Fig. 3: Endomucin expression defines two distinct BMEC populations in WBM culture.

a–c P0 WBM culture at day 14. Immunostaining of tdT⁺BMECs with antibodies for VE-Cadherin (a), CD31 (b), and Emcn (c). Confocal images of two fields in the two independent cultures. Scale bar, 100 μm. Representative of n = 3 independent experiments. d Emcn expression on gated tdT⁺BMECs in P0 WBM culture at day 14. Emcn⁺ and Emcn⁻ populations (dot plot, left) were gated and analyzed for Sca-1 expression (histogram, right). Representative of n = 5 independent experiments. Bar graph (bottom left) summarizes fold increase in mean fluorescence intensity of Sca-1 expression in Emcn + (blue) vs. Emcn- (red) cells; n = 5. Data are presented as the mean ± SEM. Gating strategies are shown in Supplementary Figure 9. Source data are provided as a Source Data file. e, f At day 14, P0 WBM cultures were serially passaged every seven days and analyzed by flow cytometry at each passage. e Dot plots show Emcn⁺ and Emcn⁻ cells on gated tdT⁺ BMEC from P0 to P2 in a representative of n = 3 independent experiments. Source data are provided as a Source Data file. f Bar graph summarizes the relative proportion of the Emcn⁺ vs. Emcn⁻ cells at P0–P2. P0 n = 10; P1 n = 8; P2 n = 8. Source data are provided as a Source Data file. g At day 14, the P0 WBM culture was serially passaged every 7–14 days (P0 to P3). Confocal images of two fields at the last day of the indicated passage. Scale bar, 100 μm. Representative of n = 5 experiments. Note that web-like networks (bottom left) were no longer present after P0.