Spatiotemporal single cell analyses reveal a transient population of retinal progenitor cells in the ciliary margin of developing human retina

Abstract

Purpose : Recent single cell (sc) RNA and ATAC-Seq studies have enabled molecular characterisation of all the retinal cell types and have provided the developmental trajectories and gene regulatory networks that lead to retinal cell specification during retinal development. Notwithstanding, these studies have not provided as yet any information on the spatial resolution of retinal progenitor cells (RPCs) or retinal neurons. Herein, we combine scRNA- and ATAC-seq with spatial transcriptomics (ST) to generate the first spatiotemporal single cell atlas of developing human retina.

Methods : 11 samples of developing human eyes and 14 samples of neural retina from 7.5-21 post-conception weeks (PCW) were obtained from the Human Developmental Biology Resource under ethics permission 08/H0906/21+5. All samples were dissociated to single cells using a neurosphere dissociation kit (Miltenyi Biotech). Approximately 10,000 cells from each sample were captured, and sequencing libraries generated using the Chromium Single Cell 3’ Library & Gel Bead Kit (version 3, 10x Genomics). 10,000 of the subsequent nuclei were captured, and sequencing libraries generated using the Chromium Single Cell ATAC Library & Gel Bead Kit (version 1, 10x Genomics). Fresh frozen sections of four fetal retina samples of 8, 10, 11 and 13PCW were used for the ST analyses performed with the Visium Spatial Gene Expression kit from 10XGenomics.

Results : Our single cell analyses demonstrate the presence of early RPCs, which transit to late RPCs, and further to transient neurogenic progenitors, that give rise to all the retinal neurons. Combining scRNA-Seq with ST of early eye samples, we demonstrate for the first time the transient presence of early RPCs in the ciliary margin zone with decreasing occurrence from 8 PCW of human development. In RPCs, we identified a significant enrichment for TEAD transcription factor binding motifs, which when inhibited led to loss of cycling RPCs and retinal identity in pluripotent stem cell derived organoids.

Conclusions : Together, these comprehensive single cell analyses shed light on the molecular mechanisms governing human retinal development and provide guidance on the generation of RPCs and differentiated retinal cell types from human pluripotent stem cells (PSCs).

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