Purchase this article with an account.
J. Wang, J. Yang, H. Klassen; GDNF Augments Proliferation and Reduces Apoptosis of Murine Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2650. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Glial cell line-derived neurotrophic factor (GDNF) is neuroprotective in the retina and retinal progenitor cells (RPCs) can deliver this cytokine, particularly as a transgene product. While GDNF alone does not sustain RPC proliferation, it may confer advantages when combined with epidermal growth factor (EGF), as we investigate here.
RPCs, originally from neonatal GFP-mice, were assessed under different conditions corresponding to the presence or absence of EGF (20 ng/ml) and/or GDNF (10 ng/ml). Growth curves were calculated and sphere formation analyzed using an IncuCyte live-cell imaging system. Apoptosis was assessed via caspase-3 activity assay; marker expression via quantitative RT-PCR. Western blotting was quantified by densitometry.
RPCs exhibited exponential growth in either EGF or EGF+GDNF, with the latter condition showing a measurable advantage. Live-cell monitoring of both conditions revealed that initial aggregates formed as early as 5.5 hr from collision and adherence of dissociated RPCs, as opposed to clonal proliferation. Spheres subsequently enlarged in size and number in both conditions, with more reaching threshold criteria for cross-sectional area in the EGF+GDNF condition. Caspase-3 activity levels were markedly higher in the absence of EGF (p < .05) and the no-growth-factor condition was higher than GDNF alone (p < .05). EGF and EGF+GDNF conditions were equivalent. Expression of most progenitor-associated markers remained stable following 5 days treatment, as did the majority of precursor and lineage-related markers, although with EGF+GDNF there were marginal increases in Ki-67 (1.07 fold), hes5 (1.03 fold), mash1 (1.12 fold) and vimentin (1.20 fold). At the protein level, Ki-67 was increased in EGF+GDNF by 1.19±0.0095 fold.
The addition of GDNF to EGF-based proliferation medium augments the proliferation of RPCs in association with increased Ki-67 expression. GDNF reduces RPCs apoptosis compared to complete growth factor withdrawal. These data support the feasibility of generating GDNF-producing RPCs for use in therapeutic models of retinal disease.
This PDF is available to Subscribers Only