Figure 1.

Effect of Wnt signaling modifiers in the growth and proliferative capacity of limbal cell cultures. (A–C) Representative micrographs of limbal explant biopsies and their outgrowths in culture medium complemented with DMSO carrier (A, control; Cntrl); 10 μM IWP2 (B); or 3 μM CHIR99021 (C; CHIR). Inserts, top right: Photographs of outgrowth cultures stained with Commassie blue R solution. Main frame: phase contrast micrographs obtained with 4x and 20x objectives. Bar = 50 µm. Inserts are 2x larger views of a field in each micrograph. (D) Average areas of outgrowth, and cell densities in limbal explant outgrowth after 8 days in control or complemented medium. (E) Comparison of relative cell yields in limbal explant outgrowth and isolated cell cultures. (F) Immunostain of Ki67 in both culture systems. Representative confocal images and a bar graph of average Ki67 fluorescence intensity stain (Ki67_i) normalized by the average DAPI intensity for 3 independent cultures are shown. (G) Relative size (FCS) and granularity (SSC) of cell recovered from limbal explant outgrowth or isolated cell cultures. Color density flow cytometry plots with different color themes were used for each culture modality. Quadrant (LLQ, ULQ, LRQ and URQ) analysis identified quadrants showing statistically significantly difference (asterisks) with the control values in either the IWP2 or the CHIR99021 complemented cultures. Control, Cntrl; CHIR99021, CHIR, *p < 0.05, **p < 0.01 vs. control (n = 9 from 3 donors).