Journal: Nature Communications

Article Title: Chemical reprogramming of fibroblasts into retinal pigment epithelium cells for vision restoration

doi: 10.1038/s41467-025-67104-w

Figure Lengend Snippet: a Representative immunostaining showing that MEF-derived ciRPE cells express ZO-1, Pax6, Rpe65, Mitf, Best1 and Cralbp. Scale bars, 50 μm. b Representative Z-stack confocal micrographs showing ciRPE cells with typical polarized expression of RPE markers. ZO-1 (green) demonstrates apical localization (top), while Best1 (red) shows basolateral localization (bottom). Scale bars, 10 μm. c Representative transmission electron microscopy image of ciRPE cells showing apical microvilli (yellow arrows), melanin granules (red arrows) and tight junctions (black arrows). Scale bars, 1 μm. d Representative confocal micrograph showing phagocytosis of POSs (green) by ciRPEs. The apical sides of ciRPE cells are stained with ZO-1(violet), whereas nuclei are counterstained with DAPI (blue). Scale bars, 50 μm. e Apical and basal secretion of PEDF and VEGF by MEFs, ciRPE, and pRPE cells cultured on Transwells. Each group was compared to the ciRPE group within apical and basal compartments ( n = 6 independent biological samples per group). f Representative morphological images showing dome structures formed by ciRPE cells during in vitro culture. The red arrows indicate the dome morphology observed under different phase-contrast microscopy conditions. Scale bars, 50 μm. g TEER measurements of MEFs, ciRPE, and pRPE cells over time in culture ( n = 6 independent biological samples per group). Data are mean ± SD. One-way ANOVA was used to assess statistical significance. Three independent experiments were performed with similar results and representative results are shown. Source data are provided as a Source Data file.

Article Snippet: Detailed procedures were followed according to the instructions of the PEDF ELISA kit (CUSABIO, CSB-E08820m and CSB-E08818h) and the VEGF ELISA kit (CUSABIO, CSB-E04756m and CSB-E11718h).

Techniques: Immunostaining, Derivative Assay, Expressing, Transmission Assay, Electron Microscopy, Staining, Cell Culture, In Vitro, Microscopy

Journal: Nature Communications

Article Title: Chemical reprogramming of fibroblasts into retinal pigment epithelium cells for vision restoration

doi: 10.1038/s41467-025-67104-w

Figure Lengend Snippet: 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.

Article Snippet: Detailed procedures were followed according to the instructions of the PEDF ELISA kit (CUSABIO, CSB-E08820m and CSB-E08818h) and the VEGF ELISA kit (CUSABIO, CSB-E04756m and CSB-E11718h).

Techniques: Purification, Microscopy, Quantitative RT-PCR, Expressing, Immunostaining, Derivative Assay, Control, Blocking Assay, Quantitative Proteomics, Two Tailed Test