Fig. 6: Generation of human ciRPE cells from HEFs by chemical reprogramming.

a Candidate small molecules identified by preliminary screening with scRCF for reprogramming HEFs into OV. b Schematic diagram of the protocol for the reprogramming of HEFs into hciRPE cells, along with representative morphological changes at indicated time points. HM represents the HEF medium, while RM represents the reprogramming medium and DM refers to the differentiation/maturation medium. Scale bar, 300 μm. c FACS purification of reprogrammed BEST1-EGFP ⁺ hciRPE cells. d Representative optical microscopy and TEM images of hciRPE cells showing melanin granules (red arrows). Scale bars, 1 μm. e qRT-PCR analysis showing the expression of RPE-associated genes at the indicated time points during reprogramming (n = 3 independent biological samples per group). f Representative immunostaining analysis showing positive expression of ZO-1, RPE65, MITF and BEST1 in the BEST1-EGFP^- HEFs-derived hciRPE cells. Scale bar, 20 μm. g PCA of samples from day 0, day 12, day 24 and day 38 (hciRPE) of celluar reprogramming, and the control primary hRPE cells. h Heatmap showing differentially expressed genes in HEF to hciRPE cell reprogramming samples at indicated time points. The number above heatmap indicates independent biological replicates. Representative genes (left side of the heatmap) and associated GO (right side of the heatmap) for each block are shown. Red and blue indicate upregulated and downregulated genes, respectively. Differential expression was analyzed using the R package limma (v3.58.1) following normalization with edgeR (v4.0.16). Significantly changed genes were defined by |log₂ fold change| > 1.5 and adjusted p < 0.01 (Benjamini-Hochberg correction). i Polarized secretion of VEGF and PEDF from the apical and basal sides of hciRPE cells grown on Transwells (n = 6 independent biological samples per group). j TEER in hciRPE cells for 30 days. p values indicate comparisons between adjacent time points (n = 5 independent biological samples per group). Data are mean ± SD. Statistical analyses were performed using one-way ANOVA (e) and unpaired, two-tailed Student’s t-test (i, j). Three independent experiments were performed with similar results and representative results are shown. Source data are provided as a Source Data file.