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. Author manuscript; available in PMC: 2019 Jan 14.
Published in final edited form as: Nat Biotechnol. 2018 Apr 16;36(5):432–441. doi: 10.1038/nbt.4127

Figure 4.

Figure 4

Functional vasculature and decreased apoptosis in grafts. (a) Illustration of the imaging approach for live two-photon microscopy imaging of the organoid graft. (b) Serial macroscopic tracking of grafts showing dynamics of blood vessel perfusion by the recipient vascular system. Dotted area indicates the graft. (c) Grafts co-immunostained for the endothelial markers CD31 and hNuclei at the indicated time points. (d) Organoids have an elevated degree of cell death that is rescued after grafting. Top, TUNEL staining of grafted organoids and stage-matched cultured organoids at the indicated stage. Left panels show staining obtained from two different organoids and organoid grafts at day 31 and 5 dpi, respectively. Bottom, quantification of TUNEL+/DAPI+ cells in grafted organoids and nearly stage-matched cultured organoids of indicated ages. Values are represented as mean ± s.e.m., (n = 3, except for 102-d organoid, n = 4, and 233 dpi n = 2); unpaired two-tailed t-test was used to compare mean difference between each group. Day 31 vs. 5 dpi (t = 0.03656, df = 4, P = 0.9726, not significant), day 53 vs. 14 dpi (t = 14.67, df = 4, P = 0.0001), day 102 vs. 50 dpi (t = 5.943, df = 5, P = 0.0019), and day 279 vs. 233 dpi (t = 6.267, df = 3, P = 0.0082). (e–g) In vivo two-photon imaging of blood vessels via dextran infusion as viewed through the cranial window. Organoids were implanted and TexasRed-dextran was injected at different time points of post-implantation (30 dpi in e, 120 dpi in f). (e) maximum projection of a 300-μm stack taken in a 30-dpi graft (Supplementary Video 2). (f) top view of a three-dimensional reconstruction of a 500-μm Z section in the organoids from a 120-dpi grafted animal (Supplementary Video 3). (g) Single z-plane obtained from 120-dpi graft and acquired at 141-μm depth below the organoid surface, showing blood flow in the vasculature network (Supplementary Video 4). Nuclei were counterstained with DAPI. Scale bars: 1 mm in b, 50 μm in c,d, and 100 μm in e–g. **P < 0.01, ***P < 0.001.