Exogenous factors induce rod and cone photoreceptor-specific progenitors from adult mouse 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 and FGF2/heparin (T+RA+FH) increases the number of rod progeny to 95%, while coco (BMP/Wnt/TGFβ triple-inhibitor) increases cone progeny to greater than 60%. RSC progeny produce 10% rods and <1% cones when differentiated in 1%FBS+FH. We hypothesized that exogenous factors act on RSC progeny in an instructive, rather than permissive, manner to bias rod and cone differentiation through the enrichment of lineage-specific progenitors – no markers for these cells have been identified and literature is divided on their existence in vivo.

Methods : RSCs were clonally isolated from the CE of 4-6 week old mice. We used limiting dilutions (<1 clone/well) of a fluorescent retrovirus to label single progenitor clones in vitro. In addition, single cell sorting isolated non-pigmented and pigmented cells in wells, which were then treated with rod- and cone-inducing factors. Phenotype was assessed by ICC. Gene expression of RSC-derived and endogenous photoreceptors was compared by RNAseq.

Results : Retroviral labeling revealed enrichment in rod-only clones between 1%FBS (13%) to T/RA (>70%), without affecting clone size or overall cell survival. This strongly argues against selective survival of rod progenitors or differential survival of post-mitotic rods within a clone. In 1%FBS, clones derived from single non-pigmented progenitors were mostly non-rod and mixed clones, with few rod-only clones (n=4 of 28). In T+RA, all clones derived from non-pigmented progenitors (n=34) were rod-only clones, while those from pigmented progenitors (n=47 of 48) were almost all non-rod clones. Survival rates of non-pigmented cell derived clones were similar in T+RA, coco and 1%FBS. Differentiation in coco gave rise to >95% cone-only clones, likely by suppression of alternative fates. When cells primed in T+RA or coco are exposed to the opposite conditions, a critical period for the instruction of lineage-specific progenitors is revealed. We used RNAseq on RSC-derived and endogenous rods/cones isolated by FACS from photoreceptor-specific reporter mice to compare gene expression, as well as identify putative rod and cone lineage-specific progenitors.

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

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.