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. 2019 Sep 5;116(39):19530–19540. doi: 10.1073/pnas.1908099116

Fig. 2.

Fig. 2.

Lgr5+ hepatocytes are long-lived and maintain their own lineage under homeostasis condition. (A) Schematic illustration of the genetic Lgr5-rtTA/TetO-Cre/R26-tdTomato mouse model for lineage tracing. Lgr5-rtTA-IRES-GFP mice were crossed with TetO-Cre mice and R26-tdTomato reporter strain to generate doxycycline (DOX)-inducible triple transgenic model. (B) Schematic illustration showing the experimental strategy for doxycycline induction and time points of analysis in lineage tracing of Lgr5-rtTA/TetO-Cre/R26-tdTomato mice. Representative whole-mount fluorescence images of the left lateral and caudate liver lobes showing identical expression pattern of tdTomato in the mice harvested immediately after doxycycline induction and at 18 mo after treatment. (C) Confocal imaging analysis of Lgr5-rtTA/TetO-Cre/R26-tdTomato mice chased for 4 d and 3, 6, 12, and 18 mo. Liver sections were stained with GFP and RFP antibodies. Triple transgenic mice without doxycycline treatment were used as control (No DOX). At least three mice were used for each experimental condition. (D) Representative confocal images for costaining of GFP, RFP, and Ecad on the liver sections of Lgr5-rtTA/TetO-Cre/R26-tdTomato mice chased for 18 mo. The tdTomato+ hepatocytes are mutually exclusive with periportal Ecad+ hepatocytes, indicating zone-restriction of progeny of Lgr5+ cells. (E) Quantification of the percentage of RFP+ in proportion to GFP+ hepatocytes in mice traced for 4 d, 6 mo, and 12 mo after doxycycline induction. An average of 300 cells per site were counted. n = 10 sites on the liver from three mice. Error bars represent mean ± SEM, *P < 0.05 is calculated by using multiple t test. No significant expansion was observed in comparisons of 4 d versus 6 mo (P = 0.07) and 4 d versus 12 mo (P = 0.12). (F) Representative FACS analysis of total hepatocytes from the Lgr5-rtTA/TetO-Cre/R26-tdTomato mice harvested immediately after doxycycline induction and chased for 18 mo. Lgr5-rtTA/TetO-Cre/R26-tdTomato mice without doxycycline treatment (No DOX) showed no tdTomato+ hepatocytes. (G) Bar graph showing no significant changes in percentage of tdTomato+ cells detected by FACS analysis of total hepatocytes from the Lgr5-rtTA/TetO-Cre/R26-tdTomato mice harvested immediately after doxycycline induction at P28 and chased for 18 mo. Error bars represent mean ± SEM, *P < 0.05 is calculated by using multiple t test. (Scale bars: B, 200 μm; C and D, 100 μm.)