Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury

Abstract

Hepatocytes are replenished gradually during homeostasis and robustly after liver injury1, 2. In adults, new hepatocytes originate from the existing hepatocyte pool3,4,5,6,7,8, 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 diseases9,10,11. 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 (TERTHigh 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 TERTHigh hepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERTHigh hepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERTHigh 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.

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Fig. 1: Identification of a hepatocyte subpopulation with elevated Tert and telomerase activity.
Fig. 2: TERTHigh hepatocytes repopulate the liver in homeostasis and show downregulation of metabolic genes.
Fig. 3: TERTHigh hepatocytes drive clonal expansion by a self-renewal mechanism.
Fig. 4: TERTHigh hepatocytes are critical for liver regeneration.
Fig. 5: A distributed model to explain hepatocyte renewal.

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Acknowledgements

This work was supported by grants from the NIH (NCI CA197563 and NIA AG056575; S.E.A.), the Emerson Foundation (S.E.A.), the DFG (C.R.G), and California TRDRP (P.N.). We thank members of the Artandi laboratory, R. Nusse, P. Beachy, M. Kay and M. Krasnow for critical comments.

Reviewer information

Nature thanks S. Forbes, K. Zaret and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors

Contributions

S.L. and S.E.A. conceived the study. S.L., E.M.N. and S.E.A. designed the experiments. S.L. and C.R.G. created the Tert knock-in line. S.L. and E.M.N. performed the lineage tracing and EdU incorporation experiments. L.C. performed the TRAP assay. S.L. and P.N. performed histological analysis. S.L. and S.W. performed the AAV experiments. S.L. and A.G. performed RNA-seq analyses. S.L. and S.E.A. analysed the data and wrote the paper.

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Correspondence to Steven E. Artandi.

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Extended data figures and tables

Extended Data Fig. 1 Generation and characterization of the TertCreERT2/+ knock-in line.

a, TertCreERT2 targeting strategy and Southern blot strategy. bc, 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 Fig. 1. eg, TertCreERT2/+Rosa26mTmG/+ mouse ES cells, which respond to Cre-mediated recombination by switching from membrane Tomato to membrane EGFP expression (e), showed either membrane Tomato (f, overlaid on bright-field image) or membrane EGFP (g, overlaid on bright-field image) in response to 500 nM 4-hydroxy tamoxifen (4-HT). h, Hepatocytes from TertCreERT2/+Rosa26LSL-Tomato/+ livers before and after FACS enrichment. i, Tamoxifen dose–response curve for TertCreERT2/+Rosa26LSL-Tomato/+ livers (n = 3 mice for each group; horizontal bar shows mean). j, Quantification of the TRAP assay shown in Fig. 1e by densitometry. kn, 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 cells, 167 cells examined) in TertCreERT2/+Rosa26LSL-Tomato/+ livers after 3-day trace with DAPI (blue) staining. Experiments repeated twice for bd, f, g, kn. Scale bars, 100 µm in g, h, 50 µm in kn. Source data

Extended Data Fig. 2 Ploidy and nuclear profiles of the TERTLow and TERTHigh lineages.

ac, Ploidy analysis by Hoechst incorporation and FACS in TERTLow (a) and TERTHigh (b) hepatocytes. c, Quantification showed no significant difference between TERTLow and TERTHigh cells regarding ploidy (n = 5 mice, each represented by unique dot shapes). df, Nucleus count by Tomato (red), phalloidin (green) and DAPI (blue) in livers traced for 3 days (d) and 6 months (e). f, Quantification showed no significant difference between TERTLow and TERTHigh cells in binucleus fractions (n = 4 mice for each group, each represented by unique dot shapes). Experiments repeated twice. Scale bar, 50 µm. Source data

Extended Data Fig. 3 Characterization of the lineage expansion of TERTHigh hepatocytes.

ae, Immunofluorescence performed on TertCreERT2/+Rosa26LSL-Tomato/+ livers after one-year trace showed that only TERTHigh hepatocytes gave rise to hepatocytes. fi, Repeated injections (f) showed that TERTHigh cells formed 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; horizontal bars show mean). j, Heat map showing differentially regulated genes among all TERTLow and TERTHigh samples. Class 1 and class 2 refer to genes significantly downregulated and upregulated in TERTHigh samples, respectively. Genes assigned to DAVID-generated annotation clusters shown on the right. Experiments repeated twice. Scale bar, 200 μm. Source data

Extended Data Fig. 4 Zonal pattern of TERTHigh lineage hepatocytes.

ad, Stitched images of immunofluorescence for Tomato protein (red) and GS (green) in liver sections from TertCreERT2/+Rosa26LSL-Tomato/+ mice treated with tamoxifen and traced for three days (a), three months (b), six months (c) or one year (d). eg, FACS-isolated and cytospun hepatocytes from TertCreERT2/+Rosa26LSL-Tomato/+ mice treated with tamoxifen and traced for three days were stained for CPS1 (red) and GS (green) in TERTLow (e) and TERTHigh hepatocytes (f), and quantified for the GS+ fraction of all cells (g, n = 3 mice; horizontal bars show mean). Experiments repeated three times. Scale bars, 200 μm. Source data

