Methods for differentiation

Abstract

Described herein are methods relating to the differentiation of stem cells to more differentiated phenotypes, e.g. to terminally differentiated cell types and/or precursors thereof. In some embodiments, the methods relate to contacting the stem cells with differentiation factors and halting the cell cycle, thereby increasing the rate of differentiation.

1 . A method of differentiating a stem cell, the method comprising:
 i) contacting the stem cell with one or more ectopic differentiation factors; and   ii) inhibiting the cell cycle of the stem cell;   wherein steps i) and ii) occur within 15 days of each other.   
     
     
         2 .- 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein steps i) and ii) occur simultaneously. 
     
     
         17 . The method of  claim 1 , wherein the differentiation factor is a terminal transcription factor. 
     
     
         18 . The method of  claim 17 , wherein the terminal transcription factor is selected from Table 1. 
     
     
         19 . The method of  claim 17 , wherein the stem cell is to be differentiated to a skeletal muscle phenotype and is contacted with the terminal transcription factor MyoD. 
     
     
         20 . The method of  claim 17 , wherein the stem cell is to be differentiated to a spinal motor neuron phenotype and is contacted with a terminal transcription factor selected from the group consisting of: Ngn2; Isl1; and Lhx3. 
     
     
         21 . The method of  claim 17 , wherein the stem cell is to be differentiated to a spinal motor neuron phenotype and is contacted with the terminal transcription factors Ngn2; Isl1; and Lhx3. 
     
     
         22 . The method of  claim 17 , wherein the stem cell is to be differentiated to a cardiomyocyte phenotype and is contacted with the terminal transcription factor Gata5. 
     
     
         23 . The method of  claim 17 , wherein the stem cell is to be differentiated to a hepatocyte or hepatoblast phenotype and is contacted with the terminal transcription factor Hnf4α. 
     
     
         24 . The method of  claim 1 , wherein the cell cycle is inhibited by one or more of the following:
 reducing or removing growth factors; reducing serum levels; reducing serum levels below 5%; contacting the cell with a PI3K inhibitor; contacting the cell with an E2F family transcription factor inhibitor; contacting the cell with a Myc inhibitor; contacting the cell with a MAPK inhibitor; contacting the cell with a MEK1/2 inhibitor; contacting the cell with a CDK inhibitor; contacting the cell with an Id inhibitor; contacting the cell with a Rb agonist; contacting the cell with a Ink family agonist; contacting the cell with a Cip/Kip family agonist; and culturing the cell in a media lacking a factor selected from the group consisting of:
 LIF; Bmp; Fgf; Activin; or TGFβ. 
   
     
     
         25 .- 27 . (canceled) 
     
     
         28 . The method of  claim 24 , wherein the PI3K inhibitor is LY294002. 
     
     
         29 . The method of  claim 24 , wherein the E2F transcription factor inhibitor is HLM006474. 
     
     
         30 . The method of  claim 24 , wherein the Myc inhibitor is JQ1 or 10058-F4. 
     
     
         31 . The method of  claim 24 , wherein the MAPK inhibitor is PD98059. 
     
     
         32 . The method of  claim 24 , wherein the CDK inhibitor is a CDK4 or CDK2 inhibitor. 
     
     
         33 . The method of  claim 24 , wherein the CDK inhibitor is p16, p15, p18, p19, p21, p27, or p57. 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein the stem cell is an embryonic stem cell. 
     
     
         36 . The method of  claim 1 , wherein steps i) and ii) result in a population of cells comprising one or more terminally-differentiated cell types. 
     
     
         37 . The method of  claim 1 , wherein steps i) and ii) result in a population of cells comprising no more than 2 terminally-differentiated cell types. 
     
     
         38 . The method of  claim 1 , wherein steps i) and ii) result in a population of cells of which at least 50% are terminally-differentiated cells. 
     
     
         39 .- 41 . (canceled)