Fig. 1: The developmental gradient of PV-SMC along the hilum-periphery axis suggests a potential correlation with the earliest differentiation of cholangiocyte progenitor cells.

A Wholemount immunofluorescence analysis of OPN⁺ cholangiocytes and PV-SMC at E15.5. SM22⁺ vascular SMCs delineates the PV thoroughly, with a gradient of αSMA expression from high to low at the periphery, suggesting variable SMC maturation stages in BD development. Scale bars: 200 μm. Two-photon imaging was performed independently on biological replicates (n = 2) and the representative image is shown in this panel. Confocal microscope imaging also performed (n = 3). B Immunotaining of PV-SMC and ductal plates in human fetal liver slice at GW15, based on the placental villi morphology. Ductal plates highlighted with dotted lines. Three to four PV areas per section (n = 2) were examined. Scale bar: 50 µm. C scRNA-seq analysis of mesenchymal cell populations in human fetal liver at GW9 ~ 12, identifying five distinct clusters. Dot plot shows cluster 1 contains ACTA2(gene coding αSMA)⁺TAGLN (gene coding SM22)⁺JAG1⁺ cells. ACTA2⁺, TAGLN⁺, and JAG1⁺ cells are highlighted on each feature plot, whereas TAGLN⁺JAG1⁺ and ACTA2^low (expression level < 4) cells are highlighted in the lower right feature plot indicating TAGLN⁺ ACTA2^low JAG1⁺ are enriched in cluster 1. D Schematic illustration of stepwise induction process of SMCs from hiPSCs. E Differentiation of human iPSC-derived smooth muscle cells in-vitro visualized through bright field imaging and immunostaining for αSMA and SM22. Scale bars: 200 µm (phase contrast) and 100 µm (fluorescence). Experiments were repeated independently more than three times with similar result. F qPCR analysis of the expression level of SM22, aSMA, JAG1 of hiPSC-derived cells throughout different stages of SMCs in-vitro differentiation process. hiPSC (n = 10), imSMC (n = 13), and SMCs (n = 11) were cultured, independently. The boxes indicate mean expression levels while error bars represent standard deviations (SD). (SM22 expression of imSMC vs SMC, Mann-Whitney test, Statistical significance: ns, P-value of 0.2066; αSMA expression of imSMC vs SMC, Mann-Whitney test, Statistical significance: *P-value of 0.0148; JAG1 expression of imSMC vs SMC, Mann-Whitney test, Statistical significance: **P-value of 0.0070).