Fig. 5

Characterization of luminal vasculature in mLO(-DAPT/D3). A Stereo-projection of confocal images of mLOs expressing the EC and hepatocyte markers (CD31 and HNF1β) on day 16 of differentiation. B Orthogonal projections (x/y, x/z, and y/z) of confocal z-stacks obtained from mLOs with vascular networks. Orthogonal views (x/z and y/z) represent planes of intersection at the position of the cross-line (x/y). Note that luminal structures (boxes #1 and #2) were created by surrounding CD31 + ECs. Two additional vascular lumens are seen in the same image (boxes #3 and #4). Scale bar, 50 μm. C Time-lapse confocal image sequence of mLOs after in vitro live perfusion of UEA-I-rhodamine (red). UEA-I-rhodamine was perfused into vessels in mLOs and propagated to vascular networks over time (s, seconds). High-magnification images at the periphery of the organoids are shown separately in the right panel. Arrowheads indicate rhodamine signal. Scale bar, 100 μm. D Immunofluorescence images of CD31 in mLOs after in vitro live perfusion of UEA-I-rhodamine. Scale bar, 100 μm. E TEM images showing the vascular lumens (VL) created by a single EC (left) and multiple ECs (right). N nucleus. Scale bars, 5 μm