Genomic characterization of sub-populations in human pluripotent stem cell-derived retinal progenitor cells that drive retinal layer structure

The mechanisms of layer formation of retinal spheroids were investigated using Rax::GFP-positive retinal progenitor cells (RPCs) obtained from human embryonic stem cell-derived retinal organoids. Single-cell RNA sequencing revealed that well-layered spheroids exhibited a transient activation of canonical WNT2B–FZD7 signaling followed by a temporary expression of non-canonical WNT5A, processes associated with the formation of organized retinal layers. Despite structural differences in vitro, both non-layered and well-layered retinal spheroids of differentiation day 60 successfully developed into each cone and rod subtypes of photoreceptors and established synaptic connections with host bipolar cells after transplantation in retinal degeneration model rats, resulting in light-evoked functional recovery.　Additionally, part of the presumable RPCs differentiated into non-retinal lineages, including ciliary marginal zone-like, retinal pigment epithelium-, and spinal cord-like tissues in vitro, reflecting the developmental plasticity of RAX-positive cells. These findings suggest that canonical and non-canonical WNT signaling pathways sequentially orchestrate early retinal morphogenesis, while environmental factors within the host retina strongly drive the alignment and functional integration of graft photoreceptors.