Twist2-lineage mesenchymal-hepatocytes are generated postnatally.

(A). Results of lineage tracing using Col1a2-CreERT, Gli1-CreERT, Acta2-Cre, Vim-CreERT, and Twist2-Cre mouse lines crossed with the Rosa-tdTomato reporter line. Male mice over 2 months of age were used. For CreERT lines, TAM was injected at 1 month of age, and the mice were sacrificed 1.5 months later. Scale bar, 50 µm.

(B). Illustrative immunostaining results of liver sections of adult tdTomatoTwist2 mice for CD31, CD45, K19, Vimentin, PDGFRα, or Hnf4α. Scale bar, 50 µm.

(C). The percentages of Tomato+ cells in MCs cultures. Liver MCs were prepared from adult TomatoTwist2mice and plated and cultured overnight. Tomato+ cells were counted (6 views) under microscope and normalized to the number of DAPI+ cells. n=3. Scale bar, 50 µm.

(D). Tracing of Twist2-lineage hepatocytes in TomatoTwist2 mice of various ages. Right panel: quantitation data. n=3 for each age group. Scale bar, 100 µm.

EpCAM+ progenitors generate Twist2-lineage hepatocytes and MCs.

(A). t-SNE analysis of Tomato+ liver cells (combined cells from P1 and P14 TomatoTwist2 mice).

(B). Violin plots showing expression of marker genes for MCs, hepatocytes and EpCAM+ cells.

(C). Heatmap analysis of the top 200 genes expressed in various Tomato+ subgroups.

(D). Comparison of Tomato+ subgroups from the livers of P1 and P14 mice.

(E). Trajectory analysis of Twist2-lineage subpopulations.

(F). t-SNE analysis of EpCAM and Twist2 expression from public scRNA-seq datasets.

(G). GO analysis of hepatomesenchyme compared to MCs.

(H). GO analysis of hepatomesenchyme compared to hepatocytes.

(I). Illustrative immunostaining results for Twist2 on liver sections of P14 and P90 TomatoTwist2 mice. Scale bar, 50 µm.

(J). Illustrative immunostaining results for Twist2 and Hnf4α on sections of E10.5 embryos. Scale bar, 50 µm.

(K). A model explaining how early progenitors generate Twist2 lineage mesenchymal-hepatocytes in mice.

Midzone location and polyploidy features of mesenchymal-hepatocytes.

(A). Location of Tomato+ hepatocytes in the liver of adult TomatoTwist2 mice. Scale bar, 100 µm.

(B). Illustrative image showing the tracing results of adult male mTmGTwist2 mice. Scale bar, 100 µm.

(C). Illustrative immunostaining results for CYP2e1 (zone 3) and CDH1 (zone 1) in liver sections from adult TomatoTwist2 mice. Scale bar, 100 µm.

(D). Flow cytometry analysis of Tomato+ and Tomato- hepatocyte populations for polyploidy. n=3. Data were presented as mean ±SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

Transcriptome profiling of the mesenchymal-hepatocytes.

(A). KEGG analysis of bulk RNA-seq data from Tomato+ and Tomato- hepatocytes.

(B). GO analysis of bulk RNA-seq data from Tomato+ and Tomato- hepatocytes.

(C). Heatmaps showing genes in the ECM, cell cycle, angiogenesis, cell migration, and metabolism modules of Tomato+ and Tomato- hepatocytes.

(D). FPKM values for Alb in Tomato+ and Tomato- hepatocytes. n=3, Data were presented as mean ± SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(E). Analysis of p-ERK, p-4EBP1, and Hes1 on liver sections with immunohistochemical staining. Two-month-old normal mice were used. Arrows: pathway signals. Scale bar, 50 µm. Right panels; quantitation data. n=3. Data were presented as mean ± SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

(F). Illustrative immunostaining results for Ki67+ cells among Tomato+ and Tomato- hepatocytes of P14 mice. Hepatocytes were counted based on cell morphology and size. n=3. Scale bar, 50 µm. Data were presented as mean ±SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

Mesenchymal-hepatocytes show greater regenerative ability than conventional hepatocytes.

