Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue

Abstract

To establish human pluripotent stem cells having properties close to human ES cells comprising the genome of the patient per se that can circumvent immunological rejection of transplanted cells from cells derived from a postnatal human tissue. It was found that human pluripotent stem cells can be induced by introducing three genes of Oct3/4, Sox2 and KIf 4, or three genes of Oct3/4, Sox2 and KIf 4 plus the c-Myc gene or a histone deacetylase (HDAC) inhibitor to undifferentiated stem cells present in various human postnatal tissues in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation.

1 . A human pluripotent stem cell having an in vitro long-term self-renewal ability and the pluripotency of differentiating into ectoderm, mesoderm and endoderm, that was induced from an undifferentiated stem cell present in a human postnatal tissue in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation. 
     
     
         2 . The human pluripotent stem cell according to  claim 1  induced from an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture, or a subculture in a low serum concentration. 
     
     
         3 . The human pluripotent stem cell according to  claim 1  induced by the forced expression of each of three genes of Oct3/4, Sox2 and Klf4 in an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture or to a subculture in a low serum concentration. 
     
     
         4 . The human pluripotent stem cell according to  claim 1  induced by the forced expression of each of four genes of Oct3/4, Sox2, Klf4 and c-Myc in an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture or to a subculture in a low serum concentration. 
     
     
         5 . The human pluripotent stem cell according to  claim 1  induced by combining the forced expression of each of three genes of Oct3/4, Sox2 and Klf4 and a histone deacetylase inhibitor treatment in an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture or to a subculture in a low serum concentration. 
     
     
         6 . The human pluripotent stem cell according to  claim 1  induced by combining the forced expression of each of three genes of Oct3/4, Sox2 and Klf4 and a MS-275 treatment in an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture or to a subculture in a low serum concentration. 
     
     
         7 . The human pluripotent stem cell according to any one of  claims 2  to  6  wherein FGF-2 is further used in the culture of said undifferentiated stem cell. 
     
     
         8 . The human pluripotent stem cell according to any one of  claims 2  to  6  wherein PDGF and FGF are further used in the culture of said undifferentiated stem cell. 
     
     
         9 . The human pluripotent stem cell according to any one of  claims 2  to  8  wherein the culture of said undifferentiated stem cell is further conducted in a lower density. 
     
     
         10 . The human pluripotent stem cell according to any one of  claims 1  to  9  wherein said human pluripotent stem cell is positive for Nanog. 
     
     
         11 . The human pluripotent stem cell according to any one of  claims 1  to  10  wherein said human pluripotent stem cell is positive for alkaline phosphatase staining. 
     
     
         12 . The human pluripotent stem cell according to any one of  claims 1  to  11  wherein said human pluripotent stem cell is positive for Tert. 
     
     
         13 . The human pluripotent stem cell according to any one of  claims 1  to  12  wherein said human pluripotent stem cell comes to have teratoma-forming potential when it is transplanted into a test animal. 
     
     
         14 . The human pluripotent stem cell according to any one of  claims 1  to  13  wherein said human postnatal tissue is a tissue immediately after birth. 
     
     
         15 . The human pluripotent stem cell according to any one of  claims 1  to  13  wherein said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal tissue or an umbilical cord tissue. 
     
     
         16 . The human pluripotent stem cell according to any one of  claims 1  to  13  wherein said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal skin or a blood vessel derived from the umbilical cord. 
     
     
         17 . The human pluripotent stem cell according to any one of  claims 1  to  16  wherein said human pluripotent stem cell further has an in vitro potential of differentiating into a primordial germ cell. 
     
     
         18 . An undifferentiated stem cell present in a human postnatal tissue, in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation and which can be induced into a human pluripotent stem cell having an in vitro long-term self-renewal ability and the pluripotency of differentiating into ectoderm, mesoderm and endoderm by the forced expression of each of three genes of Oct3/4, Sox2 and Klf4. 
     
