Endogenous retrovirus transcription as a marker for primate naive pluripotent stem cells

Abstract

One or more type 7 long terminal repeat (LTR7) nucleic acid sequences of type H human endogenous retroviruses (HERVH) (“LTR7/HERVH nucleic acid sequences”) can be used for identifying primate naïve pluripotent stem cells. LTR7/HERVH-associated transcription can be used as a marker for primate naive pluripotent stem cells. A reporter construct that includes LTR7/HERVH nucleic acid sequences can be used for optimizing culture conditions for naïve primate pluripotent stem cells. A cell growth medium can be used for cultivation of primate naive pluripotent stem cells, which can exhibit elevated levels of LTR7/HERVH-associated transcription in comparison to control cells.

1 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . The method according to  claim 5 , wherein the LTR7/HERVH nucleic acid sequence comprises a LBP9 binding motif. 
     
     
         4 . The method according to  claim 5 , wherein the LTR7/HERVH nucleic acid sequence comprises a binding motif for one or more transcription factors selected from the group consisting of LBP9, OCT4, NANOG and KLF4. 
     
     
         5 . An in vitro method for isolating primate naive pluripotent stem cells comprising:
 analyzing LTR7/HERVH-associated transcription, and   isolating cells in which LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein control cells are primed pluripotent stem cells or differentiated cells.   
     
     
         6 . An isolated in vitro population of primate naive pluripotent stem cells in which LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein control cells are primed pluripotent stem cells or differentiated cells. 
     
     
         7 . A nucleic acid reporter construct comprising a nucleic acid sequence region encoding one or more reporter molecules operably linked to a sequence comprising one or more LTR7/HERVH nucleic acid sequences. 
     
     
         8 . The nucleic acid reporter construct according to  claim 7 , wherein the reporter molecule is a fluorescent protein. 
     
     
         9 . The nucleic acid reporter construct according to  claim 7 , wherein the reporter molecule is an antibiotic resistance gene. 
     
     
         10 . An isolated cell comprising a reporter construct according to  claim 7 , wherein the reporter construct is present in a nucleic acid vector. 
     
     
         11 . A method for culturing and/or enriching primate naïve pluripotent stem cells, wherein said cells comprise the nucleic acid reporter construct according to  claim 7 , wherein said method comprises analyzing expression of the reporter molecule encoded by said construct. 
     
     
         12 . (canceled) 
     
     
         13 . A method for optimizing a cell growth medium for primate naïve pluripotent stem cells, comprising:
 cultivating in vitro of primate naive pluripotent stem cells in a cell growth medium for primate naive pluripotent stem cells, wherein said cells comprise a reporter construct according to  claim 7 ; 
 modifying the presence and/or concentration of one or more components or said cell growth medium and/or other culture conditions; and 
 analyzing expression of the reporter molecule encoded by said construct, comprising comparing reporter molecule expression under conditions of the modified growth medium (according to step b.) and unmodified cell growth medium. 
 
     
     
         14 . (canceled) 
     
     
         15 . A cell growth medium for cultivation of primate naive pluripotent stem cells comprising at least one or more GSK3 inhibitors and one or more Axin stabilizers. 
     
     
         16 . The cell growth medium for cultivation of primate naive pluripotent stem cells according to  claim 15 , comprising at least one or more MEK/ERK inhibitors, one or more a GSK3 inhibitors, one or more Axin stabilizers and one or more PKC inhibitors. 
     
     
         17 . The cell growth medium for cultivation of primate naive pluripotent stem cells according to  claim 15 , comprising at least one or more cytokines of the IL-6 family. 
     
     
         18 . A kit for the provision of a cell growth medium according to  claim 15 , comprising the following components in separated compartments in proximity to one another: a basal medium, comprising neurobasal medium and DMEM, and a cocktail of inhibitors, comprising at least one or more MEK/ERK inhibitors, one or more a GSK3 inhibitors, one or more Axin stabilizers and one or more PKC inhibitors, optionally comprising at least one or more cytokines of the IL-6 family. 
     
     
         19 . A method of culturing, maintaining and/or enriching LTR7-expressing primate naive pluripotent stem cells comprising obtaining the cell growth medium according to  claim 15 , wherein LTR7/HERVH-associated transcription is elevated in the LTR7-expressing primate naive pluripotent stem cells in comparison to control cells, wherein control cells are primed pluripotent stem cells or differentiated cells, from a stem cell population. 
     
     
         20 . A method for enriching LTR7-expressing primate naive pluripotent stem cells, in which LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein the control cells are primed pluripotent stem cells or differentiated cells, from a stem cell population, the method comprising culturing a stem cell population in the cell growth medium of  claim 15 . 
     
     
         21 . The method according to  claim 4 , wherein the LTR7/HERVH nucleic acid sequence comprises a binding motif for each of LBP9, OCT4, NANOG and KLF4. 
     
     
         22 . The isolated cell according to  claim 10 , wherein the nucleic acid vector comprises transposon sequences.