Figure 2.

Enrichment and transplantation of optogenetically-engineered cones. (A) Schematics of the general transplantation strategy. Human iPSC-derived retinal organoids were Papain-dissociated and Jaws-enriched expressing cells were transplanted subretinally into blind rd1 mice. (B) Representative histogram of flow cytometry analysis performed to determine Jaws-expressing cells within the retinal organoids (N = 8 biological replicates, n = 10,000 cells gated). (C) Fundus imaging analysis of representative rd1 mice retinae after 14 days of transplantation. (D,E) Representative fluorescent fundus images of rd1 mice in panel (C) showing Jaws-expressing cells 14 (D) and 21 (E) days after subretinal transplantation. (F) 2-Photon laser image of GFP+ cells in a rd1 transplanted retina. (G) Histological analysis of 7-week-old mice retina showing complete absence of photoreceptors layers, corresponding to the time of transplantation. Remaining cells are immunoreactive for bipolar specific marker PKCα. (H,I) IF analysis of vertical cryosections of rd1 mice retinae after transplantation of Jaws-enriched expressing cells (GFP). (H) Transplanted Jaws-cones depicted as GFP+ cells in rd1 mice were also immunoreactive for human marker HNA (red). (I) Transplanted cones overlie host PKCα bipolar cells and expressed synaptic marker Synaptophysin (Syn, white arrows) All images are representative of at least five different transplants. Animal details for each transplantation are provided in Table 1. Scale bars: (F and I) = 10 μm, (G) = 25 μm, (H) = 100 μm. INL, inner nuclear layer, RGC, retinal ganglion cells; SRS, subretinal space.