Methods for obtaining retinal progenitors, retinal pigmented epithelial cells and neural retinal cells
View Patent ↗A method for in vitro obtaining human retinal progenitors, includes the steps of (i) placing an adherent culture of human pluripotent stem cells in a pro-neural medium; and (ii) maintaining this culture in the pro-neural medium until the appearance of pigmented cells and/or of neuroepithelial-like structures. Additional steps can be performed to obtain RPE cells and/or precursors of the neural retina.
1. A method for in vitro obtaining human retinal progenitors, comprising the steps of:
(i) placing an adherent culture of human pluripotent stem cells in a neural cell culture medium, said neural cell culture medium being composed of a nutrient medium supplemented with a medium supplement which comprises at least insulin and wherein the pluripotent stem cells form a colony-type monolayer; and
(ii) maintaining this culture in said neural cell culture medium until the appearance of pigmented cells and/or of neuroepithelial-like structures.
2. The method of claim 1 , wherein said neural cell culture medium is devoid of at least one of the following differentiation factors: noggin, Dkk-1 and IGF-1.
3. The method of claim 2 , wherein said neural cell culture medium is devoid of noggin, Dkk-1 and IGF-1.
4. The method of claim 1 , wherein, in step (i), the colony-type monolayer reaching at least 80% confluence.
5. The method of claim 1 , wherein step (ii) is performed during at least 7 days.
6. The method of claim 1 , for obtaining retinal pigmented epithelial cells (RPE cells), wherein said method further comprises the steps of
(iii RPE ) collecting, from the culture obtained in step (ii), at least one pigmented cell; and
(iv RPE ) culturing the pigmented cell(s) obtained in step (iii RPE ).
7. The method of claim 6 , wherein the culture in step (iv RPE ) is carried out in an adherent culture system.
8. The method of claim 1 , for obtaining neural retinal cells, wherein said method further comprises the steps of:
(iii NR ) collecting, from the culture obtained in step (ii), cells from at least one neuroepithelial-like structure; and
(iv NR ) culturing the cells obtained in step (iii NR ).
9. The method of claim 8 , wherein at least one neuroepithelial-like structure is collected in step (iii NR ).
10. The method of claim 8 , wherein the culture in step (iv NR ) is carried out in a non-adherent culture system.
11. The method of claim 8 , wherein in step (iv NR ), the culture medium is supplemented with FGF2 during at least 5 days.
12. The method of claim 8 , wherein the culture in step (iv NR ) is performed under shaking conditions.
13. The method of claim 8 , for obtaining photoreceptor precursors, wherein step (iv NR ) is performed during at least 21 days.
14. The method of claim 13 , wherein in step (iv NR ), a Notch inhibitor is added to the culture medium during at least 1 to 5 days.
15. The method of claim 13 , further comprising a step of cell sorting of photoreceptor precursors through binding of the cell surface marker CD73.
16. The method according to claim 7 , for obtaining both RPE cells and precursors of the neural retina, wherein steps (iii RPE ) and (iv RPE ) are performed in parallel with the steps of steps of:
(iii NR ) collecting, from the culture obtained in step (ii), cells from at least one neuroepithelial-like structure; and
(iv NR ) culturing the cells obtained in step (iii NR ).
17. The method of claim 1 , wherein the neural cell culture medium is composed of a nutrient medium supplemented with a medium supplement which comprises components selected from a group comprising insulin, transferrin, progesterone, putrescine sodium selenite and combination thereof.
18. The method of claim 1 , wherein the neural cell culture medium is composed of a nutrient medium supplemented with a medium supplement which comprises insulin, transferrin, progesterone, putrescine and sodium selenite.