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: 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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    • "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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    • "We were particularly interested to determine if R-spondin2 and Wnt3a, two Wnt pathway effectors, would support in vitro expansion and morphogenesis of gallbladder cells, similar to Lgr5+ liver stem cells (Huch et al., 2013). Given the importance of the Wnt-dependent pathway for stem cell expansion, we reasoned that clonogenic gallbladder cells could possibly be expanded under the previously defined organoid culture conditions (Huch et al., 2013; Barker et al., 2009; Sato et al., 2009). Every EpCAM+ CD44+CD13+ single sorted cell formed cysts that grew into large organoids that expressed CD44 and CD13. "
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    ABSTRACT: There are currently no reports of identification of stem cells in human gallbladder. The differences between human gallbladder and intrahepatic bile duct (IHBD) cells have also not been explored. The goals of this study were to evaluate if human fetal gallbladder contains a candidate stem cell population and if fetal gallbladder cells are distinct from fetal IHBD cells. We found that EpCAM+CD44+CD13+ cells represent the cell population most enriched for clonal self-renewal from primary gallbladder. Primary EpCAM+CD44+CD13+ cells gave rise to EpCAM+CD44+CD13+ and EpCAM+CD44+CD13- cells in vitro, and gallbladder cells expanded in vitro exhibited short-term engraftment in vivo. Last, we found that CD13, CD227, CD66, CD26 and CD49b were differentially expressed between gallbladder and IHBD cells cultured in vitro indicating clear phenotypic differences between the two cell populations. Microarray analyses of expanded cultures confirmed that both cell types have unique transcriptional profiles with predicted functional differences in lipid, carbohydrate, nucleic acid and drug metabolism. In conclusion, we have isolated a distinct clonogenic population of epithelial cells from primary human fetal gallbladder with stem cell characteristics and found it to be unique compared to IHBD cells. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
    Stem Cell Research 02/2015; 18(3). DOI:10.1016/j.scr.2014.12.003 · 3.91 Impact Factor
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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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