Induction of rod and cone photoreceptor-specific progenitors from retinal stem cells

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. RSC progeny produce 10% rods and <1% cones when differentiated in 1%FBS. We hypothesized that exogenous factors act on RSC progeny in an instructive, rather than permissive, manner to bias rod and cone differentiation through enrichment of lineage-specific progenitors – no markers exist for these cells and literature is divided on their existence in vivo.

Methods : RSCs were isolated from adult mouse and human donor eyes. We used limiting dilutions (<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. This strongly argues against selective survival of rod progenitors or differential survival of post-mitotic rods within a clone. In 1%FBS, single non-pigmented progenitors gave rise to mostly non-rod and mixed clones, with 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. When early progenitors primed in T+RA for brief periods (3-days) are exposed to coco the rod fate bias is unaffected, while T+RA instructs progenitors to a rod fate in the presence of coco. We used RNAseq on RSC-derived and endogenous rods versus cones isolated from photoreceptor-specific reporter mice to compare gene expression. Transcriptomes were compared with embryonic neural retinal precursors, which show similar rod differentiation in T+RA and increased cone differentiation in coco. Pathway analysis showed clustering of stem cell-derived and endogenous cones, as well as candidate markers for photoreceptor-specific progenitors. After 4-6 weeks of coco, 60% of adult human RSC progeny differentiated into cones; both the time-course and number of cones is similar to rates from mouse RSC progeny.

Conclusions : Our study suggests a critical role for exogenous signals instructing early lineage decisions in fate-restricted retinal progenitors.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.