Abstract
The source of new hepatocytes in the uninjured liver has remained an open question. By lineage tracing using the Wnt-responsive gene Axin2 in mice, we identify a population of proliferating and self-renewing cells adjacent to the central vein in the liver lobule. These pericentral cells express the early liver progenitor marker Tbx3, are diploid, and thereby differ from mature hepatocytes, which are mostly polyploid. The descendants of pericentral cells differentiate into Tbx3-negative, polyploid hepatocytes, and can replace all hepatocytes along the liver lobule during homeostatic renewal. Adjacent central vein endothelial cells provide Wnt signals that maintain the pericentral cells, thereby constituting the niche. Thus, we identify a cell population in the liver that subserves homeostatic hepatocyte renewal, characterize its anatomical niche, and identify molecular signals that regulate its activity.
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Acknowledgements
These studies were supported by the Howard Hughes Medical Institute and a grant from the Reed-Stinehart foundation. R.N. is an investigator with the Howard Hughes Medical Institute. B.W. was supported by F32DK091005. We thank D. M. Bissell and T. Desai for comments on the manuscript, V. Waehle for assistance in preparing RNA samples for RNA-seq, M. Britton for RNA-seq analysis, and P. Lovelace for assistance with FACS.
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B.W. carried out the experiments. D.Z. performed qRT–PCR analysis. M.F. performed in situ hybridization. C.Y.L. performed RNA-seq analysis. B.W. and R.N. designed the study, analysed data and wrote the paper. All authors discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Leakiness in Axin2-CreERT2;Rosa26-mTmGflox mice is not observed in animals injected with corn oil.
a, c, No GFP labelling is seen in Axin2-CreERT2;Rosa26-mTmGflox mice after a single dose of corn oil and traced for 2 days (a) or 365 days (c). b, d, No GFP labelling is seen after five consecutive daily doses of corn oil and traced for 7 days (b) or 365 days (d). All animals were 8-week-old Axin2-CreERT2;Rosa26-mTmGflox mice. Images are representative images from n = 5 mice per condition and time point. CV, central vein; PV, portal vein. Scale bars, 100 μm.
Extended Data Figure 2 Axin2 expression remains restricted to pericentral cells.
a–c, In situ hybridization for Axin2 in 120-day trace (a), 240-day trace (b) and 365-day trace (c) Axin2-CreERT2;Rosa26-mTmGflox mice. Representative in situ images are from n = 5 animals per time point. CV, central vein; PV, portal vein. Scale bars, 100 μm.
Extended Data Figure 3 Descendants of Axin2+ cells replaced 30% of the area of the liver.
Tiled image of entire liver section of a 365-day trace Axin2-CreERT2;Rosa26-mTmGflox mice. Image is representative of n = 5 animals at this time point. Scale bar, 2,500 μm.
Extended Data Figure 4 FACS sorting gates for GFP+ cells in Axin2-rtTA;TetO-H2B-GFP mice.
Eight-week-old Axin2-rtTA;TetO-H2B-GFP mice were labelled with doxycycline for 7 days and chased for various lengths of time. Hepatocytes were enzymatically dispersed and sorted by FACS. a–c, Successive gating shows sequential selection of all hepatocytes (a), single cells by forward scatter (b), and side scatter (c). d, Dead cells were excluded by propidium iodide labelling. e, GFP-positive cells were gated and either sorted for RNA-seq analysis or further graphed as histograms for GFP intensity analysis (see Fig. 3g).
Extended Data Figure 5 Axin2 gene dosage and tamoxifen have no effect on pericentral hepatocyte proliferation rate.
Wild-type and Axin2CreERT2+/− mice were given EdU daily for 7 days. A subset of wild-type and Axin2CreERT2+/− mice was given 4 mg of tamoxifen per 25 g body weight daily for 5 days. Pericentral hepatocytes were identified by Hnf4a+/GS+ staining. All other hepatocytes were identified by Hnf4a+/GS− antibody staining. The EdU-positive rates within the two hepatocyte populations as a percentage of total HNF4a+ cells were essentially the same regardless of Axin2 gene dosage or tamoxifen administration. n = 5 animals per group. Data represent mean ± s.e.m. *P > 0.05.
Extended Data Figure 6 Axin2+ hepatocytes proliferate rapidly.
Axin2-rtTA;TetO-H2B-GFP mice were given doxycycline for 7 days. a, 56 days after cessation of doxycycline, very few GFP+ cells are seen around the central vein. b, After 84 days, no GFP+ cells are seen. Images are representative of n = 4 animals per time point.
Extended Data Figure 7 FACS sorting gates for GFP+ cells in Axin2-CreERT2;Rosa26-mTmGflox mice for ploidy analysis.
Eight-week-old Axin2-CreERT2;Rosa26-mTmGflox mice were labelled with five daily doses of tamoxifen and traced for 7 days. Hepatocytes were enzymatically dispersed and sorted by FACS. a–c, Successive gating show sequential selection of all hepatocytes(a), single cells by forward scatter(b), and side scatter (c). d, Dead cells were excluded by propidium iodide labelling. e, GFP-positive cells were gated and graphed as histograms for Hoechst staining (see Fig. 4).
Extended Data Figure 8 FACS sorting gates for endothelial cells.
Eight-week-old wild-type C57B6 mice were used for endothelial cell isolation. Livers were enzymatically digested, hepatocytes were removed by centrifugation and nonparenchymal cells were antibody stained and sorted by FACS. a–c, Successive gating showed sequential selection of non-parenchymal cells by size (a), single cells by forward scatter (b), and side scatter (c). d, Dead cells were excluded by DAPI labelling. e, endothelial cells were identified by CD31-phycoerythrin-positive staining. f, Sinusoidal endothelial cells (SEC) were identified as CD34-FITC+VEGFR3-APC+ while central vein endothelial cells (CEC) were identified as CD34-FITC+VEGFR3-APC−.
Extended Data Figure 9 Histology of VE-cadherin-CreERT2;Wlsflox/flox animal versus control.
a, Control (VE-cadherin-CreERT2;Wlsflox/+) animals given five daily doses of tamoxifen and traced for 7 days after the last tamoxifen dose. Haematoxylin and eosin staining of the liver shows normal histology. b, Wls-knockout animals (VE-cadherin-CreERT2;Wlsflox/flox) also showed normal liver histology. Images are representative images from n = 5 animals per group. Insets show central veins. Scale bars, 100 μm.
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Wang, B., Zhao, L., Fish, M. et al. Self-renewing diploid Axin2+ cells fuel homeostatic renewal of the liver. Nature 524, 180–185 (2015). https://doi.org/10.1038/nature14863
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DOI: https://doi.org/10.1038/nature14863
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