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Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation

ABSTRACT: Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here we conduct single-cell transcriptomic profiling of a hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, in which cells rapidly acquired a rostral embryo patterning signature, before converging towards the RPE lineage. At intermediate steps, we identified and examined the potency of a NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

ORGANISM(S): Oryctolagus cuniculus Homo sapiens

PROVIDER: GSE164092 | GEO | 2021/02/04

REPOSITORIES: GEO

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Project description:Background: Age-related macular degeneration (AMD) is caused by the degeneration of the macular photoreceptors. This is preceded by development of subretinal protein aggregates (drusen) and degeneration of the macular retinal pigment epithelium (RPE). The underlying pathogenesis remains unknown, but the immune system is suspected to play a key role in it. Aging of the immune system, immunosenescence, includes changes in T cell sub-populations and results in low-level chronic inflammation. Because AMD exclusively occurs in patients over 55 years of age, we hypothesize that aging of the T cell compartment may be implicated in AMD pathogenesis. Methodology and principal findings: Using an in vitro co-culture system, we investigated the effects of activated T cells on a human RPE cell line (ARPE-19). Differential gene expression in the RPE cells of complement factor genes was identified using microarrays, and selected gene transcripts from the alternative complement pathway were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and protein expression of complement component C3, factors B, H, H-like 1, CD46, CD59, and clusterin, in a dose-dependent manner. Conclusions: RPE cells responded to inflammatory assault caused by exposure to T cell-derived cytokines by upregulating expression of many complement factors from the alternative pathway. This may have implications for RPE ability to deal with complement attack, and opsonization and removal of debris from the RPE-choroidal interface. We speculate that regulation of the complement system in response to inflammatory assault may play a vital role in RPE pathology and drusen biogenesis. Experiment Overall Design: 10 samples investigating variations of co-cultures of RPE cells and T-cells. Variable parameters include the cell type, the co-culturing of the counter-part cell type and the orientation of the system (apical vs basolateral).

Project description:Background: Different sources of retinal pigment epithelial cells (RPE) were transplanted to treat Age-related Macular Degeneration. But the mechanisms of therapeutic effect are unclear till now, and GMP level fetal RPE (fRPE) need further evaluation for treatment. Methods: Modified GMP fRPE primary culture system was founded. These RPE were tested on Mer-/- mice and sodium iodate induced retinopathy primate model for safety and feasibility. Five dry-AMD patients and one Stargardt’s Disease patient were enrolled, and received sub retinal transplantation of hfRPE with three dosage cohorts(1×105，2×105，5×105). ETDRS, optic coherent tomographic imaging (OCT), color fundus (CF), fluorescent angiography (FFA), autofluorescence, microperimetry examination were conducted before surgery, and during follow-up period. The studies are registered with ClinicalTrials.gov, numbers NCT02868424. Findings: GMP level fRPE from modified primary culture system has good proliferative capacity and full functions of RPE in vitro. Human fetal RPE can survive long period in sub-retinal space in mice and monkey models of RPE degeneration, and showed therapeutic effect on Mer-/- mice. During our nine-months follow-up of six clinical cases, no endophthalmitis, no vitreous hemorrhage, no proliferative vitreoretinopathy was observed, and epiretinal memrbrane was the only complication. Among the patients, four of them achieved increase in ETDRS visual acuity (VA), of which the greatest improvement was 18 letters, and one kept steady while the other had 9 letters decreased VA. Color fundus photos and OCT demonstrated the existence of the fRPE cells, and improved retinal sensitivity as well as the changes observed in auto fluorescence also can tell the situation of grafted cells to some extent. Interpretation: GMP level fRPE from modified culture system exhibited satisfactory results on in vitro and preclinical experiments. Preliminary clinical trial proved safety of the fRPE subretinal transplantation, and substantiated it a promising therapy for dry AMD including the converted from exudative AMD.

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Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation

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