Method for generating Retinal Pigment Epithelium (RPE) cells from Induced Pluripotent Stem Cells (IPSCs)
First Claim
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1. 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 Wnt inhibitor CK1-7, 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), CK1-7, 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; and
(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 3 and TRPMI 1 or TRPMI 3, thereby producing human RPE cells.
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Abstract
High efficiency methods for producing retinal pigment epithelial cells from induced pluripotent stem cells (iPSCs) are disclosed herein. The iPSCs are produced from somatic cells, including retinal pigment epithelial (RPE) cells, such as fetal RPE stem cells. In some embodiments, the iPSC include a tyrosinase promoter operably linked to a marker. Methods are disclosed for using the RPE cells, such as for treatment. Methods for screening for agents that affect RPE differentiation are also disclosed.
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Citations
27 Claims
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1. A method for producing human retinal pigment epithelial (RPE) cells, comprising:
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(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 Wnt inhibitor CK1-7, 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), CK1-7, 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; and (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 3 and TRPMI 1 or TRPMI 3, thereby producing human RPE cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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