Method for promoting differentiation of pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-2021115397-A1
Application number	US-201917050312-A
Country	US
Kind code	A1
Filing date	Apr 25, 2019
Priority date	Apr 26, 2018
Publication date	Apr 22, 2021
Grant date	—

Abstract

In order to improve the efficiency of inducing differentiation of pluripotent stem cells, provided is a method for promoting differentiation of pluripotent stem cells, the method including a step of culturing pluripotent stem cells in a medium, wherein the medium is a medium containing a) an insulin-like growth factor, b) an insulin analogue preparation containing no zinc, c) an insulin analogue preparation containing a low concentration of zinc, or d) a compound exhibiting an insulin-like action.

First claim

1 . A method for promoting differentiation of pluripotent stem cells, comprising: a step of culturing pluripotent stem cells in a medium, wherein the medium is a medium comprising a) an insulin-like growth factor, b) an insulin analogue preparation containing no zinc, c) an insulin analogue preparation containing a low concentration of zinc, or d) a compound exhibiting an insulin-like action.

2 . The method for promoting differentiation of pluripotent stem cells according to claim 1 , wherein the medium for culturing pluripotent stem cells is a medium comprising no insulin.

3 . The method for promoting differentiation of pluripotent stem cells according to claim 1 , wherein the medium for culturing pluripotent stem cells is a medium comprising zinc at a concentration of 1 μM or less or a medium comprising no zinc.

4 . The method for promoting differentiation of pluripotent stem cells according to claim 1 , wherein the medium for culturing pluripotent stem cells is a medium allowing the pluripotent stem cells to differentiate into endoderm cells.

5 . The method for promoting differentiation of pluripotent stem cells according to claim 1 , comprising: a step of culturing pluripotent stem cells in an undifferentiated maintenance medium, wherein the undifferentiated maintenance medium is a medium comprising no methionine.

6 . The method for promoting differentiation of pluripotent stem cells according to claim 1 , wherein the medium for culturing pluripotent stem cells is a medium comprising an activin receptor-like kinase-4,7 activator and comprising zinc at a concentration of 1 μM or less or no zinc.

7 . The method for promoting differentiation of pluripotent stem cells according to claim 6 , wherein the activin receptor-like kinase-4,7 activator is human activin A, and a concentration thereof is 6 to 150 ng/mL.

8 . A medium for culturing pluripotent stem cells, comprising: a) an insulin-like growth factor, b) an insulin analogue preparation comprising no zinc, c) an insulin analogue preparation comprising a low concentration of zinc, or d) a compound exhibiting an insulin-like action.

9 . The medium according to claim 8 , comprising no insulin.

10 . The medium according to claim 8 , comprising zinc at a concentration of 1 μM or less or no zinc.

11 . The medium according to claim 8 , wherein the medium is a medium allowing the pluripotent stem cells to differentiate into endoderm cells.

12 . The medium according to claim 8 , wherein the medium for culturing pluripotent stem cells is a medium comprising an activin receptor-like kinase-4,7 activator and comprises zinc at a concentration of 1 μM or less or no zinc.

13 . The medium according to claim 12 , wherein the activin receptor-like kinase-4,7 activator is human activin A, and a concentration thereof is 6 to 150 ng/mL.

14 . A method for inducing differentiation of pluripotent stem cells into insulin-producing cells, comprising steps (1) to (6) below:
(1) a step of culturing pluripotent stem cells in a medium comprising no methionine; (2) a step of culturing the cells obtained in step (1) in the medium according to claim 12 ; (3) a step of culturing the cells obtained in step (2) in a medium comprising an FGF and a hedgehog signaling inhibitor; (4) a step of culturing the cells obtained in step (3) in a medium comprising a retinoic acid receptor agonist, a hedgehog signaling inhibitor, and a BM′ signaling inhibitor; (5) a step of culturing the cells obtained in step (4) in a medium comprising a TGF-βI type activin receptor-like kinase-4,5,7 inhibitor, a protein kinase C activator, and a BMP signaling inhibitor; and (6) a step of culturing the cells obtained in step (5) in a medium comprising nicotinamide.

15 . The method for inducing differentiation into insulin-producing cells according to claim 14 , wherein each medium in step (1) to (6) is a xeno-free medium.

