Method for inducing differentiation of pluripotent stem cells into germ cells

Patent metadata

Field	Value
Publication number	US-10563171-B2
Application number	US-201615740454-A
Country	US
Kind code	B2
Filing date	Jun 29, 2016
Priority date	Jun 29, 2015
Publication date	Feb 18, 2020
Grant date	Feb 18, 2020

Abstract

The invention provides a method for inducing human primordial germ cell-like (PGC-like) cells from human pluripotent stem cells, with high efficiency and high reproducibility, and a cell surface marker for identifying human PGC-like cells. In particular, the invention provides a method for producing a human PGC-like cell from a human pluripotent stem cell, includes a step of producing a mesoderm-like cell by culturing a human pluripotent stem cell in a culture medium comprising activin A and a GSK3β inhibitor, and a step of culturing the mesoderm-like cell in a culture medium containing BMP. The invention also provides a method for producing an isolated human PGC-like cell, which includes the aforementioned two steps and the additional step of selecting a cell positive to at least one cell surface marker selected from the group consisting of PECAM (CD31), INTEGRINα6 (CD49f), INTEGRINβ3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81.

First claim

The invention claimed is:

1. A method for producing a human primordial germ cell-like (PGC-like) cell from a human pluripotent stem cell, comprising the following steps I) and II):
I) a step of producing a mesoderm-like cell by culturing a human pluripotent stem cell in a culture medium comprising activin A and a GSK3β inhibitor,
II) a step of culturing the mesoderm-like cell obtained in step I) in a culture medium containing BMP.

2. The method according to claim 1 wherein the culture in said step I) is performed for less than 60 hr.

3. The method according to claim 2 wherein the culture in said step I) is performed for 42 hr.

4. The method according to claim 1 wherein said GSK3β inhibitor is CHIR99021.

5. The method according to claim 1 wherein the culture medium in said step I) further comprises a fibroblast growth factor receptor (FGFR) inhibitor.

6. The method according to claim 5 wherein said FGFR inhibitor is PD173074.

7. The method according to claim 1 wherein the culture medium in said step I) does not contain bFGF or BMP.

8. The method according to claim 1 wherein the culture medium in said step II) further comprises at least one cytokine selected from the group consisting of SCF, EGF and LIF.

9. The method according to claim 1 wherein said pluripotent stem cell is a pluripotent stem cell cultured under serum-free and feeder-free conditions.

10. The method according to claim 9 wherein the culturing under said feeder-free conditions is culturing on laminin511 or laminin511 fragment.

11. The method according to claim 1 further comprising III) a step of selecting a cell positive to at least one cell surface marker selected from the group consisting of PECAM (CD31), INTEGRINα6 (CD49f), INTEGRINβ3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81 from the cells obtained in said step II).

12. The method according to claim 11 wherein said step III) is a step of selecting a double positive cell of INTEGRINα6 (CD49f) and EpCAM.

13. The method according to claim 1 wherein said human pluripotent stem cell is a human iPS cell.

14. A reagent kit for inducing differentiation of a human pluripotent stem cell into a human primordial germ cell-like (PGC-like) cell comprising the following (1) and (2):
(1) a reagent for inducing a human pluripotent stem cell into a mesoderm-like cell comprising activin A and a GSK3β inhibitor,
(2) a reagent for inducing a mesoderm-like cell into a human primordial germ cell-like (PGC-like) cell comprising BMP.

15. The kit according to claim 14 wherein said GSK3β inhibitor is CHIR99021.

16. The kit according to claim 14 wherein the induction reagent of said (1) further comprises a fibroblast growth factor receptor (FGFR) inhibitor.

17. The kit according to claim 16 wherein said FGFR inhibitor is PD173074.

18. The kit according to claim 14 wherein the induction reagent of said (2) further comprises at least one cytokine selected from the group consisting of SCF, EGF and LIF.

19. The kit according to claim 14 further comprising (3) a reagent for isolating a human primordial germ cell-like (PGC-like) cell comprising an antibody to at least one cell surface marker selected from the group consisting of PECAM (CD31), INTEGRINα6 (CD49f), INTEGRINβ3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81.

20. The kit according to claim 19 wherein the isolation reagent of said (3) comprises an antibody to INTEGRINα6 (CD49f) and an antibody to EpCAM.

Assignees

• Univ Kyoto

Inventors

• Saitou Mitinori
• Sasaki Kotaro
• Yokobayashi Shihori

Classifications

• A61K35/545

  Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells · CPC title

• G01N2333/705

  Assays involving receptors, cell surface antigens or cell surface determinants · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title