Wnt pathway stimulation in reprogramming somatic cells with nuclear reprogramming factors

Abstract

The invention provides compositions and methods of use in reprogramming somatic cells. Compositions and methods of the invention are of use, e.g., for generating or modulating (e.g., enhancing) generation of induced pluripotent stem cells by reprogramming somatic cells. The reprogrammed somatic cells are useful for a number of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a pluripotent state and/or enhances the speed and/or efficiency of reprogramming. Certain of the compositions and methods relate to modulating the Wnt pathway.

1 .- 34 . (canceled) 
     
     
         35 . A method of identifying an agent useful for modulating the reprogramming of mammalian somatic cells comprising:
 (a) culturing a population of mammalian somatic cells in medium containing an agent that modulates activity of a Wnt pathway and a candidate agent;   (b) determining, after a suitable period of time, whether cells having one or more characteristics of ES cells are present after maintaining the cells and their progeny in culture for a suitable time period, wherein the candidate agent is identified as being useful for modulating the reprogramming of mammalian somatic cells to an ES-like state if cells having one or more characteristics of ES cells are present at levels different than would be expected had the medium not contained the candidate agent.   
     
     
         36 . The method of  claim 35 , wherein the characteristics are selected from: colony morphology, expression of an endogenous gene expressed selectively by ES cells, expression of a detectable marker operably linked to expression control sequences of a gene expressed selectively by ES cells, ability to differentiate into cells having characteristics of endoderm, mesoderm, and ectoderm when injected into immunocompromised mice, and ability to participate in formation of chimeras that survive to term. 
     
     
         37 . (canceled) 
     
     
         38 . The method of  claim 35 , wherein the medium is Wnt-conditioned medium. 
     
     
         39 . The method of  claim 35 , wherein the medium is Wnt3a-conditioned medium. 
     
     
         40 . The method of  claim 35 , wherein the agent that modulates activity of a Wnt pathway is Wnt3a protein. 
     
     
         41 . The method of  claim 35 , wherein the candidate agent is a small molecule. 
     
     
         42 . The method of  claim 35 , wherein the method comprises identifying an agent useful for enhancing the reprogramming of mammalian somatic cells, wherein the candidate agent is identified as being useful for enhancing the reprogramming of mammalian somatic cells to an ES-like state if cells having one or more characteristics of ES cells are present at levels greater than would be expected had the medium not contained the candidate agent. 
     
     
         43 . The method of  claim 35 , wherein step (b) comprises determining whether cell colonies having one or more characteristics of ES cell colonies are present after maintaining the cells and their progeny in culture for a suitable time period, wherein the candidate agent is identified as being useful for modulating the reprogramming of mammalian somatic cells to an ES-like state if cell colonies having one or more characteristics of ES cell colonies are present at levels different than would be expected had the medium not contained the candidate agent. 
     
     
         44 . (canceled) 
     
     
         45 . A method of identifying an agent useful for reprogramming mammalian somatic cells to an ES-like state comprising:
 (a) contacting a population of mammalian somatic cells with an agent that increases Wnt pathway activity and a candidate agent;   (b) maintaining the cells in a cell culture system for a suitable period of time; and   (c) determining whether cells having one or more characteristics of ES cells are present in said culture system, wherein the agent is identified as being useful for reprogramming mammalian somatic cells to an ES-like state if cells having one or more characteristics of ES cells are present at levels greater than would be expected had the cells not been contacted with the candidate agent.   
     
     
         46 . The method of  claim 45 , wherein the characteristics are selected from: colony morphology, expression of an endogenous gene expressed selectively by ES cells, expression of a detectable marker operably linked to expression control sequences of a gene expressed selectively by ES cells, ability to differentiate into cells having characteristics of endoderm, mesoderm, and ectoderm when injected into immunocompromised mice, and ability to participate in formation of chimeras that survive to term. 
     
     
         47 . The method of  claim 45 , wherein the agent that increases Wnt pathway activity is Wnt3a protein. 
     
     
         48 . The method of  claim 45 , wherein the candidate agent is a small molecule. 
     
     
         49 . (canceled) 
     
     
         50 . The method of  claim 45 , wherein step (b) comprises determining cell colonies having one or more characteristics of ES cell colonies are present after maintaining the cells and their progeny in culture for a suitable time period, wherein the candidate agent is identified as being useful for modulating the reprogramming of mammalian somatic cells to an ES-like state if cell colonies having one or more characteristics of ES cell colonies are present at levels different than would be expected had the medium not contained the candidate agent. 
     
     
         51 .- 53 . (canceled) 
     
     
         54 . A method of identifying a Wnt pathway modulator useful for modulating the reprogramming of mammalian somatic cells to a pluripotent state comprising:
 (a) culturing a population of mammalian somatic cells in medium containing the Wnt pathway modulator;   (b) determining, after a suitable period of time, whether cells having one or more characteristics of ES cells are present after maintaining the cells and their progeny in culture for a suitable time period, wherein the Wnt pathway modulator is identified as being useful for modulating the reprogramming of mammalian somatic cells to an ES-like state if cells having one or more characteristics of ES cells are present at levels different than would be expected had the medium not contained the Wnt pathway modulator.   
     
     
         55 . The method of  claim 54 , wherein the method comprises (i) testing at least 10 Wnt pathway modulators; and (ii) identifying one or more of the Wnt pathway modulators as having significantly greater effect on reprogramming speed or efficiency than at least 50% of the other Wnt pathway modulators tested. 
     
     
         56 . The method of  claim 54 , wherein the method comprises testing at least 20 Wnt pathway modulators. 
     
     
         57 . The method of  claim 54 , wherein the method comprises identifying one or more of the Wnt pathway modulators as having significantly greater effect on reprogramming speed or efficiency than at least 75% of the other Wnt pathway modulators tested. 
     
     
         58 . The method of  claim 54 , wherein the Wnt pathway modulators are small molecules. 
     
     
         59 . The method of  claim 54 , wherein the Wnt pathway modulator is identified as being useful for increasing the speed or efficiency of reprogramming cells to an ES-like state if, after a suitable time period, cells having one or more characteristics of ES cells are present in numbers greater than would be expected had the medium not contained the Wnt pathway modulator. 
     
     
         60 .- 65 . (canceled)