Lineage specification and molecular characterization of photoreceptor-specific progenitors

Abstract

Purpose : Adult retinal stem cells (RSCs) give rise to all retinal cell types. Clonal RSC progeny treated with taurine / retinoic acid (T+RA) produce 95% rod progeny, while coco (BMP/Wnt/TGFβ triple-inhibitor) induces 60% cones. We hypothesized that rod and cone lineage-specific progenitors, specified using exogenous factors, display unique transcriptome signatures that can identify stage-specific molecular markers. No markers exist for these lineage-specific progenitors and literature is divided on their existence in vivo.

Methods : RSCs were isolated from adult mouse and human donor eyes. We used limiting dilution (<1 clone/well) of a fluorescent retrovirus, or single cell/well sorting to isolate individual progenitor clones.

Results : Retroviral labeling showed enrichment in rod-only clones between 1%FBS (13%) to T/RA (>70%), without affecting clone size or cell survival, demonstrating induction of a rod-specific progenitor. In 1%FBS, single non-pigmented progenitors gave rise to few rod-only clones (n=4/28). In T+RA, all clones from non-pigmented progenitors (n=34) were rod-only clones. Survival rates of non-pigmented cell derived clones were similar in T+RA, coco and 1%FBS. Coco permitted differentiation from single non-pigmented progenitors to >95% cone-only clones, likely by suppression of alternative fates. We compared gene expression between RSC-derived and endogenous rods / cones with RNAseq. Embryonic neural retinal precursors (E14) show similar rod differentiation in T+RA (>95%) and increased cone differentiation in coco (>90%). We profiled the transcriptome at different time points during differentiation from embryonic retinal precursors. Principal component analysis shows a distinct progression of the rod and cone lineages derived from fate-specific progenitors. Pathway analysis showed clustering of stem cell-derived and endogenous cones, as well as candidate progenitor markers. Preliminary data suggests SOX15 may be a unique marker of a cone restricted progenitor cell, and we are analyzing whether SOX15 is expressed in a subset of embryonic retinal progenitors. After 4-6 weeks of coco, 60% of adult human RSC progeny differentiated into cones; both the time-course and efficiency is similar to mouse RSC progeny.

Conclusions : Exogenous signals instruct early lineage decisions in fate-restricted retinal progenitors, and we identify potentially new candidate markers for these cells.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.