Method for controlling differentiation of pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-11649433-B2
Application number	US-201816482160-A
Country	US
Kind code	B2
Filing date	Jan 31, 2018
Priority date	Jan 31, 2017
Publication date	May 16, 2023
Grant date	May 16, 2023

Abstract

The present invention relates to a method for controlling differentiation of pluripotent stem cells, which method comprises selecting a laminin or a fragment thereof based on binding affinity for the pluripotent stem cells and inducing differentiation of the pluripotent stem cells in the presence of the laminin or a fragment thereof. Here, the binding affinity for cells can be assessed by time-course observation of the survival rate and motility of the cells. According to the present invention, a cell population containing any desired proportion of differentiated cells can be produced from pluripotent stem cells in a simple manner. The cell population obtained by this production method is very useful for cell therapy-based treatment strategies for diseases.

First claim

The invention claimed is:

1. A method for producing corneal epithelial cells, the method comprising differentiating human iPS cells to corneal epithelial cells by:
(a) culturing said human iPS cells in a culture medium in contact with a laminin 332E8 fragment to generate a first cell population that has been contacted with said laminin332E8 fragment;
(b) culturing said first cell population in a serum free medium in contact with a laminin 332E8 fragment to generate a second cell population;
(c) culturing said second cell population of differentiated cells in a corneal differentiation medium, which lacks epidermal growth factor (EGF) or fibroblast growth factor 2 (FGF2) but comprises keratinocyte growth factor (KGF) in contact with a laminin 332E8 fragment to produce a third cell population; and
(d) culturing said third cell population of differentiated cells in a corneal epithelial cell maintenance medium, which comprises keratinocyte growth factor (KGF) in contact with a laminin 332E8 fragment to produce corneal epithelial cells.

2. The method of claim 1 further comprising detecting the presence of a marker for corneal epithelial cells with the corneal epithelial cells produced by said method.

3. The method of claim 2 , wherein the marker is E-cadherin, TP63, PAX6, K12, K14, or SSEA4/ITGB4.

4. The method of claim 1 further comprising:
(e) isolating the corneal epithelial cells produced by the method;
(f) seeding 1×10 5 or more of said corneal epithelial cells isolated in step (e) on a plate, and
(g) culturing the seeded cells of step (f) on the plate to confluency to form a corneal epithelial cell sheet.

5. The method of claim 4 , wherein the corneal epithelial cells are isolated by FACS.

6. The method of claim 1 , wherein steps (a), (b), (c) and (d) are performed in a vessel coated with said laminin332E8 fragment.

7. The method of claim 1 , wherein steps (a), (b), (c) and (d) are performed by contacting said iPS cells with a carrier comprising said laminin332E8 fragment.

8. The method of claim 1 , wherein the concentration of said laminin332E8 fragment used in said method is about 0.25-2.0 μg/cm 2 .

9. The method of claim 1 , wherein step (a) is performed for 8-10 days.

10. The method of claim 1 , wherein step (b) is performed for 4 weeks.

11. The method of claim 1 , wherein step (c) is performed for 4 weeks.

12. The method of claim 1 , wherein step (d) is performed for 2-5 weeks.

13. The method of claim 1 , further comprising analyzing for the presence or absence of a marker indicating the presence of an undifferentiated populations of cells in the corneal epithelial cells produced by said method.

14. The method of claim 13 , wherein said marker is LIN28A.

Assignees

• Univ Osaka

Inventors

• Nishida Kohji
• Sekiguchi Kiyotoshi
• Hayashi Ryuhei
• Shibata Shun

Classifications

• A61K35/545

  Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells · CPC title

• A61K38/1825

  Fibroblast growth factor [FGF] · CPC title

• C12N5/0619 Primary

  Neurons · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title

• C12N2501/998

  Proteins not provided for elsewhere · CPC title