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: 42.35). 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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Available from: Sylvia f boj, Feb 20, 2015
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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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    ABSTRACT: Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell 01/2015; 160(1-2):299-312. DOI:10.1016/j.cell.2014.11.050 · 33.12 Impact Factor
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    • "Subsequently, expansion of Lgr5 + adult stem cells of stomach and liver tissue has also been accomplished by using Rspo1 (Barker et al. 2010; Huch et al. 2013). Our previous study revealed that MaSCs can be expanded in vitro under serum-free and feeder-free defined conditions when supplemented with Wnt3A (Zeng and Nusse 2010). "
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    ABSTRACT: Signals from the niche play pivotal roles in regulating adult stem cell self-renewal. Previous studies indicated that the steroid hormones can expand mammary stem cells (MaSCs) in vivo. However, the facilitating local niche factors that directly contribute to the MaSC expansion remain unclear. Here we identify R-spondin1 (Rspo1) as a novel hormonal mediator in the mammary gland. Pregnancy and hormonal treatment up-regulate Rspo1 expression. Rspo1 cooperates with another hormonal mediator, Wnt4, to promote MaSC self-renewal through Wnt/β-catenin signaling. Knockdown of Rspo1 and Wnt4 simultaneously abolishes the stem cell reconstitution ability. In culture, hormonal treatment that stimulates the expression of both Rspo1 and Wnt4 can completely substitute for exogenous Wnt proteins, potently expand MaSCs, and maintain their full development potential in transplantation. Our data unveil the intriguing concept that hormones induce a collaborative local niche environment for stem cells.
    Genes & Development 09/2014; 28(20). DOI:10.1101/gad.245142.114 · 12.64 Impact Factor
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    • "Initiating CDE diet feeding or other injuries at this time—as done in the original Sox9, Opn, Hnf1b and GFAP studies, but not in the recent Sox9 study or our Ck19 study—may have resulted in marker gene activation in injured hepatocytes that unspecifically expressed the genes whose promoters were used to drive Cre. Our results also appear to be at odds with previous reports of isolation of cells from adult mouse liver that express BEC markers and can be expanded and differentiated into both hepatocytes and BECs in vitro (Dorrell et al., 2011; Huch et al., 2013; Shin et al., 2011). These differences suggest that removing liver cells from their in vivo environment may confer properties of plasticity not representative of in vivo biology. "
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    ABSTRACT: Hepatocytes provide most liver functions, but they can also proliferate and regenerate the liver after injury. However, under some liver injury conditions, particularly chronic liver injury where hepatocyte proliferation is impaired, liver stem cells (LSCs) are thought to replenish lost hepatocytes. Conflicting results have been reported about the identity of LSCs and their contribution to liver regeneration. To address this uncertainty, we followed candidate LSC populations by genetic fate tracing in adult mice with chronic liver injury due to a choline-deficient, ethionine-supplemented diet. In contrast to previous studies, we failed to detect hepatocytes derived from biliary epithelial cells or mesenchymal liver cells beyond a negligible frequency. In fact, we failed to detect hepatocytes that were not derived from pre-existing hepatocytes. In conclusion, our findings argue against LSCs, or other nonhepatocyte cell types, providing a backup system for hepatocyte regeneration in this common mouse model of chronic liver injury.
    Cell Reports 08/2014; 8(5). DOI:10.1016/j.celrep.2014.07.003 · 7.21 Impact Factor
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