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

Abstract

The Wnt target gene Lgr5 marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon1, stomach2 and hair follicles3. A 3D culture system allows long-term clonal expansion of single Lgr5+ stem cells into transplantable organoids that retain many characteristics of the original epithelial architecture2, 4, 5. A crucial component of the culture medium is the Wnt agonist Rspo16, the recently discovered ligand of Lgr57, 8. Here we show that Lgr5-LacZ is not expressed in healthy adult liver, yet that small Lgr5-LacZ+ cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signaling. As shown by lineage tracing using a novel Lgr5-ires-CreERT2 knock-in allele, damage-induced Lgr5+ cells generate hepatocytes and bile ducts in vivo. Single Lgr5+ cells from damaged liver can be clonally expanded as organoids in Rspo1-based culture medium over multiple months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into FAH−/− mice. These findings imply that previous observations on Lgr5+ stem cells in actively self-renewing tissues extend to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

R-spondin1 is a novel hormone mediator for mammary stem cell self-renewal

“…Steered by the prominent role of Wnt signaling in stem cell maintenance, Sato et al (2009;Barker et al 2010) used Rspo1 to augment Wnt signaling and successfully expand Lgr5 + intestine stem cells in culture. 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).…”

“…Rspo1 has been implicated in multiple stem cell in vitro culture systems, such as intestine stem cells and liver stem cells (Sato et al 2009;Huch et al 2013), highlighting its critical role as a niche factor. However, it remains elusive which cells produce Rspo proteins in these organs.…”

“…2012). Rspo1 has been implicated in many adult stem cell in vitro expansion systems, such as the intestine, stomach, and liver (Kim et al 2005;Sato et al 2009;Barker et al 2010;Huch et al 2013). However, it remains unclear in vivo which cells produce Rspo proteins in these organs.…”

R-spondin 2 promotes proliferation and migration via the Wnt/β-catenin pathway in human hepatocellular carcinoma

“…A number of previous studies have indicated that RSPOs may potentiate Wnt signaling via stimulation of the leucine-rich repeat-containing G-protein coupled receptors (LGR) 4, LGR5 and LGR6 (16)(17)(18). Wnt proteins, comprising a large family of extracellular, lipid-modified glycoproteins, are crucial for embryonic development and cell proliferation, regulation, differentiation, survival and tissue homeostasis in adults (19,20).…”

Transforming growth factor‐β in liver cancer stem cells and regeneration

“…Currently, at least three stem cell populations are known to exist in both mouse27 and human adult liver28: 1) pericentral Axin2 + hepatocytes that can regenerate liver in normal homeostasis29; 2) periportal cells positive for Lgr530; 3) Prom1 + liver stem cells that are located within or adjacent to the Krt19 + bile duct epithelium 31. Human periportal cells also label for octamer 3/4 (Oct3/4), β2 spectrin (β2SP), and TBRII in both human and mouse liver regeneration 27, 28, 32.…”