Method for controlling differentiation of pluripotent stem cells
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.
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.