Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury

Abstract

Hepatocytes are replenished gradually during homeostasis and robustly after liver injury^{1, 2}. In adults, new hepatocytes originate from the existing hepatocyte pool^3-8, but the cellular source of renewing hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases^9-11. Here we identify a subset of hepatocytes that expresses high levels of telomerase and show that this hepatocyte subset repopulates the liver during homeostasis and injury. Using lineage tracing from the telomerase reverse transcriptase (Tert) locus in mice, we demonstrate that rare hepatocytes with high telomerase expression (TERT^High hepatocytes) are distributed throughout the liver lobule. During homeostasis, these cells regenerate hepatocytes in all lobular zones, and both self-renew and differentiate to yield expanding hepatocyte clones that eventually dominate the liver. In response to injury, the repopulating activity of TERT^High hepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERT^High hepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERT^High hepatocytes combined with chemical injury causes a marked increase in stellate cell activation and fibrosis. These results provide support for a 'distributed model' of hepatocyte renewal in which a subset of hepatocytes dispersed throughout the lobule clonally expands to maintain liver mass.

Extended Data Figure 1. Generation and characterization of the Tert^CreERT2/+ knock-in line

a-d, Tert^CreERT2 targeting strategy and southern-blot strategy (a). Southern-blots using a 5′probe (b), a NeoR probe (c), and a 3′probe (d). KI, knock-in cells. WT, wild-type cells. For gel source data, see Supplementary Figure 1. e-g, Tert^CreERT2/+;Rosa26^mTmG/+ mouse embryonic stem cells showed either membrane Tomato (f, overlaid on bright-field image), or membrane EGFP(g, overlaid on bright-field image) in response to 500nM 4-hydroxy tamoxifen (4-HT). h, hepatocytes from Tert^CreERT2/+; Rosa26^LSL-Tomato/+ livers before and after FACS-enrichment. i, Tamoxifen dose response of Tert^CreERT2/+; Rosa26^LSL-Tomato/+ livers (n = 3 mice for each group, cross-bar: mean). j, Quantification of the TRAP assay shown in Fig.1e by densitometry. k-n, Co-immunofluorescence for Tomato (red) and CD45 (k, blood cells, 202 cells examined), CD68 (l, Kupffer cells, 179 cells examined), GFAP (m, stellate cells, 158 cells examined) and PECAM (n, endothelial, 167 cells examined) (each in green) in Tert^CreERT2/+; Rosa26^LSL-Tomato/+ liver, 3-day trace, with DAPI (blue). Experiments repeated twice for b-d, f-g, k-n. Scale bars, 100 μm in g,h, 50 μm in k-n.

Extended Data Figure 2. Ploidy and nuclear profiles of the TERT^Low and TERT^High lineages

a-c, Ploidy analysis by Hoechst incorporation and FACS in TERT^Low (a) and TERT^High (b) hepatocytes. Quantification showed no significant differences between TERT^Low and TERT^Highcells regarding ploidies (c, n = 5 mice, each represented by unique dot-shapes). d-f, Nuclei count by Tomato(red), phalloidin(green) and DAPI(blue) in livers traced by 3 days (d) and 6 months (e). Quantification showed no significant differences between TERT^Low and TERT^Highcells in binuclei fractions (f, n = 4 mice for each group, each represented by unique dot-shapes). Experiments repeated twice. Scale bar, 50 μm.

Extended Data Figure 3. Characterization of the lineage expansion of TERT^High hepatocytes

a-e, IF performed on Tert^CreERT2/+; Rosa26^LSL-Tomato/+ livers after one-year trace showed TERT^High hepatocytes exclusively gave rise to hepatocytes. f-i, Repeated injections showed TERT^High cells as a constant proportion of the liver. Lineage expansion over one injection (g) and three injections (h) was quantified (i, n = 3 mice for each group; cross-bar: mean). j, Heat map showing differentially regulated genes among all TERT^Low and TERT^High samples. Class I and Class 2 refer to genes significantly down-regulated and up-regulated in TERT^High samples, respectively. Genes assigned to DAVID- generated annotation clusters shown on the right. Experiments repeated twice. Scale bar, 200 μm.

