Figure 3
Development of clinical-grade photoreceptor precursor cells.
Morphological and immunocytochemical analysis of 3D organoids after 11–12 and 13–16 weeks of differentiation. (A–C) After 11–12 weeks of differentiation, organoids have many areas of retinal-like folds (A; light micrograph) and retain highly organized F-actin-positive structures (inset in A; green). Organoids express the phototransduction molecule, recoverin (B; red) and the photoreceptor precursor-specific transcription factor, CRX (B; gray), a marker of committed photoreceptor precursor cells that is functionally downstream of OTX2 (Fig. 2). Some recoverin-positive cells within organoids at this stage also begin to express NRL, a rod photoreceptor-specific transcription factor (C; gray). Recoverin labeling can also be observed along neuronal-like processes of photoreceptor precursor cells. (D–G) Organoids after 13–16 weeks of differentiation are typically full of neural rosettes (D; light micrograph) and the majority of OTX2-positive cells (inset of D; gray) are now post-mitotic, Ki67-negative cells (inset of D; red). By this stage of development, many neuronal cells that are TUJ1-positive (E; red) are now positive for NRL (E; gray). Many recoverin-positive cells (F and inset; red) within neural rosettes co-express NRL (F and inset; gray) and NR2E3 (F and inset; green), which is functionally downstream of NRL in rod photoreceptor development and is transcriptionally regulated by NRL. Some organoids retain their lamination and resemble mature retina with independent layers of recoverin-positive photoreceptors (G; red) and HuC/D-positive inner retinal amacrine- and ganglion-like cells (G; green). 3D reconstrutction of a z-stack through a laminated eyecup further demonstrates the separate layers of photoreceptor-like cells and inner retinal neuron-like cells (G, inset). Small panels to the right of (B,C,E,F) show individual fluorophores. Scale bars A,D = 400 μm; B,C,E–G = 50 μm.
