Method of inducing differentiation from pluripotent stem cells to germ cells

Patent metadata

Field	Value
Publication number	US-9938496-B2
Application number	US-201113816681-A
Country	US
Kind code	B2
Filing date	Jul 28, 2011
Priority date	Aug 13, 2010
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

This invention provides a method of producing an epiblast-like cell (EpiLC) from a pluripotent stem cell, which comprises culturing the pluripotent stem cell in the presence of activin A; a method of producing a primordial germ cell-like (PGC-like) cell a pluripotent stem cell, which comprises culturing the EpiLC obtained by the method above in the presence of BMP4 and LIF. Also provided are a cell population containing PGC-like cells as obtained by the method, and reagent kits for the EpiLC- and PGC-like cell-induction from a pluripotent stem cell.

First claim

The invention claimed is:

1. A method of producing a primordial germ cell-like (PGC-like) cell from a human or mouse pluripotent stem cell, comprising the steps of:
I) culturing a human or mouse pluripotent stem cell in the presence of activin A, bFGF, and 0.3 to 3 w/w % KSR for 48±12 hours to obtain an epiblast-like cell (EpiLC),
II) culturing the EpiLC obtained in step I) in the presence of BMP4 and LIF, to obtain a PGC-like cell,
wherein the pluripotent stem cell is a human or mouse induced pluripotent stem cell (iPSC) or a human or mouse embryonic stem cell (ESC).

2. The method of claim 1 , wherein the human or mouse PGC-like cell shows elevated gene expression of Blimp1 and/or Stella (Dppa3) compared to the EpiLC before inducing differentiation.

3. The method of claim 1 , wherein the human or mouse PGC-like cell is capable of contributing to normal spermatogenesis.

4. The method of claim 1 , wherein the culture in the step II) is performed in the presence of further SCF and/or BMP8b and/or EGF.

5. The method of claim 1 , wherein the culture in the step II) is performed under serum-free conditions.

6. The method of claim 1 , wherein the culture in step II) is performed in the presence of BMP4, LIF, SCF, BMP8b and EGF.

7. The method of claim 1 , which further comprises:
III) a step for selecting a Blimp1-positive cell from the cells obtained in step II).

8. The method of claim 1 , which further comprises:
III) a step for selecting a SSEA1- and Integrin-β3-double positive cell from the cells obtained in step II).

9. The method of claim 1 , wherein the ESC is acquired by a method in which a mammalian inner cell mass in the blastocyst stage is cultured or by a method in which an early embryo prepared by somatic cell nuclear transfer is cultured.

10. The method of claim 1 , wherein the concentration of activin A in step I) is 10 to 30 ng/mL.

11. The method of claim 10 , wherein the concentration of bFGF in step I) is 7.5 to 20 ng/mL.

12. The method of claim 1 , wherein the concentration of bFGF in step I) is 7.5 to 20 ng/mL.

Assignees

• Saitou Mitinori
• Hayashi Katsuhiko
• Univ Kyoto

Inventors

• Saitou Mitinori
• Hayashi Katsuhiko

Classifications

• C12N2501/16

  Activin; Inhibin; Mullerian inhibiting substance · CPC title

• C12N5/0606 Primary

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C12N2501/125

  Stem cell factor [SCF], c-kit ligand [KL] · CPC title

• C12N5/0611

  Primordial germ cells, e.g. embryonic germ cells [EG] · CPC title

• C12N5/0602 Primary

  Vertebrate cells · CPC title