Extended Data Figure 4. Zonal pattern of TERT^High lineage hepatocytes

a-d, Stitched images of IF for Tomato protein (red) and GS (green) in liver sections from Tert^CreERT2/+; Rosa26^LSL-Tomato/+ mice treated with tamoxifen and traced for three days (a), three months (b), six months (c) or one year (d). e-g, FACS-isolated and cytospunhepatocytes from Tert^CreERT2/+; Rosa26^LSL-Tomato/+ mice treated with tamoxifen and traced for three days were stained for CPS1 (red) and GS (green) in TERT^Low (e) and TERT^High hepatocytes (f), and quantified for the GS⁺ fraction of all cells (g, n = 3 mice; cross-bars: mean). Experiments repeated three times. Scale bars, 200 μm.

Extended Data Figure 5. Distribution of proliferating hepatocytes in Tert^+/+ and Tert^CreERT2/+ livers in homeostasis and under injury hepatocytes

a-f, Livers were stained with anti-Ki-67 antibody with standard immunohistochemistry. Ki-67⁺ nuclei were indicated by brown colours in uninjured livers (a, b), and CCl₄ (10 μL/10 g weight) injured livers (c, d), with haematoxylin counterstain in light blue. Green chromogen was used to indicate Ki-67⁺ nuclei in DDC (0.1%) treated livers (e, f). Hepatocyte nuclei were distinguished by size and morphology. Examples of Ki-67⁺ hepatocytes nuclei were shown in box insets. g, Quantification of Ki-67⁺ hepatocytes and their distribution along the central-portal axis. The position index (P.I.) was determined by the distance to the most adjacent central vein (x), the distance to the most adjacent portal vein (y), and the distance between the central and portal veins (z), following the law of cosines. h-j,Two-sided Kolmogorov-Smirnov tests were performed to analyse the distribution of Ki-67⁺ hepatocytes along the central-portal axis. Histograms (bin-width = 0.1) and shaded curves of the kernel density estimation with Gaussian approximation were shown. Bars and error-bars: mean±SEM. No significant differences between Tert^+/+ and Tert^CreERT2/+ livers were found in uninjured livers (h, n = 4 mice for each group;each mouse represented by unique dot-shapes;p = 0.58), in CCl₄ injured livers (i, n = 3 mice for each group; each mouse represented by unique dot-shapes, p = 0.32), or in DDC injured livers (j, n = 3 mice for each group; each mouse represented by unique dot-shapes;p = 0.98). Experiments repeated twice. CV, central vein; PV, portal vein; P.I., position index. Scale bar, 200 μm.

Extended Data Figure 6. EdU incorporation assays

a, Scheme of experiments. b-g, EdU incorporation in livers of Tert^CreERT2/+; Rosa26^LSL-Tomato/+ mice treated with tamoxifen, traced for three days, then treated with EdU in drinking water for 7 days (1 mg/mL). The overlay image (b), as well as individual images of HNF4A (c), DAPI (d), EdU (e) and Tomato (f) were shown. Quantification of EdU incorporation in hepatocytes (g, n = 5 mice, each represented by unique dot-colours). Dashed boxes, EdU⁺HNF4A⁺Tomato⁺ cells. h-k,EdU incorporation in livers of Tert^+/+ (h) and Tert^CreERT2/+(i) mice were compared. Co-immunofluorescence for GS (red) and CK19 (white) was overlaid with EdU (green) and DAPI (blue). Quantification of the distribution of EdU⁺ hepatocytes (j, “pericentral”: in GS⁺ zones; “periportal”: 0-2 cell layers adjacent to the portal veins space or CK19⁺ bile ducts; “mid-lobular”: neither “pericentral” nor “periportal”. Dot-colours represent individual mice) and total EdU⁺ hepatocytes in Tert^+/+ and Tert^CreERT2/+ livers (k) (n = 5 mice for Tert^+/+livers, and n = 4 mice for Tert^CreERT2/+livers). Experiments repeated twice. Scale bar, 50 μm in d, 200 μm in i.

