Fig. 6: Dependency of bile duct formation within BVLO on JAG1/NOTCH which augmented by TGFB signal.

A Representative phase-contrast and epifluorescence image of LO and BVLO containing KO-HE. KO-HE form a mesh-like network in BVLO, suppressed by A8301, L685458, or DAPT. Each experiment was repeated independently three times with similar result (Scale bar: 500 µm). B Imunofluorescence of LO, BVLO, or BVLO treated with A8301, L685458 or DAPT (scale bar: 200 µm). C Quantification of BD lumen number and CK19⁺ area ratio in LO or BVLO treated with A8301, L685458 or DAPT. Culture was independently repeated for LO (n = 4), BVLO (n = 6), BVLO + A8301 (n = 5), BVLO + L685458 (n = 6), and BVLO + DAPT (n = 6). (Kruskal-Wallis test followed by Steel multiple comparison test, CK19⁺ area with statistical significance **, **, **, **P-value of 0.0095; 0.0043; 0.0022; 0.0022), lumen number with statistical significance **,**,**,**,**P-value of 0.0070; 0.0079; 0.0022; 0.0022. Error bars are SEM. D Immunofluorescence of JAG1 from planar-cultured imSMCs (Scale bar: 50 µm). E qPCR of JAG1 from planar-cultured SMCs. Sample number for control (n = 5), A8301 (n = 3), TGFβ1 (n = 9), and TGFβ + A8301 (n = 3). (Kruskal-Wallis with Steel multiple comparison test, TGFβ1:10 ng/ml vs TGFβ1:0 ng/ml, and TGFβ1:10 ng/ml vs TGFβ1:10 ng/ml+A8301:1 µM, Statistical significance: *P-value of 0.0400; 0.0400). Error bars are SEM. F Schematic illustration of BVLO generation using JAG1 knock out-imSMC. G Bright field image of JAG1 knock-out iPSC-derived imSMCs (Scale bar 200 µm). H Immunofluorescence analysis of BVLO vs. two clones of JAG1 Knock-out imSMC BV[LO]. Cholangiocyte markers (CK19, CK7), epithelial junction and polarity markers (ZO1, ECAD, fACTIN, CLDN4, RDX). Culture was repeated three times, independently. For each set of culture, two BVLOs incorporating the BV with JAG1 + / +, JAG1−/− clone 1, and JAG1−/− clone 2 MCs were generated. Three to four fields from each organoid sample were analyzed. Scale bar 50 µm. I Quantification of CK7⁺ area, and lumen number in BVLO vs. JAG1 knock-out imSMC BV[LO]. Three to four fields in two different sections of each organoid sample were analyzed for quantification. (Kruskal-Wallis with Steel multiple comparison test: *P-value of 0.0022 and 0.0022). Error bars represent SEM. J Schematic illustration of BVLO generation using JAG1 Knock out-HE cells. K Immunofluorescence of BVLO containing JAG1 Knock-out HE cells (Scale bar: 100 µm). Cholangiocyte (CK19, CK7), epithelial markers (ZO1). Culture was repeated independently two times with similar result.