Fig. 3.

rHGF/rRspo1 induces Lgr5⁺ liver stem cells upon CCL4 damage. a Schematic outline upon 1XCCL4 damage for b–f. b, c The Lgr5 expression was measured by qRT-PCR analysis using Lgr5 primer. Eight-week-old C57BL/J mice were injected with CCL4 and different growth factors (0.1 μg/mice/time), as indicated in a, and the livers were harvested and analyzed. d–f Eight-week-old Lgr5-GFP mice were injected with CCL4 and rHGF or/and rRspo1 proteins (0.1 μg/mice/time), as indicated in a, Lgr5 expression was measured using an immunofluorescent assay with anti-GFP antibody (d), Lgr5⁺ cell numbers were analyzed by FACS assay e, f. g. Schematic outline upon chronic CCL4 damage for h–j. h, i Eight-week-old Lgr5-GFP mice were injected with CCL4 and rHGF or/and rRspo1 proteins (0.1 μg/mice/time), as indicated in g, and Lgr5 expression was measured using an immunofluorescent assay with anti-GFP antibody on day 40 (h), Lgr5⁺ cell numbers were analyzed by FACS assay i. j, single Lgr5⁺ liver stem cells were isolated from liver fibrosis model with rHGF plus rRspo1 treatment described as g on day 40 (rHGF/rRspo1) or from 1XCCl4-injured liver treated with PBS on day 5 (PBS), which was cultured to grow into organoids. The represented organoid pictures were shown, and the organoids number and organoids size were analyzed. For b and c, n = 5 mice/group. Scale bars, 200 μm (d, g). For b, c, triplicates for each condition were analyzed. The results are shown as mean ± s.d. of three independent experiments. *p < 0.05. For f, i, j, the results are shown as mean ± s.d. of three independent experiments. **p < 0.01