Method for induction/differentiation into photoreceptor cell
The present invention provides a method of producing primate retinal progenitor cells, comprising culturing primate embryonic stem cells as suspended aggregates in a serum-free medium, and obtaining retinal progenitor cells from the culture. The present invention further provides a method of producing photoreceptor precursor cells, comprising culturing isolated retinal progenitor cells differentiated from embryonic stem cells, under adhesive conditions, in the presence of a gamma secretase inhibitor, and obtaining a photoreceptor precursor from the culture.
1. A method of producing photoreceptor precursors, comprising
(a) culturing mammalian embryonic stem cells as suspended aggregates in a medium such that the ES cells differentiate into retinal progenitor cells indicated by the expression of a retinal progenitor cell maker selected from the group of Pax6, Rx and Mitf;
(b) culturing the retinal progenitor cells differentiated from the embryonic stem cells under adhesive conditions in the presence of a gamma secretase inhibitor to produce photoreceptor precursors indicated by the expression of the photoreceptor precursor marker selected from the group Crx and rhodopsin;
(c) isolating photoreceptor precursors from the culture.
2. The method of claim 1 , wherein the gamma secretase inhibitor is N—[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).
3. A method of producing cone photoreceptors, comprising
(a) culturing mammalian embryonic stem cells as suspended aggregates in a medium such that the ES cells differentiate into retinal progenitor cells indicated by the expression of a retinal progenitor cell maker selected from the group of Pax6, Rx and Mitf;
(b) culturing the retinal progenitor cells differentiated from the embryonic stem cells under adhesive conditions in the presence of a gamma secretase inhibitor to produce photoreceptor precursors indicated by the expression of the photoreceptor precursor marker selected from the group Crx and rhodopsin;
(c) culturing the photoreceptor precursor cells under adhesive conditions in a medium comprising a gamma secretase inhibitor and one of aFGF, bFGF, taurine, Shh or RA to produce cone photoreceptors indicated by the expression of a photoreceptor maker selected from the group rhodopsin, red/green opsin, blue opsin and Recoverin; and
(d) isolating cone photoreceptors from the culture.
4. The method of claim 3 , wherein the gamma secretase inhibitor is N—[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).
5. The method of claim 1 , wherein the culturing of the embryonic stem cells as suspended aggregates is performed in a serum-free medium containing at least one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.
6. The method of claim 3 , wherein the culturing of the embryonic stem cells as suspended aggregates is performed in a serum-free medium containing at least one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.
7. The method of claim 6 , wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242.
8. The method of claim 7 , wherein the Nodal signal inhibitor is Lefty-A or SB-431542.
9. The method of claim 6 , wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).
10. The method of claim 9 , wherein the Wnt signal inhibitor is Dkk1, CKI-7, or D4476.
11. The method of claim 6 , wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242, and wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{442,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).
12. The method of claim 11 , wherein the mammalian embryonic stem cells are human embryonic stem cells.
13. The method of claim 4 , wherein the culturing of the embryonic stem cells as suspended aggregates is performed in a serum-free medium containing at least one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.
14. The method of claim 13 , wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242, and wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).
15. The method of claim 14 , wherein the Nodal signal inhibitor is Lefty-A or SB-431542, and wherein the Wnt signal inhibitor is Dkk1, CKI-7, or D4476.
16. The method of claim 3 , wherein the mammalian embryonic stem cells are human embryonic stem cells.
17. The method of claim 5 , wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242.
18. The method of claim 17 , wherein the Nodal signal inhibitor is Lefty-A or SB-431542.
19. The method of claim 5 , wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).
20. The method of claim 19 , wherein the Wnt signal inhibitor is Dkk1, CKI-7, or D4476.
21. The method of claim 5 , wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242, and wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).
22. The method of claim 21 , wherein the mammalian embryonic stem cells are human embryonic stem cells.
23. The method of claim 2 , wherein the culturing of the embryonic stem cells as suspended aggregates is performed in a serum-free medium containing at least one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.
24. The method of claim 23 , wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242, and wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-1-yl}benzamide (D4476).
25. The method of claim 24 , wherein the Nodal signal inhibitor is Lefty-A or SB-431542, and wherein the Wnt signal inhibitor is Dkk1, CKI-7, or D4476.
26. The method of claim 1 , wherein the mammalian embryonic stem cells are human embryonic stem cells.