Methods for producing mammalian pluripotent stem cell-derived endodermal cells

• US 10,202,579 B2
• Filed: 11/28/2013
• Issued: 02/12/2019
• Est. Priority Date: 11/29/2012
• Status: Active Grant

• Current Assignee: Takara Bio Europe AB
• Original Assignee: Takara Bio Europe AB
• Inventors: Kuppers-Munther, Barbara, Edsbagge, Josefina
• Primary Examiner(s): Tichy, Jennifer M. H.
• Application Number:
  US14/648,336
• Publication Number:
  US 20160304834A1
• Time in Patent Office: 1,902 Days
• Field of Search: 435366
• CPC Class Codes: C12N 2501/06 Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/70 Enzymes
• 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 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• 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
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

The present invention relates to the directed differentiation of mammalian pluripotent stem cells, especially human pluripotent stem (hPS) cells, into endodermal cells. In particular, the present invention relates to the treatment of mammalian pluripotent stem cells, especially hPS cells, with a DNA demethylating agent while undergoing differentiation into endodermal. The inventors have, as disclosed herein, found that exposing differentiating mammalian pluripotent stem cells, especially hPS cells, to a DNA demethylating agent leads to an improved morphology and improved yield of endodermal cells. The treatment with a DNA de-methylating agent also leads to a significant down-regulation of expression of the stem cell marker Oct4 and to an improved expression of endoderm specific markers, notably sox17, cxcr4 and hhex.

Claims

1. A method for producing definitive endodermal cells (DE cells) from mammalian pluripotent stem cells, the method comprising:

differentiating mammalian pluripotent stem cells under directed differentiation conditions to obtain a cell composition which is characterized in that at least 70% of the cells are DE cells, and

exposing the mammalian pluripotent stem cells to a nucleoside-analogue type DNA demethylating agent while undergoing differentiation, wherein the exposing of the mammalian pluripotent stem cells to the nucleoside-analogue type DNA demethylating agent first takes place on day 2 of differentiation, and wherein the cell composition obtained shows increased gene expression of sox17, cxcr4 and hhex compared to a cell composition obtained without exposure to the nucleoside-analogue type DNA demethylating agent.

2. The method according to claim 1, wherein the mammalian pluripotent stem cells are human pluripotent stem cells, primate pluripotent stem cells, mouse pluripotent stem cells, rat pluripotent stem cells, canine pluripotent stem cells, feline pluripotent stem cells, porcine pluripotent stem cells, bovine pluripotent stem cells or equine pluripotent stem cells.

3. The method according to claim 1, wherein the mammalian pluripotent stem cells are not human pluripotent stem cells.

4. The method according to claim 1, wherein the mammalian pluripotent stem cells are mammalian embryonic stem cells.

5. The method according to claim 1, wherein the mammalian pluripotent stem cells are mammalian induced pluripotent stem cells.

6. The method according to claim 1, wherein the mammalian pluripotent stem cells are human pluripotent stem (hPS) cells.

7. The method according to claim 6, wherein the human pluripotent stem cells are human embryonic stem (hES) cells.

8. The method according to claim 7, wherein the human embryonic stem cells have been obtained without destruction of a human embryo.

9. The method according to claim 6, wherein the human pluripotent stem cells are human induced pluripotent stem (hiPS) cells.

10. The method according to claim 1, wherein the nucleoside-analogue type DNA demethylating agent is a cytidine analogue.

11. The method according to claim 1, wherein the nucleoside-analogue type DNA demethylating agent is selected from the group consisting of:

5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine), zebularine, Pseudoisocytidine, 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacyti dine, 2′

-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine, 2′

,2′

-Difluoro-deoxycytidine (gemcitabine), or Cytosine-beta-D-arabinofurasonide.

12. The method according to claim 1, wherein the nucleoside-analogue type DNA demethylating agent is a cytidine analogue selected from the group consisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine), 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine, 2′

-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and 2′

,2′

-Difluoro-deoxycytidine (gemcitabine).

13. The method according to claim 1, wherein the nucleoside-analogue type DNA demethylating agent is selected from the group consisting of:

5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine), zebularine, and combinations thereof.

14. The method according to claim 1, wherein said differentiation conditions for obtaining DE cells are characterized by culturing the mammalian pluripotent stem cells in a differentiation medium comprising activin and optionally one or more growth factors or serum.

15. The method according to claim 1, wherein said differentiation conditions for obtaining cells of the definitive endoderm are further characterized by culturing the mammalian pluripotent stem cells for up to 10 days in differentiation medium.

16. The method according to claim 1, wherein the exposing of the mammalian pluripotent stem cells to the nucleoside-analogue type DNA demethylating agent takes place on day 2 to day 3 or on day 2 to day 4 of differentiation.

17. The method according to claim 1, wherein the exposing of the mammalian pluripotent stem cells to the nucleoside-analogue type DNA demethylating agent takes place on day 2 to day 3 of differentiation.

18. The method according to claim 1, wherein the nucleoside-analogue type DNA demethylating agent is 5-aza-2-deoxycytidine (decitabine).

19. The method according to claim 18, wherein 5-aza-2-deoxycytidine is employed at a concentration in the range of 1 nM to 500 nM.

20. The method according to claim 18, wherein 5-aza-2-deoxycytidine is employed at a concentration in the range of 5 nM to 100 nM.

21. The method according to claim 1, wherein the nucleoside-analogue type DNA demethylating agent is 5-azacytidine (azacitidine).

22. The method according to claim 21, wherein 5-azacytidine is employed at a concentration in the range of 1 nM to 1 μ

M.

23. The method according to claim 1, wherein the nucleoside-analogue type DNA demethylating agent is 5-aza-2-deoxycytidine (decitabine) which is employed at a concentration in the range of 5 nM to 100 nM, and wherein the exposing of the mammalian pluripotent stem cells to 5-aza-2-deoxycytidine takes place on day 2 to day 3 or on day 2 to day 4 of differentiation.

24. The method according to claim 1, wherein the nucleoside-analogue type DNA demethylating agent is 5-aza-2-deoxycytidine (decitabine) which is employed at a concentration in the range of 5 nM to 100 nM, and wherein the exposing of the mammalian pluripotent stem cells to 5-aza-2-deoxycytidine takes place on day 2 to day 3 of differentiation.