Figure 4. cGMP patient-specific organoids recapitulate photoreceptor cell lineage.

Immunocytochemical analysis of 3D organoids from a patient with mutations in NR2E3 and a patient with normal NR2E3 alleles. (A) NR2E3 organoids develop normally and are initially indistinguishable from control organoids (Fig. 2). At 5–7 weeks post-differentiation NR2E3 organoids contain polarized (Phalloidin; green) neural epithelial cells that express PAX6 (inset; red) and OTX2 (inset; gray) and are actively proliferating (Ki67; red). (B) After 13 weeks of differentiation, NR2E3 organoids have cells that express the inner retinal-specific marker, HuC/D (green) and the early photoreceptor-specific lineage transcription factor, RORβ (red and inset). (C) After 15–16 weeks of differentiation, presumptive photoreceptors within NR2E3 mutant organoids are CRX- (gray and inset) and recoverin-positive (red). (D) Also around 15–16 weeks, NR2E3 organoids show robust labeling for SW Opsin, which is specific for blue (S) cones (D and insets; red). These SW Opsin-positive cells are NRL-negative (D and insets; gray) and have strands of SW Opsin emanating from the cell nucei towards outersegment-like cone-shaped membranes (D; insets). (E) Quantification of the percentage of SW Opsin-positive cells in two patients with NR2E3-associated enhanced S-cone syndrome (NR2E3-1 vs Control; p < 0.01 and NR2E3-2 vs Control; p < 0.001) compared to an unaffected control eyecup at 15–16 weeks post-differentiation. (F) Age-matched organoids from a patient with normal NR2E3 alleles are not positive for SW Opsin (E). Further, an eyecup from an individual with normal NR2E3 alleles after 21 weeks of differentiation has only a few SW Opsin-positive cells (E and inset; red) amongst many NRL-positive presumptive rod photoreceptor cells (E; gray). Scale bars: A and inset of E,F = 100 μm; all others = 50 μm.