In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
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Wnt靶基因Lgr 5标志着Wnt驱动的自我更新组织（如小肠和结肠、胃和毛囊）中活跃分裂的干细胞。3D培养系统允许单个Lgr 5+干细胞长期克隆扩增成可移植的类器官，这些类器官保留了原始上皮结构的许多特征。培养基的一个关键成分是Wnt激动剂Rspo 1，这是最近发现的Lgr 5的配体。在这里，我们发现Lgr 5-LacZ在健康成人肝脏中不表达，但小的Lgr 5-LacZ+细胞在损伤后出现在胆管附近，与Wnt信号的强烈激活相一致。如使用新的Lgr 5-ires-CreERT 2敲入等位基因的谱系追踪所示，损伤诱导的Lgr 5+细胞在体内产生肝细胞和胆管。来自受损肝脏的单个Lgr 5+细胞可以在基于Rspo 1的培养基中克隆扩增为类器官，持续数月。这种克隆类器官可以在体外诱导分化，并在移植到FAH−/−小鼠后产生功能性肝细胞。这些发现意味着先前对活跃自我更新组织中的Lgr 5+干细胞的观察延伸到具有低自发增殖率的组织中的损伤诱导的干细胞。
The Wnt target gene Lgr5 marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. 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 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, 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.