Method of inducing differentiation from pluripotent stem cells to germ cells

Patent metadata

Field	Value
Publication number	US-2016010056-A1
Application number	US-201414771287-A
Country	US
Kind code	A1
Filing date	Feb 28, 2014
Priority date	Mar 1, 2013
Publication date	Jan 14, 2016
Grant date	—

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

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.

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

3 . The method according to claim 1 , wherein the 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 the nucleic acid(s) encoding the exogenous transcription factor(s) is 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 reagent for inducing an isolated epiblast or EpiLC into a PGCLC comprising 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; (v) Prdm14; or nucleic acid(s) encoding the transcription factor(s).

11 . The reagent according to claim 10 , wherein the nucleic acid(s) encoding the transcription factor(s) is/are in a form capable of being conditionally expressed in the epiblast or EpiLC.

12 . An isolated epiblast or EpiLC comprising nucleic acid(s) encoding 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;
wherein the nucleic acid(s) is/are in a form capable of being conditionally expressed in the epiblast or EpiLC.

13 . An isolated PSC comprising nucleic acid(s) encoding 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;
wherein the nucleic acid(s) is/are in a form capable of being conditionally expressed in an EpiLC differentiated from the PSC.

14 . A kit for inducing an isolated epiblast or EpiLC into a PGCLC comprising the epiblast or EpiLC according to claim 12 ; and a reagent that allows the epiblast or EpiLC to express the exogenous transcription factor(s).

15 . A kit for inducing an isolated PSC into a PGCLC comprising the PSC according to claim 13 ; a reagent for inducing the PSC into an EpiLC comprising ActA and optionally bFGF and/or KSR; and a reagent that allows the EpiLC to express the exogenous transcription factor(s).

16 . 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 .

17 . The method according to claim 16 , which further comprises:
III) the step for selecting a Blimp1-positive cell from the cells obtained in the step II).

18 . A method of producing a variety of cell types derived from epiblast which comprises utilizing the PGCLC cell population obtained by the method according to claim 16 as a cell source.

Assignees

• Univ Kyoto

Inventors

• Nakaki Fumio
• Saitou Mitinori

Classifications

• C12N5/0611 Primary

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

• C12N2510/00

  Genetically modified cells · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• C12N2501/16

  Activin; Inhibin; Mullerian inhibiting substance · CPC title

• C12N2501/60

  Transcription factors · CPC title