Robust generation of photoreceptor-dominant retinal organoids from porcine induced pluripotent stem cells.

Outer retinal degenerative diseases (RDDs) and injuries leading to photoreceptor (PR) loss are prevailing causes of blindness worldwide. While significant progress has been made in the manufacture of human pluripotent stem cell (hPSC)-derived PRs, robust production of pluripotent stem cell (PSC)-PRs from swine, a popular preclinical large animal model, would provide an avenue to collect conspecific functional and safety data to complement human xenograft studies. Toward this goal, we describe the highly efficient generation of PR-dominant porcine induced PSC (piPSC)-derived retinal organoids (ROs) using modifications of our established hPSC-RO differentiation protocol. Porcine iPSC-ROs were characterized using immunocytochemistry (ICC) and single-cell RNA sequencing (scRNA-seq), which revealed the presence and maturation of major neural retina cell types, including PRs and retinal ganglion cells, which possess molecular signatures akin to those found in hPSC-ROs. In late piPSC-ROs, a highly organized outer neuroepithelium was observed with rods and cones possessing outer segments and axon terminals expressing pre-synaptic markers adjacent to dendritic terminals of bipolar cells. The existence of piPSC lines and protocols that support reproducible, scalable production of female and male ROs will facilitate transplant studies in porcine models of retinal injury and RDDs unconfounded by immunological and evolutionary incompatibilities inherent to human xenografts.