Figure 4: HSCs are the principal source of myofibroblasts in cholestatic liver fibrosis.

(a–c) Liver sections of 14 day bile duct-ligated (BDL) mice (a, ZsGreen n=3, tdTom/Col-GFP n=4), 9-week-old Mdr2^ko mice (b, ZsGreen n=4, tdTom/Col-GFP n=5), or 0.1% DDC-diet-treated mice (c, ZsGreen n=4, tdTom/Col-GFP n=3) were stained with desmin to demonstrate co-localization of HSC marker desmin and LratCre-induced ZsGreen by confocal microscopy (upper panel). Confocal microscopy was employed to show co-localization of Col-GFP reporter, marking activated myofibroblasts and LratCre-induced tdTomato expression (lower panel). (d) Flow cytometric images from 2-week-old BDL mice (upper panel) and 9-week-old Mdr2_ko (lower panel) mice co-expressing LratCre, tdTomato and Col-GFP. Images next to fluorescence-activated cell sorting (FACS) plots show sorted cells freshly after plating. (e) Quantification of Col-GFP-expressing cells, derived from LratCre-labelled tdTomato-positive HSCs, was performed in liver sections (14 days BDL: n=4; Mdr2^ko n=5; 0.1% DDC diet: n=3) or in non-parenchymal cell fractions using flow cytometry (14 days BDL: n=4, Mdr2^ko n=6). (f) Images demonstrating small and large bile ducts surrounded by Col-GFP-positive and LratCre-negative portal fibroblasts. (g) Quantitative PCR analysis of FACS-sorted unplated LratCre-labelled tdTom-positive and Col-GFP-positive cells (HSC, n=5 isolates), and tdTom-negative and Col-GFP-positive cells (PFLCs, n=5 isolates). (h) Representative images of HSCs and PFLCs show morphologically distinct cell populations. Scale bars, 100 μm (a–c), 10 μm (d), 100 μm (f) and 50 μm (h). Data are shown as means±s.d. *P<0.05; **P<0.01; ***P<0.001 (determined by Student’s t-test).