Fig. 2

Combinatorial injection of Noggin and anti-sFRP2 with and without growth factors have differential effects on CE proliferation and retinal stem cell expansion. A Schematic of the intravitreal injection paradigm followed by endpoint clonal retinal stem cell (RSC) sphere forming assay from primary ciliary epithelium (CE) and/or immunohistochemistry (IHC). Mice received one intravitreal injection per day for three days followed by endpoint analysis at Day 4, 10 or 31. Injections consisted of: C = PBS control, N = Noggin, S = anti-sFRP2, NS = Noggin + anti-sFRP2 combined, FI = FGF2 + Insulin combined, FINS = FGF2 + Insulin + Noggin + anti-sFRP2 combined. B, C Quantification of Pax6⁺EdU⁺ co-labeled cells relative to total CE area in eyes treated with PBS vehicle or indicated factors. B Day 4 Pax6⁺EdU⁺ cells. (p < 0.001; N = 5–6 eyes per group). c Day 4 Pax6⁺EdU⁺ cells. (p = 0.037; N = 3–6 eyes per group). D-G Pax6 immunostaining and EdU labeling in the ciliary epithelium and peripheral retina of eyes injected with PBS vehicle or indicated factors at Day 4 and Day 31. Hoechst stain was used to label all nuclei. White arrows indicate Pax6⁺EdU⁺ double-positive cells. Straight dashed line indicates the border between the ciliary epithelium (CE) and the neural retina (NR). Dashed line box indicates high magnification inset. H Quantification of EdU cell number in the CE normalized by area in eyes treated with the indicated conditions. (p < 0.001; N = 5–6 eyes per group). I-K Percent of total EdU-positive cells in the CE that co-labeled for cell-type-specific markers in eyes treated with the indicated conditions. i % Pax6⁺EdU⁺ CE cells. (p < 0.001; N = 5–6 eyes per group). J % ERG⁺EdU⁺ endothelial cells. (p < 0.001; N = 5–6 eyes per group). K % CD68⁺EdU⁺ microglia/macrophage cells. N = 5–6 eyes per group. L-N Quantification of RSC sphere frequency normalized to naïve un-injected control following intravitreal injection of the indicated factors. L Day 4. N = 5–12 eyes per group. M Day 10. (p = 0.003; N = 5–12 eyes per group). N Day 31. (p = 0.001; N = 4–6 eyes per group). Each data point represents a single eye. Statistics: One-way ANOVAs with Holm-Sidak posthoc tests. Data are means ± SEMs. * = p < 0.05