Extended Data Figure 7. Single-molecule RNA FISH on wild-type hepatocytes

a, Experiment performed on wild-type hepatocytes isolated by FACS and cytospun. b, Control experimentby omitting the detection probe for Tert. c, Quantification by foci counts (n = 3 mice, each represented by unique dot-shapes. Bars and error-bars are mean+SEM). Experiments repeated three times. Scale bar, 50 μm.

Extended Data Figure 8. Responses of Tert^+/+ and Tert^CreERT2/+livers to injuries

a-b, H&E analysis on uninjured livers. c-d, H&E analysis on livers 3 days post CCl₄ injection. Artificial white dotted lines encircle the damage pericentral area. e-f, H&E analysis on livers 7 days post CCl₄ injection. g-h, H&E analysis on livers 1 month post DDC treatment. CV, central vein; PV, portal vein. Experiments repeated five times. Scale bar, 200 μm.

Extended Data Figure 9. Progeny of TERT^High hepatocytes can adopt ductal fate after DDC injury

a-d, IF analysis on Tert^CreERT2/+; Rosa26^LSL-Tomato/+ livers treated with tamoxifen and DDC, and traced for 1 month. The overlay image (a), as well as individual images of Tomato (b), CK19 (c), and DAPI (d) were shown. e. Quantification of the percentage of CK19⁺Tomato⁺ cells in all Tomato⁺ cells (n = 5 mice, mean±SEM:10.0±1.2%) f. Quantification of the percentage of CK19⁺Tomato⁺ cells in all CK19⁺ cells (n = 5 mice, mean±SEM:6.1±1.0%). Bars: mean. Experiments repeated three times. Scale bar, 50 μm.

Extended Data Figure 10. Characterization of AAV.lsl.DTA and AAV.flex.DTA

a-d, Epifluorescence of EGFP, as well as DAPI staining from livers 4 days after injection with AAV.GFP (a), AAV.lsl.DTA (b), AAV.flex.DTA (c), and uninjected control (d) shown. e, Diagram of AAV.flex.DTA, and recombination events that lead to DTA expression. f, Survival effects of AAV.TBG viruses. Combined injection of “AAV.lsl.DTA+AAV.Cre” (red line) or “AAV.flex.DTA+AAV.Cre” (green line) lead to a narrow window of complete mortality between 4.5-6 days; in contrast, “AAV.GFP+AAV.Cre”, AAV.lsl.DTA, or AAV.flex.DTA injection did not result in mortality. Between 4-6 mice were used for each regimen. Survived mice were monitored for up to 2 months. g-j,H&E on liver sections from mice injected with AAV.lsl.DTA alone (g), AAV.flex.DTA alone (h), “AAV.lsl.DTA+AAV.Cre” (i), and “AAV.flex.DTA+AAV.Cre” (j). k-r, Livers injected with AAV.flex.DTA and Tamoxifen showed reduction of TERT^High cells (k, l), as well as increases in collagen deposit (m, n), activated stellate cells (o, p) and ductal cells (q, r). Experiments repeated three times for a-d, and twice for g-j, k-r. Scale bars, 200 μm.

Figure 1. Identification of a hepatocyte subpopulation with elevated Tert and telomerase activity

a-b, Immunofluorescence (IF) analysis on Tert^CreERT2/+; Rosa26^LSL-Tomato/+ liverstreated with single-dose of tamoxifen and analysed 3 days post injection. Tomato (red), HNF4A (green), CK19 (white), and DAPI (blue) shown. c-g, Analysis of telomerase expression in FACS-sorted hepatocytes. Representative FACS plot (d), TRAP assay (e, B: Buffer only) and quantitative reverse-transcription PCR(f, position of primer pairs) (g, fold-changes.n = 3 mice, each indicated by unique dot shapes; cross-bar: mean) shown. Experiments repeated three times for b, more than five times for d, and twice for e and g. Scale bars, 50 μm.

