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Fig. 4 | Stem Cell Research & Therapy

Fig. 4

From: Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling

Fig. 4

Characterization of bile ducts and endothelial networks in mLOs produced by regulating Notch activity. A qRT-PCR analysis of Notch target genes (HES1 and HEY1), biliary markers (ONECUT1, SOX9, JAG1, EPCAM, GGT1, CFTR, and AQP1), and an endothelial tip cell marker (KDR) in mLOs. Data are expressed as mean ± SD (n = 3, normalized to ACTB). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by unpaired t tests. B H&E staining showing multiple duct-like structures in mLO(-DAPT/D3) (arrowheads). An enlarged image of the boxed area is shown separately in the right panel. Scale bar, 200 μm. C, D Representative immunofluorescence images showing expression of Jagged 1/HNF1β (C) and SOX9/CK19 (D) in duct-like structures of mLO(-DAPT/D3). Scale bar, 100 μm. E, F, TEM images showing the ductal lumen (DL) surrounded by biliary ductal cell-like cells (BD) in mLO(-DAPT/D3). A high-resolution image of the boxed area in E is shown separately in the right panel. MV, microvilli; N, nucleus. A TEM image of bile canaliculi (BC) with junctional complexes (JC) in mLO(-DAPT/D3) is shown in F. Scale bar, 20 μm. G, H Immunofluorescence images of cells expressing both HNF4α and A1AT (G). Albumin-/SOX9 + cells and albumin + /SOX9- cells were distributed separately in a single mLO (H). Scale bar, 20 μm. I, J Albumin (I) and A1AT (J) secretions from mLO(-DAPT/D3) on day 16 compared with primary human hepatocytes (PHH) from three different donors. The secretion level was normalized to the number of cells. **p < 0.01, ***p < 0.001, and ns (not significant) compared to -DAPT/D3 (unpaired t tests). K Comparison of a single confocal z-section (CD31-immunofluorescence staining) between mLOs produced in DM containing DAPT throughout the whole period of differentiation [16 days] and mLOs produced in DM containing DAPT for the first 3 days followed by DM lacking DAPT for the remaining 13 days of differentiation (-DAPT/D3). Serial z-scan images for the two groups of mLOs at the end of differentiation (day 16) are shown in Additional file 1: Fig. S5. Scale bar, 100 μm. L, M Quantification of CD31 + endothelial networks in mLOs for discontinuous endpoints (L), branching and average length (M) of vessels. Confocal images of vascular structures were analyzed using AngioTool and ImageJ. Each black dot represents a single z-scan image (n ≥ 40 per group, two independent experiments). **p < 0.01, ***p < 0.001, and ****p < 0.0001 by unpaired t tests

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