Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells
View Patent ↗Methods are provided for the production of photoreceptor cells and photoreceptor progenitor cells from pluripotent stem cells. Additionally provided are compositions of photoreceptor cells and photoreceptor cells, as well as methods for the therapeutic use thereof. Exemplary methods may produce substantially pure cultures of photoreceptor cells and/or photoreceptor cells.
1. A method of producing photoreceptor progenitor cells, comprising
(a) culturing pluripotent stem cells in the presence of a BMP signaling inhibitor to produce eye field progenitor cells;
(b) culturing the eye field progenitor cells under low adherence or non-adherent conditions in a neural differentiation medium in the absence of the BMP signaling inhibitor to form cell spheres;
(c) culturing the cell spheres under adherent conditions in the neural differentiation medium in the absence of the BMP signaling inhibitor to form attached cells;
(d) dissociating the attached cells and culturing the cells under low adherence or non-adherent conditions in the neural differentiation medium in the absence of the BMP signaling inhibitor to form further cell spheres;
(e) culturing the further cell spheres under adherent conditions in the neural differentiation medium in the absence of the BMP signaling inhibitor to form further attached cells;
(f) repeating steps (d) and (e) at least one additional time; and
thereby obtaining a preparation of photoreceptor progenitor cells, wherein 75%-100% of the photoreceptor progenitor cells in the preparation are PAX6(+) and CHX10(−) at the mRNA level.
2. The method of claim 1 , wherein said neural differentiation medium comprises Neurobasal medium, D-glucose, N2 supplement, B27 supplement, non-essential amino acids, and (i) glutamine, (ii) a stabilized form dipeptide from L-glutamine or L-alanyl-L-glutamine, or (iii) both glutamine and a stabilized form dipeptide from L-glutamine or L-alanyl-L-glutamine.
3. The method of claim 2 , wherein said neural differentiation medium comprises between about 400 and about 500 mg/ml D-glucose, one or both of streptomycin and penicillin, N2 supplement in a concentration of about 0.1 to 5%, B27 supplement in a concentration of about 0.05-5.0%, and non-essential amino acids in a concentration of about 0.1 mM.
4. The method of claim 3 , wherein said neural differentiation medium comprises about 2% N2 supplement.
5. The method of claim 3 , wherein said neural differentiation medium comprises about 0.05-2.0% B27 supplement.
6. The method of claim 3 , wherein said neural differentiation medium comprises about 2% B27 supplement.
7. The method of claim 1 , wherein step (a) comprises culturing the pluripotent stem cells under adherent conditions.
8. The method of claim 1 , further comprising differentiating said photoreceptor progenitor cells into photoreceptors.
9. The method of claim 1 , wherein the BMP signaling inhibitor is selected from the group consisting of Noggin polypeptide, dorsomorphin, LDN-193189, and any combination thereof.
10. The method of claim 9 , wherein the BMP signaling inhibitor is a Noggin polypeptide.
11. The method of claim 1 , wherein the photoreceptor progenitor cells express one or more of the Nr2e3, Trβ2, RORβ and NRL markers.
12. The method of claim 1 , wherein the photoreceptor progenitor cells are further characterized as mRNA transcript positive for Nr2e3, Trβ2, RORβ and NRL as detected by qPCR.
13. The method of claim 1 , wherein the photoreceptor progenitor cells are characterized by differentiation to photoreceptor cells upon treatment with retinoic acid.
14. The method of claim 1 , wherein the photoreceptor progenitor cells formed in step (c) retain plasticity to differentiate into either rods and cones.
15. The method of claim 1 , wherein the eye field progenitor cells are characterized as immunocytochemically PAX6+ and RX1+ and OCT4− and NANOG−.
16. The method of claim 15 , wherein the eye field progenitor cells are further characterized as Six3+, Six6+, Lhx2+, Tbx3+, SOX2+ and Nestin+ by immunostaining or flow cytometry.
17. The method of claim 1 , wherein the pluripotent stem cells are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.
18. The method of claim 1 , wherein the photoreceptor progenitor cells are capable of differentiating into photoreceptor cells in vitro.
19. The method of claim 18 , wherein the photoreceptor progenitor cells differentiate into photoreceptor cells in vitro in the presence of retinoic acid and taurine.
20. The method of claim 18 , wherein the photoreceptor cells express rhodopsin, opsin, recoverin, and PDE6a.
21. The method of claim 1 , wherein the neural differentiation medium further comprises transferrin and insulin.