Fig. 2: Optimized human iPSC-derived blood vessel induces tubular-like bile duct structure within hiPSC-blood vessel incorporated liver organoid (BVLO). | Nature Communications

Fig. 2: Optimized human iPSC-derived blood vessel induces tubular-like bile duct structure within hiPSC-blood vessel incorporated liver organoid (BVLO).

From: Generation of human iPSC-derived 3D bile duct within liver organoid by incorporating human iPSC-derived blood vessel

Fig. 2

A Experimental workflow for generating iPSC-derived blood vessel on collagen-1 scaffold. B Localization of Kusabira Orange (KO)-HE cells in optimized co-culture shown in merged phase-contrast (grayscale), and KO fluorescence (red) images. Scale bar, 500 µm. Culture in each condition was repeated independently more than three times with similar results. C Immunofluorescence of CK19 (cholangiocyte) and HNF4α (hepatoblast) at day 14 under different conditions. Scale bar, 100 µm. Three to four fields of view from over five independent organoid samples were analyzed. D H&E staining of co-cultured liver organoid with BV (BVLO) or without hiPSC-BV (LO). E Quantification of BD lumen number, diameter, and CK19+ ratio per field for BVLO and LO. Three to four fields of view from over five independent organoids were analyzed (Error bars: SEM, Two-tailed unpaired T-test, Statistical significance: ***, **, **P-value of 0.0004, 0.0006, 0.0058). F FACS sorting of GFP-hiPSC-MC (GFP+CD31-) or GFP-hiPSC-EC (GFP+CD31+) populations post-dissociation, followed by qPCR analysis of SMC markers (αSMA, SM22, JAG1). Living cells were identified by propidium iodide negativity and categorized by GFP and CD31 expression level (Statistical analysis using Two-tailed Mann-Whitney; n.s, n.s, n.s; P-value of 0.1143; 0.4329; 0.3429); Endothelial markers (TGFB1, TGFB2, TGFB3, CDH5) (Internal control 18S rRNA, n.s, n.s, n.s, n.s; P-value of 0.7302; 0.7857; 0.3429; 0,3429). KO-HE sorted from BVLO post-dissociation. Cell isolation for LO and BVLO was performed at various days (LO day 1 (n = 4), day 7 (n = 4), day 14 (n = 4), day 21 (n = 3); BVLO day 1 (n = 4), day 7 (n = 5), day 14 (n = 5), day 21 (n = 4)). Cholangiocyte markers (KRT7, GPBAR1, AQP1, CFTR); (Statistical significance for KRT7, ns, *P-value of 0.8571, 0.0286; GPBAR n.s, **P-value of 0.4000, 0.0079; AQP1 n.s, n.s, P-value of 0.2000, 0.0571; CFTR n.s, n.s, P-value of 0.6286, 0.1143). Error bars: SEM. G Immunostaining of cholangiocyte (CK19, SOX9, CK7, GRHL2), epithelial junction (ZO1, ECAD, FACTIN, CLDN4, βCAT, RDX) and hepatocyte (HNF4A). Over three fields of view from over five independent organoid samples were analyzed. Scale bar: 50 µm.

Back to article page