Figure 3

dUCs were expanded and enriched through serial passages in UCM. (a) Scheme of passage procedures of dUCs. Passage 0 (P0) dUCs were induced as in Fig. 1a, except that UCM was substituted for CnT-Prime. They were detached, resuspended in UCM and reseeded in fresh laminin-coated plates to obtain passage 1 (P1) dUCs. Passages were repeated to obtain P2 to P4 dUCs. (b) Confocal microscopic images of the P1 to P4 dUCs are shown. (c,d) RNA was extracted from the cells and mRNA for the indicated genes was evaluated by real-time RT-PCR. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. aHDFs. ^##P < 0.01 and ^###P < 0.001 vs. HUCs. ^†P < 0.05, ^††P < 0.01 and ^†††P < 0.001 vs. P0 dUCs. (e,f) Cells were immunostained with the indicated antibodies. % positive cells (e) and fluorescent microscopic images (f) are shown. ^**P < 0.01 and ^***P < 0.001 vs. aHDFs. ^†P < 0.05 and ^†††P < 0.001 vs. P0 dUCs. (g,h) P4 dUCs and aHDFs were cultured on inner chambers in Transwells, and permeability assay was performed using FITC-Dextran. Confocal microscopic images of the inner chamber (g) and percentage of leaked FITC-dextran (h) are shown. Values are mean +/− SD (n = 3). ^***P < 0.001 between groups.