16 . A method for inducing differentiation of pluripotent stem cells into insulin-producing cells, comprising steps (1) to (9), or (1) to (3) and (5) to (9) below:
(1) a step of culturing pluripotent stem cells in a medium comprising no methionine; (2) a step of culturing the cells obtained in step (1) in the medium according to claim 12 ; (3) a step of culturing the cells obtained in step (2) in a medium comprising an FGF and a hedgehog signaling inhibitor; (4) a step of culturing the cells obtained in step (3) in a medium comprising a KGF; (5) a step of culturing the cells obtained in step (3) or step (4) in a medium comprising a KGF, a hedgehog signaling inhibitor, a protein kinase C activator, and a retinoic acid receptor agonist; (6) a step of culturing the cells obtained in step (5) in a medium comprising a KGF, a hedgehog signaling inhibitor, a retinoic acid receptor agonist, and a BMP signaling inhibitor; (7) a step of culturing the cells obtained in step (6) in a medium comprising a TGF-βI type activin receptor-like kinase-4,5,7 inhibitor, an EGF receptor agonist, a γ-secretase inhibitor, and a retinoic acid receptor agonist; (8) a step of culturing the cells obtained in step (7) in a medium comprising a TGF-βI type activin receptor-like kinase-4,5,7 inhibitor, an EGF receptor agonist, a γ-secretase inhibitor, and a retinoic acid receptor agonist, wherein a concentration of the retinoic acid receptor agonist in the medium is lower than a concentration of the retinoic acid receptor agonist in step (7); and (9) a step of culturing the cells obtained in step (8) in a medium comprising a TGF-βI type activin receptor-like kinase-4,5,7 inhibitor.

17 . A method for differentiating pancreatic progenitor cells into pancreatic beta cells, comprising culturing pancreatic progenitor cells in a medium comprising a factor selected from the group consisting of a retinoic acid receptor agonist, a hedgehog signaling inhibitor, a KGF, a γ-secretase inhibitor, a BMP signaling inhibitor, a TGF-βI type activin receptor-like kinase-4,5,7 inhibitor, and an EGF receptor agonist, wherein the pancreatic progenitor cells are PDX1 positive or NKX6.1 positive.

18 . A method for inducing differentiation of pluripotent stem cells into insulin-producing cells, comprising steps (1) to (7) below:
(1) a step of culturing pluripotent stem cells in a medium comprising no methionine; (2) a step of culturing the cells obtained in step (1) in the medium according to claim 12 ; (3) a step of culturing the cells obtained in step (2) in a medium comprising a KGF and comprising zinc at a concentration of 1 μM or less or no zinc; (4) a step of culturing the cells obtained in step (3) in a medium comprising a KGF, a hedgehog signaling inhibitor, and a retinoic acid receptor agonist; (5) a step of culturing the cells obtained in step (4) in a medium comprising a KGF, a hedgehog signaling inhibitor, and a retinoic acid receptor agonist, wherein a concentration of the retinoic acid receptor agonist in the medium is lower than a concentration of the retinoic acid receptor agonist in step (4); (6) a step of culturing the cells obtained in step (5) in a medium comprising a TGF-βI type activin receptor-like kinase-4,5,7 inhibitor and a retinoic acid receptor agonist; and (7) a step of culturing the cells obtained in step (6) in a medium comprising a TGF-βI type activin receptor-like kinase-4,5,7 inhibitor and a retinoic acid receptor agonist, wherein a concentration of the retinoic acid receptor agonist in the medium is lower than a concentration of the retinoic acid receptor agonist in step (6).

Assignees

• Tokyo Inst Tech
• Ajinomoto Kk

Inventors

• Kume Shoen
• Shiraki Nobuaki
• Chiba Akira
• Enomoto Takayuki

Classifications

• C12N5/0676 Primary

  Pancreatic cells · CPC title

• C12N2501/105

  Insulin-like growth factors [IGF] · CPC title

• C12N2501/119

  Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10 · CPC title

• C12N2501/41

  Hedgehog proteins; Cyclopamine (inhibitor) · CPC title

• C12N2501/117

  Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12) · CPC title