(A). Illustrative immunostaining results for Ki67+ cells among Tomato+ and Tomato- hepatocytes of adult mice treated with CCl4 for 2 days. Right panels: quantitation data. n=5 for Tomato- cells and n=3 for Tomato+ cells. Scale bar, 50 µm. Data were presented as mean ± SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

(B). The percentage of Tomato+ hepatocytes in TomatoTwist2 mice was increased 2 weeks after CCl4 administration. Vimentin was stained to detect fibrosis. Right panels: quantitation data. n=5 for Veh and n=3 for CCl4. Scale bar, 100 µm. Data were presented as mean ± SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(C). Changes in diploid and polyploid hepatocytes in regenerated livers of CCl4-induced injury model mice (TomatoTwist2). See Fig. 3d for control normal mice.

(D). Immunostaining for the cumulative EdU-labelled cells among Tomato+ and Tomato- hepatocytes of adult mice (14 days after CCl4), which received EdU every day to label all divided cells. Scale bar, 50 µm. Right panel: quantitation data. n=3. Data were presented as mean ±SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(E). Immunostaining results for Ki67+ cells among Tomato+ and Tomato- hepatocytes from adult mice 2 days after PHx. Scale bar, 50 µm. Right panel: quantitation data. n=3. Data were presented as mean ± SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(F). The percentage of Tomato+ hepatocytes in TomatoTwist2 mice was increased 2 weeks after PHx. Hnf4a was stained to show hepatocytes. Scale bar, 100 µm. Right panel: quantitation data. n=4 for sham and n=3 for PHx. Data were presented as mean ±SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

Notch1 suppresses mesenchymal-hepatocyte expansion via mTOR signaling.

(A). RNA-seq data revealed that Twist2-lineage (Tomato+) hepatocytes are enriched in Notch1 pathway gene expression compared to Tomato- hepatocytes. n=3.

(B). Notch1Twist2mice had 4 instead of 7 liver lobes at birth and showed an increased liver weight-to-body weight ratio at adulthood. n=4. Data were presented as mean ± SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(C). Histological analysis revealed an increase in the number of Tomato+ hepatocytes and Ki67+ cells in adult Notch1 Twist2;TomatoTwist2 mice. Right panel: quantitation data. n=3. Scale bar, 100 µm. Data were presented as mean ± SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(D). Analysis of major mitogenic signaling pathways by Western blotting. Right panels: quantitation data. n=3. Data were presented as mean ± SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(E). RAP treatment diminished overproliferation in the livers of Notch1Twist2 mice. n=3. Scale bar, 50 µm.

(F). Quantitation data for proliferating cells. n=3. Data were presented as mean ±SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

(G). RAP treatment restored the liver weight-to-body weight ratio to close-to-normal levels in adult Notch1Twist2mice. n=4 for Veh and n=3 for Rapamycin. Data were presented as mean ± SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

VEGFR inhibitor suppresses mTOR signaling and Twist2-lineage hepatocyte expansion in Notch1Twist2 mice.

(A). Liver samples from Notch1Twist2mice showed an increase in Vegfa and Vegfb expression but not Vegfc or Vegfd expression. The values of control samples were set at 1.0. n=3. Data were presented as mean ± SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

(B). Immunostaining of Hif1α on liver sections. Arrows: Hif1α signals. Scale bar, 50 µm. Arrows indicate Hif1α signals. Right panel: quantitation data. n=3. Data were presented as mean ± SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(C). Axitinib treatment diminished overproliferation and mTOR activation in the livers of Notch1Twist2mice. n=3. Scale bar, 50 µm, 100 µm for p-S6.

