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 .- 65 . (canceled) 
     
     
         66 . A method of identifying or screening an agent useful for reprogramming a mammalian somatic cell comprising:
 (i) identifying an agent that modulates an activity of a Wnt pathway or an agent that substitutes for a reprogramming factor in reprogramming mammalian somatic cells to a pluripotent state; and   (ii) testing the effect of the agent on reprogramming a mammalian somatic cell.   
     
     
         67 . The method of  claim 66 , wherein the method in step (i) comprises identifying an agent that modulates an activity of a Wnt pathway. 
     
     
         68 . The method of  claim 67 , wherein the method further comprises in step (i):
 (a) providing a medium containing the agent;   (b) culturing a population of mammalian somatic cells in the medium; and   (c) measuring the activity of the Wnt pathway, thereby determining if the agent modulates the activity of the Wnt pathway.   
     
     
         69 . The method of  claim 67 , further comprises in step (ii) determining, after a period of time, whether cells having one or more characteristics of pluripotent cells are present after maintaining the mammalian somatic cells and their progeny in culture for the time period. 
     
     
         70 . The method of  claim 66 , wherein the mammalian somatic cells are genetically modified or transiently transfected to express Oct4, Sox2, and Klf. 
     
     
         71 . The method of  claim 66 , wherein the method in step (i) comprises identifying an agent that substitutes for a reprogramming factor in reprogramming mammalian somatic cells to a pluripotent state. 
     
     
         72 . The method of  claim 71 , wherein the method in step (i) comprises:
 (a) introducing into the mammalian somatic cell one or more reprogramming factors, wherein the reprogramming factor to be substituted is not introduced into the cell;   (b) contacting a population of mammalian somatic cells with a Wnt pathway activator selected from the group consisting of: an exogenous, soluble, biologically active Wnt protein that binds to a Wnt receptor and activates the Wnt pathway, and a small molecule GSK-3 inhibitor that activates the Wnt pathway;   (c) contacting the mammalian somatic cells with a candidate agent;   (d) maintaining the mammalian somatic cells in a cell culture system for a suitable period of time; and   (e) determining whether cells having one or more characteristics of pluripotent cells are present in said culture system, wherein the agent that substitutes for the reprogramming factor in reprogramming mammalian somatic cells is identified if cells having one or more characteristics of pluripotent cells are present at levels greater than would be expected had the mammalian somatic cells not been contacted with the candidate agent.   
     
     
         73 . The method of  claim 72 , wherein the method is for identifying an agent that substitutes for Klf in reprogramming mammalian somatic cells to a pluripotent state, and wherein the method comprises in step (a) introducing into the mammalian somatic cell reprogramming factors Sox2 and Oct4, but not Klf. 
     
     
         74 . The method of  claim 72 , wherein the method is for identifying an agent that substitutes for Sox2 in reprogramming mammalian somatic cells to a pluripotent state, and wherein the method comprises in step (a) introducing into the mammalian somatic cell reprogramming factors Klf and Oct4, but not Sox. 
     
     
         75 . The method of  claim 72 , wherein the method is for identifying an agent that substitutes for Oct4 in reprogramming mammalian somatic cells to a pluripotent state, and wherein the method comprises in step (a) introducing into the mammalian somatic cell reprogramming factors Sox2 and Klf, but not Oct4.