Extended Data Fig. 5 Distribution of proliferating hepatocytes in Tert+/+ and TertCreERT2/+ livers in homeostasis and after injury.

af, Livers were stained with anti-Ki-67 antibody by standard immunohistochemistry. ad, Ki-67+ nuclei are indicated by brown colours in uninjured livers (a, b), and CCl4 (10 μl per 10 g weight) injured livers (c, d), with haematoxylin counterstain in light blue. e, f, Green chromogen was used to indicate Ki-67+ nuclei in DDC (0.1%) treated livers. Hepatocyte nuclei were distinguished by size and morphology. Examples of Ki-67+ hepatocyte nuclei are shown in 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 (CV) (x), the distance to the most adjacent portal vein (PV) (y), and the distance between the central and portal veins (z), following the law of cosines. hj, Two-sided Kolmogorov–Smirnov tests were performed to analyse the distributions 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 are shown (mean ± s.e.m.). No significant differences were found between Tert+/+ and TertCreERT2/+ livers 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. Scale bar, 200 μm. Source data

Extended Data Figure 6 EdU incorporation assays.

a, Scheme of experiments. bg, EdU incorporation in livers of TertCreERT2/+Rosa26LSL-Tomato/+ mice treated with tamoxifen, traced for three days, then treated with EdU in drinking water for 7 days (1 mg ml−1); overlay image (b), HNF4A (c), DAPI (d), EdU (e) and Tomato (f). Dashed boxes, EdU+HNF4A+Tomato+ cells. g, Quantification of EdU incorporation into hepatocytes (n = 5 mice, each represented by unique dot colours). hk, EdU incorporation into livers of Tert+/+ (h) and TertCreERT2/+ (i) mice were compared. Co-immunofluorescence for GS (red) and CK19 (white) was overlaid with EdU (green) and DAPI (blue). j, Quantification of the distribution of EdU+ hepatocytes (pericentral, in GS+ zones; periportal, 0–2 cell layers adjacent to the portal vein space or CK19+ bile ducts; mid-lobular, neither pericentral nor periportal). Dot colours represent individual mice. k, Total EdU+ hepatocytes in Tert+/+ and TertCreERT2/+ livers (n = 5 mice for Tert+/+ livers; n = 4 mice for TertCreERT2/+ livers). Experiments repeated twice. Scale bars, 50 μm in d, 200 μm in i. Source data

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

a, Experiment performed on wild-type hepatocytes isolated by FACS and cytospun. Red foci show individual Tert mRNA molecules. Control experiment by omitting the detection probe for Tert. c, Quantification by focus counts (n = 3 mice, each represented by unique dot shapes; mean + s.e.m.). Experiments repeated three times. Scale bar, 50 μm. Source data

Extended Data Figure 8 Responses of Tert+/+ and TertCreERT2/+ livers to injuries.

a, b, Haematoxylin and eosin (H&E) staining of uninjured livers. c-d, H&E staining of livers 3 days after CCl4 injection. White dotted lines encircle the damaged pericentral area. e, f, H&E staining of livers 7 days after CCl4 injection. g, h, H&E staining of livers 1 month after DDC treatment. Experiments repeated five times. Scale bar, 200 μm.

Extended Data Figure 9 Progeny of TERTHigh hepatocytes can adopt ductal fate after DDC injury.

ad, Immunofluorescence analysis of TertCreERT2/+Rosa26LSL-Tomato/+ livers treated with tamoxifen and DDC, and traced for 1 month (a, overlay image; b, Tomato; c, CK19. d, DAPI). e, Quantification of the percentage of CK19+Tomato+ cells among all Tomato+ cells (n = 5 mice, mean ± s.e.m. 10.0 ± 1.2%) f, Quantification of the percentage of CK19+Tomato+ cells among all CK19+ cells (n = 5 mice, mean ± s.e.m. 6.1 ± 1.0%). Bars show mean. Experiments repeated three times. Scale bar, 50 μm. Source data

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

ad, Epifluorescence of EGFP and DAPI staining of livers 4 days after injection with AAV-GFP (a), AAV-lsl-DTA (b), AAV-flex-DTA (c), and uninjected control (d). 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 and AAV-Cre (red line) or AAV-flex-DTA and AAV-Cre (green line) lead to a narrow window of complete mortality between 4.5 and 6 days; by contrast, injection of AAV-GFP and AAV-Cre, AAV-lsl-DTA, or AAV-flex-DTA did not result in mortality. Between 4 and 6 mice were used for each regimen. Surviving mice were monitored for up to 2 months. g–j, H&E staining of liver sections from mice injected with AAV-lsl-DTA alone (g), AAV-flex-DTA alone (h), AAV-lsl-DTA and AAV-Cre (i), or AAV-flex-DTA and AAV-Cre (j). kr, Livers injected with AAV-flex-DTA and tamoxifen showed a reduction in TERTHigh cells (k, l), as well as increases in collagen deposition (m, n), activated stellate cells (o, p) and ductal cells (q, r). Experiments repeated three times for ad, and twice for gr. Scale bars, 200 µm. Source data

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Lin, S., Nascimento, E.M., Gajera, C.R. et al. Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury. Nature 556, 244–248 (2018). https://doi.org/10.1038/s41586-018-0004-7

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