Fig. 3: Establishing purity of Eyecyte-RPE.

A Cell proliferation status examined through FACS analysis indicates proliferation is decreased w.r.t. increased pigmentation across batches. Results show low or negligible levels of Ki67 (<5%) and pHH3 (<3%) in GMP protocol. Cell count post staining for Ki67 and pHH3 markers w.r.t DAPI indicate similar results. B Heatmaps created for cell cycle and proliferation related genes showing lower expression in GMP protocol compared to research and GLP protocols. C–E Gene expression of OCT4 across different protocols by qPCR ( < 0.01), and FACS analyses ( < 1%) indicate absence of undifferentiated pluripotent cells as an impurity in Eyecyte-RPE. RNA seq analysis confirms absence of expression of pluripotent genes across all batches. F-G qPCR and FACS analysis of OCT4 level in RPE cultures spiked with 1% and 10% iPSC (0 hr and 15 day). Absence of OCT4 points out that iPSCs did not survive in RPE cultures. H-I qPCR analyses of trilineage markers (TH, AFP, HAND2) in RPE cultures alone and when spiked with iPSC, demonstrate absence of non-RPE lineage (off-target) markers. J Heatmaps created to compare differentially expressed genes involved in EMT and neural lineage across the three protocols. Significant down-regulation is seen in GMP protocol compared to GLP and research protocols. Scale bars indicate 100 μm. A p-value < 0.05 is considered statistically significant. (* indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001).