(D). Quantitation data for proliferating cells in livers among vehicle and axitinib groups. n=3. Data were presented as mean ± SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

(E). Axitinib treatment restored the liver weight-to-body weight ratio to close-to-normal levels. n=4 for Veh and n=3 for Axitinib group. Data were presented as mean ± SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

(F). A model depicting how Twist2 lineage mesenchymal-hepatocytes regenerate the liver in mice.

Analysis of liver development and regeneration in Twist2-Cre mice.

(A). The percentages of Tomato+ MCs positive for Vimentin or PDGFRα (See immunostaining results in Fig. 1b). n=3 mice.

(B). The liver weight-to-body weight ratios of adult male Twist2-Cre and control mice. n=3. Data were presented as mean ±SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(C). Histological structures of the livers of adult Twist2-Cre and control mice. Scale bar, 50 µm.

(D). Immunostaining results for Vimentin, PCNA, p-4EBP1 and p-ERK on liver sections of adult Twist2-Cre and control mice. Quantitation data: the numbers of Vimentin+ cells per view and the percentages of cells positive for PCNA, p-4EBP1, or p-ERK. n=3. Scale bar, 50 µm. Data were shown as mean ±SD.

(E). Illustrative H/E staining results of regenerated livers from Twist2-Cre and control mice 2 weeks after CCl4-induced injury or PHx. n=3. Scale bar, 50 µm.

scRNA-seq analysis of liver Tomato+ cells from P1 and P14 mice.

(A). t-SNE analysis of Tomato+ liver cells including proerythrocytes (combined Tomato+ cells from P1 and P14 TomatoTwist2 mice).

(B). Comparison of the Tomato+ subgroups from P1 and P14 mice. Right panels: percentage of each subpopulation.

(C). t-SNE analysis showing the expression of proerythrocyte marker genes.

(D). t-SNE analysis showing the expression of Twist2 in various subpopulation.

(E). t-SNE analysis of CD31 and CD45 expression in various Twist2 lineage groups exclusive of proerythrocytes.

(F). t-SNE analysis of expression of important cell-specific marker genes for MCs, hepatocytes, and epithelial cells.

(G). Comparison of the expression of multiple marker genes in different Tomato+ subgroups. Density indicates the percentage of cells with a given score and score indicates the equivalence to a given lineage.

KEGG and GO analyses of liver Tomato+ cells of P1 or P14 mice.

(A-E). GO and KEGG analyses of various subpopulation using scRNA-seq data.

Analysis of the public domain scRNA-seq datasets of hepatocytes and MCs.

(A). Analyses of hepatocytes from normal mice (based on Alb expression levels) and their expression of Twist2 and EpCAM under normal or injury conditions (GSE125688). DDC (1 week), 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

(B). t-SNE analysis of liver mesenchymal populations from normal mice and their expression of Twist2 and EpCAM under normal or injury conditions (CCl4, 6 weeks) (GSE137720).

(C). The proportions of various subgroups in the adult mouse liver based on Alb expression (GSE125688) (derived from Fig. S4A).

(D). Heatmap showing the expression of MC markers in hepatocyte subpopulations from adult normal mice (derived from Fig. S4A).

(E). GO analyses of Alblow 2 hepatocytes compared to Albhigh (1+2) hepatocytes.

(F). GO analyses of Albhigh (1+2) compared to Alblow 2 hepatocytes.

Expression of hepatocyte marker genes in Twist2-lineage liver cells.

Analysis of hepatocyte marker gene expression in Twist2-lineage liver cells. These markers were studied to trace hepatocytes in reference 25.

Analysis of Twist2-lineage mesenchymal-hepatocytes in young and old mice.

Comparison of the percentage of Tomato+ hepatocytes and their polyploid status in 2- and 20-month-old TomatoTwist2 mice by immunostaining and flow cytometry, respectively.

Deletion of one Twist2 allele did not affect midzone hepatocytes during homeostasis or regeneration.

