Fig. 1: Genetically balanced hESC lines differentiate into RPE with individual gene-expression signatures as well as astrocytes and pigmented ciliary body cells.
a Example of a 9 cm2 culture dish, 8 weeks into RPE differentiation. b Magnification of the cells shown in (a), the dotted line marks three adjoining clusters of pigmented cells, which were picked and passaged to establish a pure RPE population. c Fluorescence imaging of an hESC line labeled with the RGB lineage tracing system, 8 weeks into RPE differentiation. d UMAP of the scRNA sequencing of hESCwt after 60 days of spontaneous differentiation, prior to colony picking, containing retinal pigment epithelium cells (RPE), cortical hem cells (CH), amacrine cells (AC), retinal ganglion cells (RGC), amnion-like cells (Amnion), retinal progenitor cells (RPC), oligodendrocyte progenitor cells (OPC) and proliferating cells (Prolif). e Heatmap for the expression of marker genes for the cell types identified in panel (d). f Brightfield imaging of a purified cell culture showing the RPE cobblestone morphology. g Example of the immunostaining of the purified RPE cell cultures for PMEL, ZO-1, PAX6, and BEST1. h UMAP of the scRNA sequencing of hESC-derived RPE, labelled by cell line. i Violin plots of the expression per cell and per cluster of undifferentiated hESC and RPE marker gene sets. j Aggregation of our single-cell RNA sequencing dataset to that of Senabout et al.18, including 127659 RPE cells derived from 79 induced pluripotent stem cell lines. k Venn diagram of the differentially expressed genes across the different hESC-derived RPE. l Heatmap of the differentially expressed genes shown in panel (k). m UMAP of the hESC-derived RPE without the cell-line-specific transcriptomic profiles. n UMAP indicating the RPE clusters, and the 4 clusters identified by Seurat and composed of cells from different lines. o Cluster 2 shows a higher expression of MKI67. p UCell scoring for each cluster in (k), with scores for neural and ocular-related cell types.
