Article

In vitro expansion of single LGR5+ liver stem cells induced by wnt-driven regeneration

1] Hubrecht Institute for Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584CT Utrecht & University Medical Centre Utrecht, Netherlands [2].
Nature (Impact Factor: 41.46). 01/2013; 494(7436). DOI: 10.1038/nature11826
Source: PubMed

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. 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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    • "In the case of chronic liver injury, resident hepatic progenitor cells become active and start differentiating to replace the injured hepatocytes. The resident progenitors, sometimes called " oval cells " or " small hepatocytes, " emerge from the bile ducts or canals of Hering (Oertel & Shafritz, 2008;Turner et al., 2011;Best et al., 2015) and may differentiate into both hepatocytes and biliary cells (Suñer et al., 2012;Huch et al., 2013). However, the complex signaling pathways (Taub, 2004;Fausto, Campbell & Riehle, 2006;Michalopoulos, 2007;Böhm et al., 2010) that regulate the fate of differentiating cells in the regenerating liver are not completely clear and the relations between " oval cells, " bile ducts cells and hepatocytes are debated in the literature (Greenbaum, 2011;Michalopoulos, 2012). "
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    • "Extensive analysis of the genetic stability of cultured organoids in vitro demonstrates that the expanded cells preserve their genetic integrity over months in culture. These results agree with our previous observations in the mouse (Huch et al., 2013b) yet are in striking contrast to recent publications in which, utilizing several lineage tracing approaches, ductal/resident stem cells have been described as not contributing to mouse liver regeneration (Schaub et al., 2014;Yanger et al., 2014;Yanger et al., 2013). Our results resemble what has been elegantly shown in zebrafish and rat models: in the event of an almost complete hepatocyte loss or blockage of hepatocyte proliferation, biliary epithelial cells convert into hepatocytes (Choi et al., 2014) (Michalopoulos, 2014). "
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