Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation - PubMed (original) (raw)

Figure 4

Characterization of the NCAM1-High sorted D30 hESC-RPE population (A) Bar graph of top genes from anticorrelation analysis at HS980 D30. Genes with a mean normalized expression <0.5 were excluded. (B) Brightfield and immunofluorescence stainings of D30 cells showing co-expression of VSX2, NCAM1, and Ki67 markers. Scale bars, 200 μm. (C) Representative FACS plot of NCAM1-CD140b sorting to distinguish distinct populations at D30. Negative gates were set based on fluorescence minus one (FMO) and hESC control samples. (D) Post-sort pellets of CD140b-High and NCAM1-High cells. (E) UMAP of NCAM1-High (pink), CD140b-High (blue), and unsorted (gray) D30 cells after CCA integration. (F) Dot plot illustrating the proportion of cells corresponding to each identified cell type in scRNA-seq samples from (E). (G) Dot plot of selected progenitor (FEZF2, CRB1, SOX2, FGF9, VSX2) and RPE (SFRP5, TTR, SLC35D3, TYR, RLBP1) genes enriched in the sorted samples. (H) Graphs showing qRT-PCR of retinal progenitor (SIX6, VSX2) and RPE (BEST1, RPE65) marker genes in populations from (E) at the moment of sort and at post-sort D30, D35, D40, D45, and D60. (I) UMAP of NCAM1-High (pink), CD140b-High (blue), and unsorted (gray) D60 cells. (J) Dot plot illustrating the proportion of cells corresponding to each identified cell type in scRNA-seq samples from (I). (K) Dot plots of early (MITF, TYRP1, PMEL, SERPINF1, DCT, ELN) and late (RLBP1, BEST1, RPE65, RGR, TTR, SFRP5) RPE genes in the LateRPE cell clusters from each sorted sample. (L) Brightfield and immunofluorescence stainings of unsorted, CD140b-High, and NCAM1-High populations 30 days after sorting (D60) showing co-expression of CD140b, BEST1, and ZO-1 markers. Scale bars, 100 μm. (M and N) Bar graphs showing PEDF secretion (M) and TEER measurements (N) of the unsorted, CD140b-High, and NCAM1-High populations at D60. ∗∗p < 0.0001 compared to Not Sorted and NCAM1-High. In (H), (M), and (N), error bars represent mean ± SEM from three independent experiments. See also Figure S3.