Method of inducing differentiation from pluripotent stem cells to germ cells

Patent metadata

Field	Value
Publication number	US-10023838-B2
Application number	US-201414771287-A
Country	US
Kind code	B2
Filing date	Feb 28, 2014
Priority date	Mar 1, 2013
Publication date	Jul 17, 2018
Grant date	Jul 17, 2018

Abstract

This invention provides a method of producing a primordial germ cell-like cell (PGCLC) from an epiblast isolated from an embryo or an epiblast-like cell (EpiLC) induced from a pluripotent stem cell (PSC), which comprises allowing the epiblast or EpiLC to express exogenous transcription factor(s) selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; ii) Blimp1 and Prdm14; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; and (v) Prdm14; thereby inducing the epiblast or EpiLC into a PGC state without acquiring transient mesodermal program.

First claim

The invention claimed is:

1. A method of producing a primordial germ cell-like cell (PGCLC) from an isolated epiblast or epiblast-like cell (EpiLC), which comprises allowing the epiblast or EpiLC to express exogenous transcription factor(s) selected from the group consisting of:
(i) Blimp1, Prdm14 and Tfap2c;
(ii) Blimp1 and Prdm14;
(iii) Blimp1 and Tfap2c;
(iv) Prdm14 and Tfap2c; and
(v) Prdm14;

thereby inducing the epiblast or EpiLC into a PGC state without acquiring transient mesodermal program, wherein the epiblast or EpiLC is induced into a PGCLC in the absence of BMP4.

2. The method according to claim 1 , wherein nucleic acid(s) encoding the exogenous transcription factor(s) is/are introduced into the epiblast or EpiLC.

3. The method according to claim 1 , wherein nucleic acid(s) encoding the exogenous transcription factor(s) has/have been introduced into the epiblast or EpiLC, in a form capable of being conditionally expressed, prior to the induction of the epiblast or EpiLC.

4. The method according to claim 3 , wherein the epiblast or EpiLC is cultured under conditions which the nucleic acid(s) encoding the exogenous transcription factor(s) is/are expressed for 1 to 5 days.

5. The method according to claim 1 , wherein the EpiLC is obtained by culturing a pluripotent stem cell (PSC) in the presence of activin A (ActA), optionally in the presence of further basic fibroblast growth factor (bFGF) and/or Knockout™ Serum Replacement (KSR).

6. The method according to claim 5 , wherein the PSC is an embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC).

7. The method according to claim 1 , wherein nucleic acid(s) encoding the exogenous transcription factor(s) is/are in a form capable of disappearing from the PGCLC.

8. The method according to claim 7 , wherein the nucleic acid(s) is/are carried on vector(s) selected from the group consisting of plasmid, episomal vector, transposon, adenoviral vector and Sendai viral vector.

9. The method according to claim 1 , wherein the EpiLC is derived from mouse or human.

10. A method of producing a PGCLC from a PSC, which comprises the following steps I) and II):
I) the step for producing an EpiLC by culturing a PSC in the presence of ActA, optionally in the presence of further bFGF and/or KSR;
II) the step for inducing the EpiLC obtained in the step I) into a PGCLC by the method according to claim 1 .

11. The method according to claim 10 , which further comprises:
III) the step for selecting a Blimp1-positive cell from the cells obtained in the step II).

12. The method according to claim 1 , wherein the epiblast or EpiLC is induced into a PGCLC in the absence of BMP4, LIF, SCF, BMP8b, and EGF.

13. The method according to claim 10 , wherein the step II) is performed in the absence of BMP4, LIF, SCF, BMP8b, and EGF.

14. The method according to claim 1 , wherein exogenous transcription factor(s) is/are selected from the group consisting of:
(i) Blimp1, Prdm14 and Tfap2c;
(iii) Blimp1 and Tfap2c;
(iv) Prdm14 and Tfap2c; and
(v) Prdm14.

15. The method according to claim 1 , wherein exogenous transcription factor(s) is/are selected from the group consisting of:
(i) Blimp1, Prdm14 and Tfap2c;
(iii) Blimp1 and Tfap2c; and
(iv) Prdm14 and Tfap2c.

16. The method according to claim 10 , wherein exogenous transcription factor(s) is/are selected from the group consisting of:
(i) Blimp1, Prdm14 and Tfap2c;
(iii) Blimp1 and Tfap2c;
(iv) Prdm14 and Tfap2c; and
(v) Prdm14.

17. The method according to claim 10 , wherein exogenous transcription factor(s) is/are selected from the group consisting of:
(i) Blimp1, Prdm14 and Tfap2c;
(iii) Blimp1 and Tfap2c; and
(iv) Prdm14 and Tfap2c.

Assignees

• Univ Kyoto

Inventors

• Nakaki Fumio
• Saitou Mitinori

Classifications

• C12N2510/00

  Genetically modified cells · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• C12N5/0611 Primary

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

• C12N2506/025

  from extra-embryonic cells, e.g. trophoblast, placenta · CPC title

• C12N2501/60

  Transcription factors · CPC title