Method for generating retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (IPSCs)

A method for producing human retinal pigment epithelial (RPE) cells, comprising:

• (a) culturing human induced pluripotent stem cells (hiPSCs) in a human embryonic stem cell culture medium comprising human basic fibroblast growth factor (bFGF) and not containing ingredients obtained from non-human animals to produce small embryoid bodies of 200-500 cells;
• (b) culturing the small embryoid bodies from step (a) in a first medium comprising retinal induction medium (RIM) and rock inhibitor (RI), wherein the RIM comprises a Wnt inhibitor, a Nodal pathway inhibitor, Noggin, and 1 to 3% v/v knockout serum replacement, wherein the Nodal pathway inhibitor is SB-431542 or SB-505124, to form embryoid bodies that have increased efficiency of RPE differentiation;
• (c) culturing the embryoid bodies that have increased efficiency of RPE differentiation from step (b) on a matrigel coated tissue culture substrate-in a second medium comprising a retinal differentiation medium that does not comprise basic fibroblast growth factor (bFGF) and comprises Dickkopf-related protein 1 (DKK1), the Wnt inhibitor, a bFGF inhibitor, and Noggin, wherein the bFGF inhibitor is PD0325901, PD98059, PD161570, or PD166285, to form differentiating RPE cells that express PAX6 and MITF;
• (d) culturing the differentiating RPE cells from step (c) in a third medium comprising retinal media comprising Activin A and Wnt3a to produce cells that have increased expression of MITF and PAX6 and increased RPE differentiation efficiency;
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• (e) culturing the cells that have increased expression of MITF and PAX6 and increased RPE differentiation efficiency from step (d) in a fourth medium comprising a RPE cell medium comprising a non-canonical Wnt 5a inducer, DKK1, SU5402, and cyclopamine to produce human RPE cells that express TYR, TYRP1, MYRIP, Cadherin 1 or Cadherin 1 and TRPMI 1 or TRPMI 3, thereby producing human RPE cells.