Endodermal cell production method, liver cell production method, pancreatic cell production method, endodermal cell induction promoter, liver cell induction promoting kit, pancreatic cell induction promoting kit, and microfluidic device

• US 10,590,389 B2
• Filed: 12/22/2015
• Issued: 03/17/2020
• Est. Priority Date: 12/24/2014
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Kamei, Kenichiro, Honda, Momoko
• Primary Examiner(s): Hixson, Christopher Adam
• Application Number:
  US15/539,546
• Publication Number:
  US 20170362568A1
• Time in Patent Office: 1,547 Days
• CPC Class Codes: C12M 1/00 Apparatus for enzymology or microbiology
• CPC Class Codes: C12M 3/00 Tissue, human, animal or plant cell, or virus culture apparatus
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/00 Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides an endodermal cell production method that can induce differentiation of pluripotent cells into endodermal cells even when the pluripotent cells are dispersed and can achieve improved endodermal cell production efficiency. The endodermal cell production method according to the present invention is a method for producing endodermal cells by inducing differentiation of pluripotent cells into the endodermal cells, including the step of: inducing differentiation of the pluripotent cells into the endodermal cells in the presence of an endodermal cell inducing factor. In the induction step, the cell density of the pluripotent cells at the start of the induction preferably is from 0.5×10⁴ to 2×10⁴ cells/cm².

Claims

1. A method for producing endodermal cells by inducing differentiation of pluripotent cells into the endodermal cells, the method comprising the step of:

inducing differentiation of the pluripotent cells into the endodermal cells in the presence of an endodermal cell inducing factor,

wherein, in the inducing step, ROCK protein activity is inhibited, and

the endodermal cell inducing factor comprises a PI₃K protein activity inhibitor.

2. The endodermal cell production method according to claim 1, wherein

in the inducing step, a cell density of the pluripotent cells at the start of the induction is from 0.5×

10⁴ to 2×

10⁵ cells/cm².

3. The endodermal cell production method according to claim 1, wherein

the pluripotent cells are dispersed.

4. The endodermal cell production method according to claim 1, wherein

the endodermal cell inducing factor comprises at least one selected from the group consisting of a TGF-β

family protein, bFGF, a GSK-3β

protein activity inhibitor, and an mTOR protein activity inhibitor.

5. The endodermal cell production method according to claim 4, wherein

the TGF-β

family protein comprises at least one selected from the group consisting of activin, BMP-4, and NODAL.

6. The endodermal cell production method according to claim 1, wherein

the inducing step is a step of inducing differentiation of the pluripotent cells into the endodermal cells in the presence of a pluripotent cell culture solution containing the endodermal cell inducing factor.

7. The endodermal cell production method according to claim 6, wherein

the pluripotent cell culture solution contains a TGF-β

family protein, bFGF, and insulin.

8. The endodermal cell production method according to claim 1, wherein

in the inducing step, the ROCK protein activity is inhibited by a ROCK protein activity inhibitory compound.

9. A liver cell production method comprising the step of:

differentiating endodermal cells into liver cells in the presence of a liver cell differentiation factor,

wherein the endodermal cells are obtained by the endodermal cell production method according to claim 1.

10. The liver cell production method according to claim 9, wherein

the liver cell differentiation factor comprises at least one selected from the group consisting of a TGF-β

family protein, FGF10, and WNT.

11. The liver cell production method according to claim 10, wherein

the TGF-β

family protein comprises at least one selected from the group consisting of activin, BMP-4, and NODAL.

12. The liver cell production method according to claim 9, further comprising the step of:

maturing the liver cells in the presence of a liver cell maturation factor.

13. The liver cell production method according to claim 12, wherein

in the maturing step, the liver cells are matured on a scaffold.

14. The liver cell production method according to claim 13, wherein

the scaffold is a coating containing an extracellular matrix protein, a coating containing a blood component or a coating containing a recombinant protein.

15. The liver cell production method according to claim 12, wherein

the liver cell maturation factor comprises at least one selected from the group consisting of OSM, HGF, dexamethasone, nicotinamide, dimethyl sulfoxide, and sodium butyrate.

16. A pancreatic cell production method comprising the step of:

differentiating endodermal cells into pancreatic cells in the presence of a pancreatic cell differentiation factor,

wherein the endodermal cells are obtained by the endodermal cell production method according to claim 1.

17. The endodermal cell production method according to claim 1, wherein

the endodermal cell inducing factor comprises a TGF-β

family protein and a PI₃K protein activity inhibitory compound, or comprises a TGF-β

family protein, a PI₃K protein activity inhibitory compound, and a GSK-3β

protein activity inhibitory compound.

18. The endodermal cell production method according to claim 1, wherein

the inducing step is a step of inducing differentiation of the pluripotent cells into the endodermal cells in the presence of a culture solution containing the endodermal cell inducing factor,

in the inducing step, the ROCK protein activity is inhibited by a ROCK protein activity inhibitory compound, and

the culture solution contains a TGF-β

family protein, a PI₃K protein activity inhibitory compound, a GSK-3β

protein activity inhibitory compound, bFGF, and insulin.

19. The endodermal cell production method according to claim 17, wherein

the TGF-β

family protein comprises activin A and BMP-4.

20. The endodermal cell production method according to claim 1, wherein

in the inducing step, the ROCK protein activity is inhibited by a ROCK protein activity inhibitory compound,

the inducing step is a step of inducing differentiation of the pluripotent cells into the endodermal cells in the presence of a first culture solution, a second culture solution, and a third culture solution, each containing the endodermal cell inducing factor,

the first culture solution contains activin A and the ROCK protein activity inhibitory compound,

the second culture solution contains activin A, BMP-4, a PI₃K protein activity inhibitory compound, a GSK-3β

protein activity inhibitory compound, and the ROCK protein activity inhibitory compound,

the third culture solution contains activin A, BMP-4, a PI₃K protein activity inhibitory compound, and the ROCK protein activity inhibitory compound, and

the inducing step comprises;

a first step of culturing the pluripotent cells in the presence of the first culture solution;

a second step of culturing cells obtained in the first step in the presence of the second culture solution; and

a third step of culturing cells obtained in the second step in the presence of the third culture solution.