     
         19 . An undifferentiated stem cell present in a human postnatal tissue, in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation and which can be induced into a human pluripotent stem cell having an in vitro long-term self-renewal ability and the pluripotency of differentiating into ectoderm, mesoderm and endoderm by the forced expression of each of four genes of Oct3/4, Sox2, Klf4 and c-Myc. 
     
     
         20 . An undifferentiated stem cell present in a human postnatal tissue, in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation and which can be induced into a human pluripotent stem cell having an in vitro long-term self-renewal ability and the pluripotency of differentiating into ectoderm, mesoderm and endoderm by combining the forced expression of each of three genes of Oct3/4, Sox2 and Klf4 and a histone deacetylase inhibitor treatment. 
     
     
         21 . An undifferentiated stem cell present in a human postnatal tissue, in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation and which can be induced into a human pluripotent stem cell having an in vitro long-term self-renewal ability and the pluripotency of differentiating into ectoderm, mesoderm and endoderm by combining the forced expression of each of three genes of Oct3/4, Sox2 and Klf4 and a MS-275 treatment. 
     
     
         22 . The undifferentiated stem cell present in a human postnatal tissue according to any one of  claims 18  to  21 , wherein said human postnatal tissue is a tissue immediately after birth. 
     
     
         23 . The undifferentiated stem cell present in a human postnatal tissue according to any one of  claims 18  to  21 , wherein said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal tissue or an umbilical cord tissue. 
     
     
         24 . The undifferentiated stem cell present in a human postnatal tissue according to any one of  claims 18  to  21 , wherein said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal skin or a blood vessel of the umbilical cord. 
     
     
         25 . The undifferentiated stem cell present in a human postnatal tissue according to any one of  claims 18  to  24 , wherein said human pluripotent stem cell further has an in vitro potential of differentiating into a primordial germ cell. 
     
     
         26 . A method of inducing a human pluripotent stem cell wherein an undifferentiated stem cell present in a human postnatal tissue, in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation, is subjected to a primary culture or a second subculture or to a third or fourth subculture in a low serum concentration at 0 to 5%, and then each of three genes of Oct3/4, Sox2 and Klf4 is subjected to forced expression. 
     
     
         27 . The method of inducing a human pluripotent stem cell according to  claim 26 , wherein each of four genes comprising each of three genes of Oct3/4, Sox2 and Klf4 plus c-MYc is subjected to forced expression. 
     
     
         28 . The method of inducing a human pluripotent stem cell according to  claim 26 , wherein the forced expression of each of three genes of Oct3/4, Sox2 and Klf4 is combined with a histone deacetylase inhibitor treatment. 
     
     
         29 . The method of inducing a human pluripotent stem cell according to  claim 26 , wherein the forced expression of each of three genes of Oct3/4, Sox2 and Klf4 is combined with a MS-275 treatment. 
     
     
         30 . The method of inducing a human pluripotent stem cell according to any one of  claims 26  to  29 , wherein said undifferentiated stem cell is cultured in the presence of FGF-2. 
     
     
         31 . The method of inducing a human pluripotent stem cell according to any one of  claims 26  to  29 , wherein said undifferentiated stem cell is cultured in the presence of PDGF and EGF 
     
     
         32 . The method of inducing a human pluripotent stem cell according to any one of  claims 26  to  31 , wherein said human postnatal tissue is a tissue immediately after birth. 
     
     
         33 . The method of inducing a human pluripotent stem cell according to any one of  claims 26  to  31 , wherein said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal tissue or an umbilical cord tissue. 
     
     
         34 . The method of inducing a human pluripotent stem cell according to any one of  claims 26  to  31 , wherein said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal skin or a blood vessel of the umbilical cord. 
     
     
         35 . A method of culturing a human pluripotent stem cell according to any one of  claims 1  to  17  in a culture medium comprising an inhibitor of Rho associated kinase as an active ingredient. 
     
     
         36 . The human pluripotent stem cell according to anyone of  claims 1  to  17  on which cell surface antigens SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, CD24, and CD90 are expressed.