METHOD OF GENERATING 2 CELL-LIKE STEM CELLS

• US 20200131473A1
• Filed: 03/20/2018
• Published: 04/30/2020
• Est. Priority Date: 03/20/2017
• Status: Active Application

• Current Assignee: IFOM Fondazione Istituto FIRC Di Oncologia Molecolare
• Original Assignee: IFOM Fondazione Istituto FIRC Di Oncologia Molecolare
• Inventors: COSTANZO, Vincenzo, ATASHPAZGARGARI, Sina, SAMADI SHAMS, Sara
• Application Number:
  US16/494,249
• Publication Number:
  US 20200131473A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2501/724 Glycosyltransferases (EC 2.4.)
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The present invention relates to 2 cell-like stem cells and particularly, although not exclusively, the generation of such cells from pluripotent stem cells.

Claims

1. A method for inducing the expression of one or more 2 cell-like genes in a pluripotent stem cell, the method comprising activating replication stress response in a pluripotent stem cell.

2. The method of claim 1, comprising inducing replication stress in the pluripotent stem cell.

3. The method of claim 2, comprising exposing the pluripotent stem cell to one or more DNA damage inducing agents.

4. The method of claim 1, comprising culturing the pluripotent stem cell in the presence of one or more agent selected from Aphidicolin, ATR, ETAA1, hydroxyurea or high O₂ or exposing the cell to UV radiation or ionising radiation.

6. The method of claim 1 or claim 5, comprising overexpressing ETAA1 or an ATR binding fragment thereof in the pluripotent stem cell.

7. The method of claim 6, wherein the pluripotent stem cell is infected with a lentivirus that expresses ETAA1 or an ATR binding fragment thereof.

8. The method of any one of the preceding claims wherein the one or more 2 cell-like genes is selected from the group comprising Eif1a-like genes (Gm5662, Gm2022, Gm4027, Gm2016, and Gm8300), MERVL, MT2_Mm, Dux, Gm4981, Zfp352, Zfp750, Tdpoz genes (Tdpozl and Tdpoz3), and Tmem92, Zscan4 genes (Zscan4a-Zscan4d), Dux, Tcstv3, GM12794, γ

H2AX, P-CHK1, GM4340, Gm12794, Afp352, Sfp750, Tdpoz, Eif1a and Tmem19.

9. The method of any one of the preceding claims wherein the cell expressing one or more 2 cell-like genes is substantially totipotent.

10. The method of any one of the preceding claims wherein the pluripotent stem cell is an induced pluripotent stem cell or an embryonic stem cell.

11. The method of any one of the preceding claims, further comprising a step of differentiating the cell into an embryonic or extra-embryonic tissue cell.

12. The method of any one of the preceding claims, further comprising isolating a cell expressing one or more 2 cell-like genes from pluripotent cells that do not express one or more 2 cell-like genes.

13. A 2 cell-like stem cell prepared by the method of any one of claims 1 to 12.

5. A method for inducing the expression of one or more 2 cell-like genes in a pluripotent stem cell, the method comprising activating the ATR pathway in the pluripotent stem cell.

14. A stem cell that expresses one or more of Zscan4 genes, Tcstv3, GM12794, Eif, muERV-L, γ

H2AX, P-CHK1, GM4340, Gm12794, Afp352, Sfp750, Tdpoz, Eif1a and Tmem19.

15. Stem cell culture medium comprising a DNA damage inducing agent, the DNA damage agent being optionally selected from Aphidicolin, hydroxyurea or high O₂.

16. Use of a DNA damage inducing agent in the culture of stem cells.

17. Stem cell culture medium comprising an ATR activating agent.

18. Use of an ATR activating agent in the culture of stem cells.

19. A population of pluripotent stem cells wherein at least 6%, 7%, 8%, 9% or 10% of the stem cells express one or more 2C like genes.

20. A pluripotent stem cell containing a plasmid encoding ETAA1 or an ATR fragment thereof.