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In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration

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Abstract

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon1, stomach2 and hair follicles3. A three-dimensional culture system allows long-term clonal expansion of single Lgr5+ stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture2,4,5. A crucial component of the culture medium is the Wnt agonist RSPO16, the recently discovered ligand of LGR57,8. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ+ cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5+ cells generate hepatocytes and bile ducts in vivo. Single Lgr5+ cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah−/− mice. These findings indicate that previous observations concerning Lgr5+ stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

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Figure 1: Liver damage induces Lgr5+ bipotential liver progenitors.
Figure 2: In vitro expansion of single Lgr5 cells from adult liver tissue.
Figure 3: Single-cell-derived hepatic organoids acquire hepatocyte fate and display hepatocyte functions in vitro.
Figure 4: Hepatocyte islands upon transplantation of clonal liver organoids into Fah−/− mutant mice.

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Gene Expression Omnibus

Data deposits

The data for the microarray analysis have been deposited to the Gene Expression Omnibus under the accession number GSE32210.

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Acknowledgements

We thank H. Begthel, A. Buijs, W. Karthaus, C. Kroon-Veenboer, M. van den Born, S. van der Brink, and L. Zeinstra for technical assistance. This work was supported by grants to M.H. (EU/236954), S.F.B. (EU/232814), and V.S.W.L. and J.H.v.E. (Ti Pharma/T3-106).

Author information

Authors and Affiliations

Authors

Contributions

Experiments were conceived and designed by M.H and H.C. Transplantation experiments were conceived and designed by M.H., C.D., M.G. and H.C. Experiments were performed by M.H., C.D., S.F.B. and V.S.W.L. A.H. helped with the transplantation experiments. M.v.d.W. and N.S. performed the sortings and K.H. the karyotyping experiments. J.H.v.E. designed and generated the Lgr5-IRES-creERT2 allele. V.S.W.L. performed the bioinformatic analysis of the microarrays and M.J.F. the Y-chromosome staining. M.H. and T.S. developed the isolation protocol. T.S. helped in the beginning phase of the project. R.G.V. helped with sorting experiments. M.H. analysed the data. M.H. and H.C. wrote the manuscript. The other authors commented on the manuscript.

Corresponding author

Correspondence to Hans Clevers.

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Competing interests

M.H. and H.C. are inventors on a patent application related to this work.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 and Supplementary Tables 4 -5. (PDF 4291 kb)

Supplementary Table 1

This file contains overlapping genes between CCl4 treated Liver and small intestine Wnt target genes published in Lau et al. (ref.8). (XLSX 63 kb)

Supplementary Table 2

This file contains overlapping genes between Lgr5-cells and small intestine Wnt target genes published in de Lau et al. (ref.8). (XLSX 33 kb)

Supplementary Table 3

This file contains overlapping genes between Liver Lgr5-cells and small intestine stem cell signature genes published in Munoz et al. (ref. 18). (XLSX 17 kb)

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Huch, M., Dorrell, C., Boj, S. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013). https://doi.org/10.1038/nature11826

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