HUMAN PLURIPOTENT STEM CELLS INDUCED FROM UNDIFFERENTIATED STEM CELLS DERIVED FROM A HUMAN POSTNATAL TISSUE

• US 20110039332A1
• Filed: 11/20/2007
• Published: 02/17/2011
• Est. Priority Date: 06/15/2007
• Status: Abandoned Application

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Masaki, Hldeki, Takahashi, Shunichi, Ishikawa, Tetsuya, Sakurada, Kazuhiro
• Application Number:
  US12/663,840
• Publication Number:
  US 20110039332A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

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.

Claims

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.

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.

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.

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.

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.

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.