Figure 3.

Tumorigenic characteristics of RB1-null hESC organoids. (A) Characterization of RB1-null-derived retinal organoids at day 150 of differentiation. Bar charts showing the percentage of Caspase-3⁺ cells (CASP3), the retinal cell types and Ki67⁺ co-expressing fractions (left) and representative immunofluorescence analysis for RXRγ, SNCG, AP2α, RHO, and Ki67, counterstained with Hoechst (right) at day 150 of differentiation. Data are presented as mean ± SEM (n = 10 sections from each biological replicate). White arrowheads point at co-localization of Ki67 with the specified retinal marker. Scale bar= 50 µm. (B) Soft agar colony formation assay showing ability of pRB-depleted retinal cells originating from day 90 RB1-null hESC retinal organoids to grow in suspension (bright field images, scale bar = 100 μm). (C) Transmission electron microscopy of photoreceptor inner segments (IS), visible mitochondria (white arrowheads) and outer limiting-like membrane (black arrows) (scale bar = 1μm). (D) Representative TEM example of mitotic cell in RB1-null organoid at day 200 of differentiation (left panel) followed by rosette-like structure (marked with dashed ellipse) shown in TEM images (middle panel) and H&E-stained sections in the right-hand side panel, followed by VSX2 and Ki67 immunofluorescence analysis; rosette-like structure marked with dashed ellipse (scale bars = 5, 2, 50, 5 µm, respectively).