Genomic characterization of human pluripotent stem cell-derived RAX-positive sub-populations that direct retinal layer structuring and non-retinal tissue

Abstract

Purpose : Cell-based regenerative therapy is considered a promising strategy to restore visual function in retinal degenerative diseases. Retinal progenitor cells (RPCs) in the early stage [differentiation day (DD) 25-30] can develop inner and outer nuclear layers after transplantation in animals. When we sorted the RAX-positive (RPC marker) and SSEA1-negative populations from the dissociated cells of such early retinal organoids (ROs) derived with different differentiation protocols, reaggregated spheroids from the sorted cells presented three characteristic phenotypes: circumferential retinal spheroids (C-spheroids) with or without layered structures, and polarized spheroids (P-spheroids) composed of structured retinal and non-retinal parts. In this study, we investigated the gene expression patterns of component cells of these spheroids and identified the possible RPC sub-populations contributing to the formation of retinal layer structures.

Methods : In the course of RO differentiation from Rax::Venus hESC (hESC-KhES-1), different timing of BMP4 treatment (DD3 and DD6) yielded ROs with different profiles of Rax::Venus cell content. These ROs were dissociated into single cells on DD25 and sorted based on Venus intensities (Venus++ or Venus+) and SSEA1-absence. We studied the phenotypes of spheroids made from the sorted cells on DD30, 34, 40, and 60, and performed single-cell RNAseq analysis at each time point to analyze gene expressions of cell clusters, ligand-receptor (L-R) interactions, and trajectory analysis.

Results : Venus++ cells in DD3-BMP4 and DD6-BMP4 ROs developed C-spheroids without and with layered structures, respectively while Venus+ cells in DD6-BMP4 ROs developed P-spheroids. The layer structure of C-spheroids started to form around DD40. Single-cell analysis of this spheroid group presented significant L-R interactions associated with WNT2B on DD30 and with WNT5A on DD34 in a small population, whereas C-spheroids without layered structures did not show such changes across all timelines. Non-retinal areas of P-spheroids expressed hindbrain marker genes suggestive of other cell origins than retinal field.

Conclusions : RAX-positive cells comprise non-retinal cells and heterogeneous retinal progenitor populations, some of which transiently secrete WNT2B and WNT5A, potentially guiding the self-organization of retinal layer structures.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.