Method for generating retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (IPSCs)
Inventors
Bharti, Kapil • Davis, Janine • Maminishkis, Arvydas • Miller, Sheldon S.
Assignees
US Department of Health and Human Services
Publication Number
US-11441184-B2
Publication Date
2022-09-13
Expiration Date
2034-01-31
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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.
Core Innovation
The invention discloses high efficiency methods for producing retinal pigment epithelial (RPE) cells from induced pluripotent stem cells (iPSCs). These iPSCs are derived from somatic cells, including retinal pigment epithelial cells such as fetal RPE stem cells. The methods involve culturing iPSCs to form embryoid bodies and subsequent culturing steps with specific media containing inhibitors and inducers of pathways like Wnt, Nodal, FGF, and Sonic to promote differentiation to RPE cells.
The background explains that the retina contains photoreceptor cells responsible for converting light into nerve impulses. The retinal pigment epithelium (RPE) is a polarized monolayer that supports the neural retina, acting as a blood-retinal barrier and maintaining visual function by nutrient transport and participating in the visual cycle. Many ophthalmic diseases involve degeneration of the retina or RPE, and previous studies showed photoreceptor rescue by transplantation of RPE cells. However, there is a need for efficient methods to produce RPE cells, such as from human stem cells, for treatment of retinal degeneration.
Claims Coverage
The patent contains one independent claim which covers a multi-step method for producing human retinal pigment epithelial cells from human induced pluripotent stem cells, detailing specific culture media, factors, and conditions at each step to achieve efficient RPE differentiation.
Stepwise culture methods employing specific inhibitors and inducers for efficient RPE differentiation
The method comprises culturing human iPSCs in a human embryonic stem cell culture medium with bFGF to produce embryoid bodies of 200-500 cells, followed by culture in retinal induction medium with Wnt and Nodal inhibitors, Noggin, and knockout serum replacement to increase RPE differentiation efficiency.
Use of specific media compositions at distinct stages for differentiation and maturation
Embryoid bodies are further cultured on Matrigel-coated substrates in retinal differentiation medium lacking bFGF but including DKK1, Wnt inhibitors, bFGF inhibitors, and Noggin to form differentiating RPE cells expressing PAX6 and MITF. These cells are then cultured in retinal media with Activin A and Wnt3a to increase MITF and PAX6 expression and RPE differentiation efficiency.
Final maturation step using defined factors to produce functional RPE cells
Cells with increased MITF and PAX6 expression are cultured in medium comprising a non-canonical Wnt 5a inducer, DKK1, SU5402 (FGF inhibitor), and cyclopamine (Sonic pathway inhibitor) to produce human RPE cells expressing markers such as TYR, TYRP1, MYRIP, Cadherin 1, and TRPM1/3.
The claims cover a detailed, multi-stage method for generating functional human RPE cells from iPSCs by employing specific culture conditions, pathway inhibitors, and inducers at sequential stages to enhance differentiation efficiency and maturity of RPE cells.
Stated Advantages
The disclosed methods increase the efficiency and quality of RPE cell production from iPSCs.
The methods produce RPE cells that express appropriate markers and exhibit functional and physiological properties similar to native RPE.
Use of pathway inhibitors and inducers in defined culture media enhances RPE differentiation and maturation, resulting in homogeneous and fully differentiated RPE cultures.
Documented Applications
Treatment of retinal degenerative diseases and injuries by transplantation of produced RPE cells.
Use as therapeutics to rescue or preserve photoreceptors and visual function in ocular diseases such as macular degeneration and retinitis pigmentosa.
Screening assays for identifying agents that modulate RPE differentiation, proliferation, or survival.
Use in personalized medicine by generating autologous or MHC-matched RPE cells from patient-derived iPSCs for therapeutic transplantation.
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