Abstract
Purpose :
Outer retinal degenerative diseases (RDDs) and damage leading to photoreceptor (PR) loss are prevailing causes of blindness. While advancements have been made in human pluripotent stem cell (hPSC)-derived PR replacement, generation of conspecific PSC-derived PRs for use in allograft studies would ameliorate issues inherent to xenografts. Previously, we described the generation of retinal organoids (ROs) from a female pig induced pluripotent stem cell (piPSC) line via modifications of our hPSC-RO differentiation protocol. Here we present a comprehensive molecular analysis of female piPSC-ROs at multiple developmental stages using immunocytochemistry (ICC) and single cell RNA-sequencing (scRNA-seq). We also describe the generation of ROs from a male piPSC line to facilitate donor cell identification in female hosts. Our goal is to develop a robust piPSC toolkit to test the efficacy of NR cell allografts in pig models to complement ongoing human xenograft studies.
Methods :
We adapted a hPSC-RO protocol to generate piPSCs-ROs. ICC was performed at d40, d80, d120 and d190 post-differentiation for expression and localization of multiple neural retina (NR)-specific markers, with an emphasis on PRs. We then performed scRNA-seq to provide in-depth transcriptional profiles of piPSC-ROs at early (d40) and late (d120) stages of differentiation.
Results :
Light microscopic imaging over a 190-day differentiation period showed characteristic early outer neuroepithelium (NE) with later development of short PR outer segments (OS). ICC revealed the presence of major NR cell types akin to those seen in early hPSC-ROs, including maturing CRX+/RCVRN+ PRs and numerous SNCG+ retinal ganglion cells (RGCs). In mature ROs, a highly organized outer NE was observed with rods (RHO+ and SAG+) and cones (M/L-OPSIN+, RP-1+, and GNAT2+) possessing ARL13b+ connecting cilia and PRPH2+ OS. Rod and cone axon terminals expressed the PR pre-synaptic marker VGLUT1 adjacent to dendritic terminals of G0α+ bipolar cells. scRNA-seq analysis confirmed presence of RGC and PR precursors in early ROs, and mature PRs and other NR cells in late ROs.
Conclusions :
We report the generation of mature NR cells, including PRs and RGCs, from both female and male piPSCs. Our piPSC-RO differentiation protocol provides an abundant source of donor PRs and other retinal cell types for allogenic transplantation in pig models of injury and RDD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.