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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    • "Such changes may complicate their use for regenerative medicine purposes (Bayart and Cohen-Haguenauer, 2013). We have recently described a culture system that allows the long-term expansion (>1 year) of single mouse adult intestine (Sato et al., 2009), stomach (Barker et al., 2010), liver (Huch et al., 2013b), and pancreas (Huch et al., 2013a) stem cells. Lgr5, the receptor for the Wnt agonists R-spondins (Carmon et al., 2011; de Lau et al., 2011), marks adult stem cells in these mouse tissues (Barker et al., 2007, 2010; Huch et al., 2013a, 2013b). "
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    • "Cell transplantation has been proposed as an alternative to liver transplantation, but the ideal donor cell remains to be identified. Liver stem cells, cultured ductal cells, and their derivatives were proposed (Huch et al., 2013, 2015; Miyajima et al., 2014). Fetal liver progenitor cells also hold promise for transplantation therapy, but concerns were raised about their safety and methods of isolation (Kisseleva et al., 2010). "
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    • "Mouse pancreatic and hepatic organoids were initiated, cultured and cryopreserved as described previously (Huch et al., 2013b) with minor modifications. Gastrin was omitted from the culture media and constitutive administration of ALK5 inhibitor SB431542 (Tocris) was added. "
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