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. 2013 Feb 14;494(7436):247-50.
doi: 10.1038/nature11826. Epub 2013 Jan 27.

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

Meritxell Huch  1 , Craig Dorrell, Sylvia F Boj, Johan H van Es, Vivian S W Li, Marc van de Wetering, Toshiro Sato, Karien Hamer, Nobuo Sasaki, Milton J Finegold, Annelise Haft, Robert G Vries, Markus Grompe, Hans Clevers
Affiliations

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

Meritxell Huch et al. Nature. .

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 colon, stomach and hair follicles. 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 architecture VSports手机版. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. 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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Conflict of interest statement

Competing Financial Interests

MH & HC are inventors on a patent application related to this work.

"VSports" Figures

Figure 1
Figure 1. Liver damage induces Lgr5+ bi-potential liver progenitors
a–b, Lgr5-LacZ mice were injected IP with corn oil (n=6) (a) or with a single dose of CCl4 in corn oil (1ml/kg) (n=6) (b). Six days later, mice were sacrificed and livers collected and processed for β-gal staining. a, Undamaged liver does not express Lgr5-LacZ. b, Upon CCl4, strong Lgr5-LacZ expression was detected in small cells near ducts. Compare Lgr5-LacZ+ cells (arrow) with neighboring hepatocytes (asterisk,*). Scale bars, 200 μm (top panels) and 30 μm (bottom panels). c, Lgr5-ires-CreERT2 mice were crossed with Rosa26R-LacZ Cre reporter mice. Offspring received a single IP injection of CCl4 and 2 days later CreERT2 activity was induced with tamoxifen (3mg/mouse) as indicated in the scheme. Representative pictures showing lineage tracing of Lgr5-cells upon CCl4 damage (n=8). Compare differences in size between LacZ+ cells (arrows) and hepatocytes (asterisk,*) at days 2 and 4 after tamoxifen induction. Magnifications on each day are the same. Scale bars, 500 μm (top panels) and 50 μm (bottom panels). d, Lgr5-ires-CreERT2 x Rosa26-LacZ offspring were fed with DDC (n=3), and 4 and 6 days later CreERT2 activity was induced with tamoxifen as indicated in the scheme. Representative pictures showing positive hepatocytes (d′) and positive ductal cells (d″). Tam, tamoxifen. Scale bars, 200 μm (d), 25 μm (d′) and 50 μm (d″).
Figure 2
Figure 2. In vitro expansion of single Lgr5 cells from adult liver tissue
a–c, Lgr5-LacZ mice were injected with corn oil or CCl4 (IP) as in Fig. 1. Six days later, liver tissue was dissociated to single cells, loaded with the fluorescent CMFDG β-galactosidase substrate and analyzed by FACS. Sorted isolated Lgr5-LacZ+ cells were cultured at a ratio of 1-single Lgr5-LacZ+ positive cell/well (clonal) as described in Supplementary Methods. a, scheme representing the protocol used. b, Representative FACS plot of dissociated single cells from CCl4-treated (CCl4+) and non-treated (no CCl4) livers. Cells were gated following sequential selection by cell-size (FSC vs SSC) and PI exclusion. Viable CMFDG+ PI− cells were selected and sorted. Representative cell is shown. c, Serial DIC images showing the outgrowth of a single Lgr5-LacZ+ cell. Original magnifications: 40x (days 0–5), 20x (day 7–11), 10x (day 19) and 4x (1 month onwards). P, passage.
Figure 3
Figure 3. Single cell derived hepatic organoids acquire hepatocyte fate and display hepatocyte functions in vitro.
a–i, Clonal Lgr5-derived cultures were grown in expansion medium (EM) and transferred to differentiation medium (DM) for 8–14 days prior to analysis. Two independent clonal cultures were analyzed. a–d, confocal images (z-stack projection) for hepatocyte specific markers. a, HNF4α (red), albumin (ALB) (green) and EpCAM (grey). b, MRP4 (green). c, ZO-1 (magenta). d, ALB (red) KRT19 (magenta) and hepatocyte surface marker OC2-2F8 (green, full description of OC2-2F8 marker in Supplementary Fig. 8). Nuclei were counterstained with Hoechst. e–f, LDL uptake was analyzed using Dil-ac-LDL fluorescent substrate (red) in cultures maintained in EM (e) or DM (f) for 14 days. Only cultures maintained in DM incorporated the substrate (red). Nuclei were counter-stained with DRAQ5. Scale bar, 50 μm. g, Glycogen accumulation was determined by PAS staining in organoids grown in EM or DM for 10 days. Graph shows the percentage of cells weakly or strongly positive for PAS. Results are shown as mean ± SEM of 10 independent sections of 10 EM or 10 DM independent organoids. h, Albumin (Alb.) secretion was measured in the 24h supernatant of clonal cultures kept in EM, or DM for 8 days (DM-8d) or 13 days (DM-13d). Results are expressed as pg of albumin per cell. i, Cyp3a activity was measured in cultures kept in DM for 13 days. Results are expressed as RLU per ml per million cells. h–i, MEFs and Hepatocytes (Hep.) and HepG2 cells were used as negative and positive controls respectively. Triplicates for each condition were analyzed. Results are shown as mean ± SEM of 3 independent experiments. ***, p<0.0001.
Figure 4
Figure 4. Hepatocyte islands upon transplantation of clonal liver organoids into FAH−/−-mutant mice
Single-cell derived liver organoids from 3 independent clones were expanded under EM and differentiated for 9 days prior to transplantation into Fah−/−/Rag2−/−/Il2Rγ−/− (FRG) mice. Clone I (n=8), clone II (n=6), clone III (n=5). a, scheme showing the transplantation protocol. b–d, Representative positive graft within the liver parenchyma. b, FAH+ area (clone III). The grafted cells were negative for Krt19 (c, ductal marker) and positive for HNF4α (d, hepatocyte marker). Scale bars, 400μm (b) and 100μm (b′–d). e, Kaplan-Meier survival curve of transplanted mice with positive engraftment (brown curve, graft+, n=5), transplanted mice without evidence of engraftment (blue curve, graft-, n=9) and non-transplanted control mice (black curve, not transp., n=4). Plot displays the cumulative survival on a linear scale. Kaplan-Meier survival analysis compares overall survival rates between two groups. Log-rank test is used to compare differences in survival. *, log-rank=0.02 (graft+ vs graft-), log-rank= 0.007 (graft+ vs not transp.).
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References

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