Figure 2. TERT^High hepatocytes repopulate the liver in homeostasis and show downregulation of metabolic genes

a-i, Lineage tracing in Tert^CreERT2/+; Rosa26^LSL-Tomato/+ micetreated with single-dose tamoxifen (b-g) or oil vehicle (i) and analysed at indicated tracing periods by IF for Tomato. Quantification of Tomato⁺hepatocyte area (h, n = 4, 5, 4, 4, 7, 5 mice for each time-point;cross-bar: mean). j-m, Co-IF for Tomato and GS inTert^CreERT2/+; Rosa26^LSL-Tomato/+ micetraced for 3 days, 6 months or one year.Quantification of Tomato⁺GS⁺ fraction of GS⁺ hepatocytes (m, n = 4, 5, 5 mice for each time-point; cross-bars, mean). n-o, RNA-seq results on FACS-purified TERT^High (Tomato⁺) or TERT^Low (Tomato^-) hepatocytes (n = 3 mice for each group). Volcano plot for enriched genes and GO terms (n,cut-offs:q< 0.05, |log₂(fold difference)|> 0.8). GSEA analysis for enriched gene-sets (o, number of genes shown for each gene set). Experiments repeated twice for time-points in b,f,g,j-l. Scale bars, 100 μm.

Figure 3. TERT^High hepatocytes drive clonal expansion by a self-renewal mechanism

a-h, 3D analysis of sparse-labelled hepatocyte clones in Tert^CreERT2/+; Rosa26^LSL-Tomato/+ mice treated with low-dose tamoxifen, traced for 3 days, 3 months or 6 months. Clone-sizes (b,c) and clone-number per volume (d) (n = 5, 3, and 4 mice, respectively; each mouse represented by unique dot-colour in c; cross-bar: mean). Co-IF for Tomato and GS to assess clone-location in 6-month trace samples (e-h). Quantification of clone size and position relative to GS⁺ cells (h).i-r, Single-molecule RNA FISH on FACS-purified TERT^High derived (l-n) and TERT^Low derived (o-q) hepatocytes. Quantification by foci counts (r, n = 3 mice per time-point, each mouse indicated by unique dot-shapes. Bars and error-bars are mean+SEM. For cells with >5 foci, p_1month-3days = 0.56, p_1year-3days = 2.7e-5, p_1year-1month = 4.3e-5). Experiments repeated twice. Scale bars, 50 μm.

Figure 4. TERT^High hepatocytes are critical in liver regeneration

a-f, Single-dose CCl₄ liver injury. Tert^CreERT2/+; Rosa26^LSL-Tomato/+ mice treated with oil vehicle (b,c) or CCl₄ (d,e) analysed by IF for Tomato and GS at day 7 post-treatment. IF Quantification (f); white lines, GS⁺ pericentral zone(n = 5 mice). g-j, DDC diet-induced injury in Tert^CreERT2/+; Rosa26^LSL-Tomato/+ mice. IF of liver for Tomato after 30 days treatment with normal diet (h) or DDC diet (i). IF quantification (j) (n = 4 mice). k-m, Ablation of TERT^High hepatocytes via AAV.lsl.dtA injection to Tert^CreERT2/+; Rosa26^LSL-Tomato/+ mice (n = 4 mice), quantification of Tomato⁺ cells (m). n-v, Genetic ablation followed by DDC injury in Tert^CreERT2/+; Rosa26^LSL-Tomato/+ mice. Livers analysed by Sirius Red for collagen (o-q), SMA for activated stellate cells (r-t), and CK19⁺ cells (u-w) (n = 4 mice). AAV.GFP injected control animals (o, r, t) and AAV.lsl. DTA injected animals (p, s, u).Cross-bars: mean.Experiments repeated at least twice. Scale bars, 100 μm.

Figure 5. A distributed modelto explain hepatocyte renewal

a, TERT^High hepatocytes generate expanding clones comprised of both TERT^High and TERT^Low hepatocytes. b, Rare TERT^High hepatocytes distributed throughout the lobule form enlarging clones during homeostasis in response to local hepatocyte loss. c, Repopulation of injured liver by clonal expansion ofTERT^High hepatocytes is accelerated during injury. Blue vessels, portal and central veins; pink vessel, hepatic artery; small green cells, cholangiocytes; large green cells, pericentral hepatocytes; red cells, TERT^High hepatocytes; black cells, damaged hepatocytes.