Production method for nerve tissue

Patent metadata

Field	Value
Publication number	US-12492373-B2
Application number	US-202117559458-A
Country	US
Kind code	B2
Filing date	Dec 22, 2021
Priority date	Oct 24, 2014
Publication date	Dec 9, 2025
Grant date	Dec 9, 2025

Abstract

The present invention provides a method for producing neural cells or a neural tissue, including the following steps (1)-(3):
(1) a first step of culturing pluripotent stem cells in the absence of feeder cells and in a medium containing 1) a TGFβ family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal transduction pathway activating substance, and 2) a factor for maintaining undifferentiated state,

(2) a second step of culturing the cells obtained in the first step in suspension to form a cell aggregate, and

(3) a third step of culturing the aggregate obtained in the second step in suspension in the presence or absence of a differentiation-inducing factor to obtain an aggregate containing neural cells or a neural tissue.

First claim

The invention claimed is:

1 . A method for producing cerebral cells or a cerebral tissue, comprising the following steps (1)-(3):
(1) a first step of culturing pluripotent stem cells in the absence of feeder cells and in a medium comprising (a) a Sonic hedgehog signal transduction pathway activating substance or a combination of a TGFβ family signal transduction pathway inhibiting substance and the Sonic hedgehog signal transduction pathway activating substance, and (b) a factor for maintaining an undifferentiated state of the pluripotent stem cells for a period of 0.5 hr-144 hr, (2) a second step of culturing the cells obtained in the first step in suspension to form a cell aggregate, and (3) a third step of culturing the aggregate obtained in the second step in suspension in the presence of a TGFβ family signal transduction pathway inhibiting substance and/or a Wnt signal transduction pathway inhibiting substance to obtain an aggregate containing cerebral cells or a cerebral tissue, wherein the factor for maintaining an undifferentiated state of the pluripotent stem cells comprises a FGF signal transduction pathway activating substance and insulin, wherein the FGF signal transduction pathway activating substance is bFGF, wherein the TGFβ family signal transduction pathway inhibiting substance is Lefty, SB431542, A-83-01 or LDN193189, wherein the Sonic hedgehog signal transduction pathway activating substance is Shh, SAG or Purmorphamine, and wherein the Wnt signal transduction pathway inhibiting substance is IWR-1-endo.

2 . The production method according to claim 1 , wherein, in the second step, the cells obtained in the first step are dispersed, and the dispersed cells are cultured in suspension.

3 . The production method according to claim 1 , wherein, in the second step, the cells are cultured in suspension in a serum-free medium containing a Sonic hedgehog signal transduction pathway activating substance.

4 . The production method according to claim 1 , wherein the TGFβ family signal transduction pathway inhibiting substance is a Nodal/Activin signal transduction pathway inhibiting substance, a TGFβ signal transduction pathway inhibiting substance, or a BMP signal transduction pathway inhibiting substance.

5 . The production method according to claim 1 , wherein the first step is performed by an adhesion culturing method.

6 . The production method according to claim 1 , wherein, in the third step, the TGFβ family signal transduction pathway inhibiting substance and/or the Wnt signal transduction pathway inhibiting substance are/is added to the medium between day 3 and day 6 after the start of the second step.

7 . The production method according to claim 1 , wherein the pluripotent stem cells are primate pluripotent stem cells.

8 . The production method according to claim 1 , wherein the pluripotent stem cells are human pluripotent stem cells.

9 . The production method according to claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells.

10 . The production method according to claim 1 , wherein uniformed aggregates are formed in the second step.

11 . The production method according to claim 1 , wherein, in the first step, the pluripotent stem cells are cultured for a period during which pluripotent-like state of the pluripotent stem cells are maintained.

12 . The production method according to claim 11 , wherein the pluripotent-like state of the pluripotent stem cells is Oct3/4 positive.

13 . The production method according to claim 1 , wherein, in the second step and/or the third step, the culture is performed in the medium free of a factor for maintaining an undifferentiated state of the pluripotent stem cells.

14 . The production method according to claim 1 , wherein the cells obtained in the first step contain Oct3/4 positive cells at a proportion of not less than 60%.

15 . The production method according to claim 1 , wherein a period for the culturing in the second step is 12 hr-6 days.

16 . A method for producing a cerebral tissue comprising the following steps (1) and (2):
(1) a step of producing an aggregate comprising a cerebral tissue by the method of claim 1 ; and (2) a step of cutting out a cerebral tissue from the aggregate obtained in step (1).

17 . The production method according to claim 1 , wherein the pluripotent stem cells are primed pluripotent stem cells.

18 . The production method according to claim 17 , wherein the primed pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

19 . The production method according to claim 1 , wherein the medium comprising the factor for maintaining an undifferentiated state of the pluripotent stem cells can maintain and/or expand the pluripotent stem cells in the absence of the TGFβ family signal transduction pathway inhibiting substance or the Sonic hedgehog signal transduction pathway activating substance.

20 . A method for producing cerebral cells or a cerebral tissue, comprising the following steps (1)-(4):
(1) a first step of maintaining and/or expanding human pluripotent stem cells comprising culturing the human pluripotent stem cells in the absence of a feeder cell in a medium comprising a factor for maintaining an undifferentiated state of the pluripotent stem cells, (2) a second step of culturing the pluripotent stem cells obtained by the first step in a medium comprising (a) a Sonic hedgehog signal transduction pathway activating substance or a combination of a TGFβ family signal transduction pathway inhibiting substance and the Sonic hedgehog signal transduction pathway activating substance, and (b) a factor for maintaining an undifferentiated state of the pluripotent stem cells in the absence of feeder cells for a period of 0.5 hr-144 hr, (3) a third step of culturing the cells obtained in the second step in suspension to form a cell aggregate, and (4) a fourth step of culturing the aggregate obtained in the third step in suspension in the presence of a TGFβ family signal transduction pathway inhibiting substance and/or a Wnt signal transduction pathway inhibiting substance to obtain an aggregate containing cerebral cells or a cerebral tissue, wherein the factor for maintaining an undifferentiated state of the pluripotent stem cells comprises a FGF signal transduction pathway activating substance, wherein the FGF signal transduction pathway activating substance is bFGF, wherein the TGFβ family signal transduction pathway inhibiting substance is Lefty, SB431542, A-83-01 or LDN193189, wherein the Sonic hedgehog signal transduction pathway activating substance is Shh, SAG or Purmorphamine, and wherein the Wnt signal transduction pathway inhibiting substance is IWR-1-endo.

21 . The production method according to claim 1 , wherein the factor for maintaining an undifferentiated state of the pluripotent stem cells further comprises a TGFβ family signal transduction pathway activating substance.

22 . The production method according to claim 20 , wherein the factor for maintaining an undifferentiated state of the pluripotent stem cells further comprises a TGFβ family signal transduction pathway activating substance.

Assignees

• Racthera Co Ltd
• Riken

Inventors

• Kuwahara Atsushi
• YAMASAKI Suguru
• Hiramine Yasushi
• Sasai Yoshiki
• Takahashi Masayo

Classifications

• G01N33/5014

  for testing toxicity · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N2501/40

  Regulators of development · CPC title

• C12N2501/155

  Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor · CPC title

• C12N2501/115

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