Methods and compositions for the rapid production of retinal pigmented epithelial cells from pluripotent cells
The invention relates to the field of cell culture, specifically, the derivation of retinal pigmented epithelial cells from pluripotent cells. The invention comprises the use of various cell culture medium supplements, medium formulations, and methods of using such medium supplements and formulations, in order to effect the rapid differentiation of pluripotent cells into retinal pigmented epithelial cells with very high yields. The invention further includes cell culture media formulations for the efficient maintenance, propagation, and maturation of cultured retinal pigmented epithelial cells.
1. A method of differentiating mammalian pluripotent stem cells into retinal pigmented epithelium cells, comprising
culturing pluripotent stem cells in a first medium comprising a basal medium supplemented with IGF1, DKK1, and noggin;
subsequently, culturing the cells in a second medium comprising a basal medium supplemented with IGF1, DKK1, noggin, and BFGF;
subsequently, culturing the cells in a third medium comprising a basal medium supplemented with IGF1, DKK1 and Activin A; and
subsequently, culturing the cells in a fourth medium comprising a basal medium supplemented with Activin A and SU5402, thereby obtaining retinal pigmented epithelium cells.
2. The method of claim 1 , wherein
the basal medium is selected from a group consisting of:
Dulbecco's Modified Eagle Medium mammalian cell culture medium, Ham's F12 medium, a 1:1 mixture of Dulbecco's Modified Eagle Medium mammalian cell culture medium and F12, Iscove's Modified Dulbecco's Medium, Neurobasal™ medium, X-Vivo™ 10 medium, Minimum Essential Medium Eagle medium, Roswell Park Memorial Institute Medium 1640, and MCDB medium.
3. The method of claim 1 , wherein
noggin is present in the first medium and the second medium at a concentration between 1 and 100 ng/ml;
IGF1 is present in the first medium, the second medium, and the third medium at a concentration between 1 and 100 ng/ml;
DKK1 is present in the first medium, the second medium, and the third medium at a concentration between 1 and 50 ng/ml;
BFGF is present in the second medium at a concentration between 1 and 20 ng/ml;
Activin A is present in the third medium and the fourth medium at a concentration between 10 and 200 ng/ml; and
SU5402 is present in the fourth medium at a concentration between 1 and 100 μM.
4. The method of claim 1 , wherein
nicotinamide is present in the first medium, the second medium, or both the first and second media.
5. The method of claim 4 , wherein
nicotinamide is present at a concentration between 1 and 100 mM.
6. The method of claim 1 , wherein
VIP is present in the fourth medium.
7. The method of claim 6 , wherein
VIP is present at a concentration between 0.1 to 100 μM.
8. The method of claim 1 , wherein
the transition between the first and second medium is made 24-72 hours after the initiation of culture in the first medium.
9. The method of claim 1 , wherein
the transition between the second and third medium is made 48-144 hours after the initiation of culture in the first medium.
10. The method of claim 1 , wherein
the transition between the third and fourth medium is made 60 to 192 hours after the initiation of culture in the first medium.