Next Generation Sequencing of Retinal Pigment Epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs).

We have previously reported on RPE differentiation protocol that mimic the morphological, molecular, and functional characteristics of RPEs (GSE140545). In the present study, we developed an improved protocol that generates clinical grade hiPSC-RPE cells with enhanced purity and maturity. NGS was performed on RPE cells generated using Research, GLP and GMP grade protocols to understand the differential gene expression of RPE maturity between the protocols. The genesets were grouped based on their spatiotemporal expression during retinogeneisis - Early RPE, committed RPE and late-stage mature RPE genes. When compared to the GLP and GMP protocols, the research protocol showed a significant up-regulation of genes related to early RPE development indicating the product comprised of a heteregnous propulation of committed, non-committed and mature RPE cells (predominantly differentiating RPE progenitor cells). Similarly, the GLP protocol showed increased expression of genes associated with both committed and mature RPEs, but the GMP protocol had significantly higher expression of late-stage and mature RPE genes than the other two protocols. Additionally, most genes related to cell cycle and proliferation were significantly down regulated in the GMP protocol compared to the research protocol which indicates that our GMP protocol generated a RPE product that is devoid of proliferating or tumor forming cells and is therefore completely safe for clinical applications. In this study, we have provided a detailed description of the modifications in our manufacturing protocol to eliminate immature cells in our final product and Chemistry, Manufacturing and Controls (CMC) studies performed during product development. Overall design: RPE cells were generated using research grade protocol had a mixture of committed and mature cells. This protocol was modified by incorporating enrichment and scale up steps to generate mature RPE cells. RPE cells generated from 3 different protocols were analyzed for their molecular profile by NGS- Research grade RPE cells, Enriched RPE cells, Enriched and scaled up mature RPE cells