Figure 3.

Functional behavior of ETA cells in 2D and 3D cultures. (A) Freshly isolated subpopulations were cultured up to 196 h after seeding and MTT assay was performed at different times. (B) Long-term assay was performed on freshly isolated keratinocyte subpopulations, as described in Materials & Methods. (C) The extent of proliferation of ETA and LTA cells left in a culture was estimated by calculation of the population doubling. (D) Reconstructed skin equivalent (SE) models generated from freshly isolated KSC, ETA, and LTA cells. After 6 or 12 days from keratinocyte seeding, skin equivalents were paraffin-embedded and sections were stained with H&E. Bar = 100 μm. Epidermal thickness was measured by D-Sight slide scanner software. (E) Immunohistochemical staining of Ki67 was evaluated as the percentage of positive cells, as described in Materials & Methods. Bar = 100 μm. (F) Sections of healthy skin and skin equivalents obtained from KSC, ETA, and LTA cells, were stained with anti-Survivin, anti-K15, and anti-Involucrin antibodies by IF. Bar = 100 μm. Stained areas were evaluated by image pixel count using Fiji-ImageJ. Data are represented as mean ± SEM. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons are represented. ns: P > .05; * .01 < P < .05; ** P < .01; *** P < .001; ****P < .0001.