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Brian G Ballios, Saeed Khalili, Kenneth Grisé, Laura Donaldson, Gilbert Bernier, Derek van der Kooy; Exogenous factors induce rod photoreceptor-specific progenitors from adult mouse retinal stem cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3588.
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© ARVO (1962-2015); The Authors (2016-present)
Adult retinal stem cell (RSCs) derived from the ciliary epithelium (CE) of mice can give rise to all retinal cell types. Taurine, retinoic acid and FGF2/heparin (T+RA+FH) added to differentiating clonal RSC colonies increases the number of rods to 90% of all progeny; RSC progeny produce 10% rods when differentiated in 1%FBS+FH (pan-retinal conditions). We hypothesized that T/RA acts on RSC progeny in an instructive, rather than permissive, manner to bias photoreceptor differentiation through the enrichment of rod-specific progenitors.
RSCs were clonally isolated from the CE of 4-6 week old mice. We used limiting dilutions (<1 clone / well) of a fluorescent retroviral construct to label individual progenitor clones in vitro. In addition, single cell sorting isolated non-pigmented and pigmented cells in wells, which were then treated with T/RA for 28 d. Survival, clone size, and phenotype were assessed by immunocytochemistry.
Clonal retroviral labeling revealed enrichment in the percentage of rod-only clones between 1%FBS (13%) to T/RA (over 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 distributed between non-rod and mixed clones, with a minority of rod-only clones (100% Rhodopsin-positive; n=4 of 28 clones). Clones derived from pigmented cells in 1%FBS never gave rise to rod-only clones. In T+RA conditions, all clones derived from non-pigmented progenitors (n=34) were rod-only clones, while those derived from pigmented progenitors (n=47 of 48) were almost all no-rod clones. Of note, one rod-only clone (the largest) was derived from a single pigmented cell in T+RA conditions, suggesting potential neural lineage plasticity in a very early, pigmented progenitor. Survival rates of non-pigmented cell derived clones were similar in T+RA and 1%FBS. Similar experiments using Wnt, BMP4 and TGFβ inhibition increases the number of RSC-derived cones to >60% of all progeny.
This study marks an important step in the characterization of a rod-specific progenitor - no markers exist and literature is divided on their existence in vivo. Our study suggests a critical role for exogenous signals instructing early lineage decisions between fate-restricted retinal progenitors.
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