Method for generating Retinal Pigment Epithelium (RPE) cells from Induced Pluripotent Stem Cells (IPSCs)
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10480031-B2
Publication Date
2019-11-19
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
This disclosure presents high efficiency methods for producing retinal pigment epithelial cells from induced pluripotent stem cells (iPSCs). The iPSCs are generated from somatic cells, including retinal pigment epithelial cells, such as fetal retinal pigment epithelial stem cells. The methods involve culturing iPSCs to form embryoid bodies, followed by sequential culturing in media comprising specific combinations of pathway inhibitors and inducers, including Wnt pathway inhibitors, Nodal pathway inhibitors, basic fibroblast growth factor inhibitors, Activin A, and Wnt3a, and factors that promote maturation such as non-canonical Wnt inducers and inhibitors of the Sonic and FGF pathways. These procedures result in the production of differentiated human retinal pigment epithelial cells characterized by expression of specific markers and physiological properties.
The problem being solved addresses the need for efficient production of retinal pigment epithelial cells that can be used for therapeutic purposes, as many ophthalmic diseases are associated with degeneration of the retina or of the RPE. Existing methods lacked efficiency and consistency in producing large numbers of functional RPE cells from stem cells. This invention provides optimized culture conditions and protocols using defined combinations of inhibitors and growth factors to significantly improve the yield and quality of RPE cells derived from human iPSCs, suitable for clinical and research applications.
Claims Coverage
The patent includes one independent claim describing a multi-step method with specific culture media and conditions to efficiently differentiate human induced pluripotent stem cells into retinal pigment epithelial cells.
Production of embryoid bodies from hiPSCs in defined animal component-free medium
Culturing human induced pluripotent stem cells in human embryonic stem cell culture medium containing human basic fibroblast growth factor but no ingredients from non-human animals to produce small embryoid bodies of 200-500 cells.
Culturing embryoid bodies in retinal induction medium with specific inhibitors
Culturing embryoid bodies in a first medium comprising retinal induction medium and rock inhibitor; the retinal induction medium includes Wnt inhibitor CK1-7, a Nodal pathway inhibitor (SB-431542 or SB-505124), Noggin, and 1 to 3% knockout serum replacement to increase efficiency of RPE differentiation.
Culturing on matrigel-coated substrate in medium lacking bFGF but including inhibitors
Culturing embryoid bodies on a matrigel-coated tissue culture substrate in a second medium comprising retinal differentiation medium without basic fibroblast growth factor but including Dickkopf-related protein 1, CK1-7, a bFGF inhibitor (such as PD0325901, PD98059, PD161570, or PD166285), and Noggin, to form differentiating RPE cells expressing PAX6 and MITF.
Culturing differentiating RPE cells in medium with Activin A and Wnt3a
Culturing differentiating RPE cells in a third medium comprising retinal media including Activin A and Wnt3a to produce cells with increased expression of MITF and PAX6 and enhanced RPE differentiation efficiency.
Maturation of RPE cells using a fourth medium with non-canonical Wnt inducers and pathway inhibitors
Culturing the cells in a fourth medium comprising an RPE cell medium with a non-canonical Wnt 5a inducer, Dickkopf-related protein 1, SU5402 (FGF inhibitor), and cyclopamine (Sonic hedgehog pathway inhibitor) to produce mature human RPE cells expressing TYR, TYRP1, MYRIP, Cadherin 1 or 3, and TRPM1 or 3.
The independent claim covers a stepwise differentiation method applying specific defined culture conditions and modulators of signaling pathways to produce mature human retinal pigment epithelial cells from hiPSCs with high efficiency and expression of characteristic RPE markers.
Stated Advantages
The method significantly improves the efficiency of differentiation of human iPSCs into retinal pigment epithelial cells.
The protocol produces RPE cells that are fully functional and exhibit gene expression profiles, physiological, and morphological features closely resembling native human RPE.
Utilization of specific pathway inhibitors and inducers enables controlled, high fidelity RPE differentiation and maturation, reducing contaminating cell types.
The disclosed method is scalable and amenable to high-throughput screening for agents that modulate RPE cell biology or serve therapeutic purposes.
Use of biodegradable scaffolds facilitates generation of polarized monolayer RPE tissues suitable for transplantation, potentially improving clinical efficacy over cell suspensions.
Documented Applications
Use of the produced retinal pigment epithelial cells for the treatment of retinal degenerative diseases and injuries, including diseases such as age-related macular degeneration, macular dystrophies, Stargardt's disease, Best disease, and retinitis pigmentosa.
Use of the RPE cells in screening assays to identify agents that modulate RPE cell proliferation, differentiation, survival, or phenotype.
Use of the differentiated RPE cells or compositions comprising these cells as pharmaceutical compositions for cell-based therapy or regenerative medicine in ophthalmology.
Generation of homogeneous, functional, and polarized RPE tissues on biodegradable scaffolds for transplantation applications and in vitro disease modeling and drug screening.
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