Methods for modulating embryonic stem cell differentiation

Abstract

Described herein is Zscan4, a gene exhibiting 2-cell embryonic stage and embryonic stem cell specific expression. Identification of nine Zscan4 co-expressed genes is also described Inhibition of Zscan4 expression inhibits the 2-cell to 4-cell embryonic transition and prevents blastocyst implantation, expansion and outgrowth. Provided herein are methods of inhibiting differentiation of a stem cell, promoting blastocyst outgrowth of embryonic stem cells and identifying a subpopulation of stem cells expressing Zscan4. Further described is the identification of Trim43 as a gene exhibiting morula-specific expression. Also provided are isolated expression vectors comprising a Zscan4 promoter, or a Trim43 promoter operably linked to a heterologous polypeptide and uses thereof. Further provided are transgenic animals comprising transgenes encoding marker proteins operably linked to Zscan4 and Trim43 promoters.

1 . A method of inhibiting differentiation of a stem cell, comprising increasing the expression of Zscan4 in the stem cell, thereby inhibiting differentiation of the stem cell. 
     
     
         2 . The method of  claim 1  wherein increasing the expression of Zscan4 comprises increasing the expression of Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e, Zscan4f, human ZSCAN4 or a combination thereof. 
     
     
         3 . The method of  claim 1 , wherein increasing the expression of Zscan4 in the cell comprises transfecting the stem cells with a nucleic acid encoding Zscan4 operably linked to a promoter. 
     
     
         4 . The method of  claim 3 , wherein the nucleic acid encoding Zscan4 comprises SEQ ID NO: 60. 
     
     
         5 . The method of  claim 3 , wherein the nucleic acid encoding Zscan4 is at least 95% identical to Zscan4c (SEQ ID NO: 19), Zscn4d (SEQ ID NO: 21) or Zscan4f (SEQ ID NO: 25). 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The method of  claim 3 , comprising transfecting the cells with a vector comprising the nucleic acid encoding Zscan 4. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein inhibiting the differentiation of the stem cell increases the viability of the stem cell. 
     
     
         11 . The method of  claim 1 , wherein inhibiting the differentiation of the stem cell prevents senescence of the stem cell. 
     
     
         12 . The method of  claim 1 , wherein the stem cell is an embryonic stem cell, an embryonic germ cell, a germline stem cell or a multipotent adult progenitor cell. 
     
     
         13 . A method of promoting blastocyst outgrowth of embryonic stem cells, comprising increasing the expression of Zscan4 in the embryonic stem cells, thereby promoting blastocyst outgrowth of the embryonic stem cells. 
     
     
         14 . The method of  claim 13 , wherein increasing the expression of Zscan4 comprises increasing the expression of Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e, Zscan4f, human ZSCAN4 or a combination thereof. 
     
     
         15 . The method of  claim 13 , wherein increasing the expression of Zscan4 in the cell comprises transfecting the stem cells with a nucleic acid encoding Zscan4 operably linked to a promoter. 
     
     
         16 . The method of  claim 15 , wherein the nucleic acid encoding Zscan4 comprises SEQ ID NO: 6064. 
     
     
         17 . The method of  claim 15 , wherein the nucleic acid encoding Zscan4 is at least 95% identical to Zscan4c (SEQ ID NO: 19), Zscn4d (SEQ ID NO: 21) or Zscan4f (SEQ ID NO: 25). 
     
     
         18 - 19 . (canceled) 
     
     
         20 . The method of  claim 15 , comprising transfecting the cells with a vector comprising the nucleic acid encoding Zscan 4. 
     
     
         21 . (canceled) 
     
     
         22 . A method of identifying a subpopulation of stem cells expressing Zscan4, comprising transfecting the cells with an expression vector comprising a Zscan4 promoter and a reporter gene, wherein expression of the reporter gene indicates Zscan4 is expressed in the subpopulation of stem cells. 
     
     
         23 . The method of  claim 22 , wherein the Zscan4 promoter is a Zscan4c promoter. 
     
     
         24 . The method of  claim 23 , wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as:
 nucleotides 1-2540 of SEQ ID NO: 28,   nucleotides 1-2643 of SEQ ID NO: 28,   nucleotides 1-3250 of SEQ ID NO: 28,   nucleotides 1-3347 of SEQ ID NO: 28, or SEQ ID NO: 28.   
     
     
         25 - 28 . (canceled) 
     
     
         29 . An isolated expression vector comprising a Zscan4c promoter operably linked to a nucleic acid encoding a heterologous polypeptide. 
     
     
         30 . The isolated expression vector of  claim 29 , wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as
 nucleotides 1-2540 of SEQ ID NO: 28,   nucleotides 1-2643 of SEQ ID NO: 28,   nucleotides 1-3250 of SEQ ID NO: 28, or   nucleotides 1-3347 of SEQ ID NO: 28.   
     
     
         31 .- 33 . (canceled) 
     
     
         34 . The isolated expression vector of  claim 29 , wherein the polypeptide is a marker, enzyme, or fluorescent protein. 
     
     
         35 .- 36 . (canceled) 
     
     
         37 . An isolated embryonic stem cell comprising the expression vector of  claim 29 . 
     
     
         38 . An isolated expression vector comprising a Trim43 promoter operably linked to a nucleic acid encoding a heterologous polypeptide. 
     
     
         39 . The isolated expression vector of  claim 38 , wherein the Trim43 promoter comprises the nucleic acid sequence set forth as SEQ ID NO: 31. 
     
     
         40 . The isolated expression vector of  claim 38 , wherein the polypeptide is a marker, enzyme, or fluorescent protein. 
     
     
         41 .- 42 . (canceled) 
     
     
         43 . An isolated embryonic stem cell comprising the expression vector of  claim 38 . 
     
     
         44 . A method of identifying a subpopulation of stem cells, wherein the stem cells express Zscan4, comprising detecting expression of one or more of AF067063, Testv1/Tcstv3, Tho4, Arginase II, BC061212 and Gm428, Eif1a, EG668777 and Pif1. 
     
     
         45 . An isolated stem cell identified according to the method of  claim 40 .