(A). Immunostaining results for Cyclin D1 in P14 Twist2-Cre and control mice. Right panel: Quantitation data: the numbers of Cyclin D1+ cells per view. n=3. Scale bar, 100 µm. Data were shown as mean ±SD.

(B). Immunostaining results for Cyclin D1 in P60 Twist2-Cre and control mice. Right panel: Quantitation data: the numbers of Cyclin D1+ cells per view. n=3. Scale bar, 100 µm. Data were shown as mean ±SD.

(C). Immunostaining results for Cyclin D1 at day 2 post CCl4 in adult Twist2-Cre and control mice. Right panel: Quantitation data: the numbers of Cyclin D1+ cells per view. n=3. Scale bar, 100 µm. Data were shown as mean ±SD.

(D). Immunostaining results for Cyclin D1 at day 14 post CCl4 in adult Twist2-Cre and control mice. Right panel: Quantitation data: the numbers of Cyclin D1+ cells per view. n=3. Scale bar, 100 µm. Data were shown as mean ±SD.

Analysis of expression of Twist2 and MET genes during liver repair.

(A). Immunostaining results for Twist2 on liver sections of normal adult mice 0 day, 2 days, or 2 weeks after CCl4. Scale bar, 50 µm.

(B). Immunostaining results for Twist2 on liver sections of normal adult mice 0 day, 2 days, or 2 weeks after PHx. Scale bar, 50 µm.

(C). qPCR results showed that Snai1, Zeb1, and Twist1 mRNA levels were not significantly altered in regenerating liver of Twist2-Cre mice 2 weeks after CCl4 compared to normal mice. n=3. Data were presented as mean ±SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

(D). qPCR results showed that Snai1 mRNA level was increased while Zeb1 and Twist1 mRNA levels were not significantly altered in regenerating livers of mice 2 weeks after PHx in Twist2-Cre mice. n=3. Data were presented as mean ±SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant.

Notch1 limits Twist2-lineage hepatocyte expansion via VEGFR-mTOR signaling.

(A). FPKM values for major Notch1 pathway genes in Tomato+ and Tomato- hepatocytes. n=3, Data were presented as mean ±SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(B). qPCR results showed a reduction in Notch1 and Hes1 in hepatocytes from Nocth1Tiwst2 mice compared to control mice. n=3. Data were presented as mean ±SD. Two-way ANOVA was applied. P-value <0.05 was considered as statistically significant. The skeletal structure of adult male Nocth1Twsit2 and control mice.

(C). The muscle phenotypes of Nocth1Twsit2 and control mice. Right panels: weight to body weight ratio and cross section areas of myofibers. n=3. Data were presented as mean ±SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(D). Immunostaining results showed reduced expression of Hes1 in regenerating liver samples of normal mice. Scale bar, 50 µm. Right panel: quantitation data. n=3. Data were presented as mean ±SD. Unpaired two-tailed Student’s t-test was applied. P-value <0.05 was considered as statistically significant.

(E). Illustrative staining results showing the numbers of Tomato+ hepatocytes in Nocth1Twist2mice and control mice after CCl4-induced injury. n=3. Scale bar, 200 µm. Data were shown as mean ±SD. * P-value <0.05 between Nocth1Twist2 mice and control mice after CCl4-induced injury.

(F). Analysis of p-Erk and p-S6 on liver sections from adult male Nocth1Twist2 and control mice. Scale bars, 50 µm for p-ERK and 100 µm for p-S6.

(G). The Nocth1Twist2 mice showed an increase in blood vessels in the liver, which were stained positive for αSMA. Scale bar, 50 µm.

Axitinib inhibits proliferation of Huh7 cells.

CCK-8 assays were performed to evaluate the effect of Axitinib on the proliferation of hepatocellular carcinoma cell line Huh7. n=5. Data were presented as mean ±SD. Two-way ANOVA was applied. Statistical significance is displayed as P<0.001 (***).