Method of inducing the differentiation of embryonic stem cells into nerve by serum-free suspension culture
The present invention provides a clinically applicable method of inducing differentiation of embryonic stem cells, particularly a method of inducing differentiation of embryonic stem cells into forebrain neurons. More specifically, the present invention provides a method of inducing differentiation of embryonic stem cells, comprising culturing the embryonic stem cells as a floating aggregate in a serum-free medium, particularly a method of inducing differentiation of the embryonic stem cells into nervous system cells such as forebrain neurons and cerebellar neurons and sensory organ cells; a floating aggregate of embryonic stem cells obtained by culturing the embryonic stem cells as a floating aggregate in a serum-free medium; and cells derived from a floating aggregate of embryonic stem cells, particularly nervous system cells such as forebrain neurons and cerebellar neuron, sensory organ cells such as retinal precursor cells, and the like.
1. A method of inducing differentiation of mammalian embryonic stem cells into retinal system cells, which method comprises
(a) producing a floating aggregate of embryonic stem cells in a serum-free medium in the absence of feeder cells, and
(b) culturing the floating aggregate in a serum-free medium comprising a Nodal signal inhibitor and a Wnt signal inhibitor in the absence of feeder cells,
thereby inducing differentiation of the embryonic stem cells into retinal system cells.
2. The method of claim 1 , wherein the Nodal signal inhibitor is Lefty-A.
3. The method of claim 1 , wherein the Wnt signal inhibitor is Dkk1.
4. The method of claim 1 , wherein the concentration of the embryonic stem cells at the start of cultivation is 1×10 4 to 5×10 5 cells/ml.
5. The method of claim 1 , wherein cultivation of the embryonic stem cells is performed in a non-cell-adhesive culture vessel.
6. The method of claim 1 , which comprises culturing the floating aggregate of the embryonic stem cells for at least 5 days.
7. The method of claim 1 , which further comprises culturing cells obtained in step (b) under adherent conditions.
8. The method of claim 1 , which further comprises adding an Shh signal promoter to the serum-free medium comprising the floating aggregate.
9. The method of claim 8 , wherein the Shh signal promoter is Shh.
10. The method of claim 8 , wherein the Shh signal promoter is added to the serum-free medium within 3 to 7 days after the start of the culture of the floating aggregate.
11. The method of claim 1 , which further comprises adding serum to the serum-free medium comprising the floating aggregate.
12. The method of claim 11 , wherein the serum is added to the serum-free medium within 3 to 7 days after the start of the culture of the floating aggregate.
13. The method of claim 1 , which further comprises adding activin to the serum-free medium comprising the floating aggregate.
14. The method of claim 13 , wherein the activin is added to the serum-free medium within 3 to 7 days after the start of the culture of the floating aggregate.
15. The method of claim 11 , wherein the floating aggregate of the embryonic stem cells is cultured in the presence of Lefty-A, Dkk1, and activin.
16. The method of claim 1 , wherein the retinal system cells are retinal precursor cells.
17. The method of claim 1 , wherein the serum-free medium is (i) a medium substantially not comprising one or more items selected from the group consisting of FGF, RA, and a BMP signal promoter, or (ii) a medium wherein one or more items selected from the group consisting of FGF, RA and a BMP signal promoter, have been substantially inactivated.
18. The method of claim 1 , wherein the serum-free medium is a medium substantially not comprising a Nodal signal promoter and/or a Wnt signal promoter.