Genomic characterization of sub-populations in human pluripotent stem cell-derived retinal progenitor cells driving retinal lamination.

The mechanism underlying retinal spheroid layer formation was investigated using Rax::GFP-positive retinal progenitor cells from human embryonic stem cell-derived retinal organoids. Single-cell RNA sequencing suggested the earlier cell-cycle exit in non-layered spheroids, while well-layered spheroids retained longer proliferative property with transiently activated canonical WNT2B-FZD7 signaling followed by temporary expression of non-canonical WNT5A. Despite structural differences in vitro, however, both non-layered and well-layered retinal spheroids on differentiation day 60 developed a layer of photoreceptors after transplantation in a retinal degeneration rat model, resulting in synaptic and functional integration. Additionally, part of the Rax::GFP-positive cells differentiated into non-retinal lineages, including ciliary marginal zone-like, retinal pigment epithelium, and spinal cord-like tissues in vitro, reflecting the heterogeneity of RAX-positive cells. These findings suggest that canonical and non-canonical WNT signaling pathways sequentially orchestrate early retinal morphogenesis, whereas environmental factors within the host retina strongly drive the alignment and functional integration of graft photoreceptors.