biochemical pathway that provides a substrate for the transfer of a methyl group to cytosine by a DNA methyltransferase;   (d) expressing in a somatic cell an agent that inhibits expression or activity of an endogenous protein other than a DNA methyltransferase, wherein the endogenous protein is needed for any step of a biochemical pathway that provides a substrate for the transfer of a methyl group to cytosine by a DNA methyltransferase; and/or   (e) culturing the somatic cells under conditions in which the somatic cells are deprived of nutrients needed for synthesis of a substrate for the transfer of a methyl group to cytosine by a DNA methyltranferase.   
     
     
         193 . The method of  claim 192 , wherein the DNA methyltransferase is selected from a group consisting of DNMT1, DNMT3a, and DNMT3b. 
     
     
         194 . The method of  claim 186 , wherein the method further comprises testing the effect of the candidate reprogramming agent on reprogramming somatic cells, or wherein the method further comprises testing the effect of the candidate reprogramming agent on reprogramming somatic cells in combination with one or more additional reprogramming agents. 
     
     
         195 . The method of  claim 186 , wherein the method is for identifying an agent that reprograms somatic cells to a less differentiated state in combination with one or more additional agents selected from a group consisting of Oct-4, Sox-2, C-Myc, and Klf4.