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

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 Rspo, 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 findings on Lgr5+ stem cells in actively self-renewing tissues extend to damage-induced stem cells in a tissue with a low rate of spontaneous self-renewal. We first generated arrays from multiple wildtype tissues including muscle, white adipose tissues, brown adipose tissues, liver and pancreas. Then we generated arrays from liver derived cultures maintained in different conditions, and compared the expression profile with the corresponding parental tissues and other non-related tissues.Wnt靶基因Lgr5可标记Wnt驱动的自我更新组织中的活跃分裂干细胞，这类组织包括小肠、结肠、胃及毛囊。现有三维培养（3D culture）系统可实现单个Lgr5+干细胞的长期克隆扩增，使其形成可移植的类器官（organoids），该类器官保留了原上皮结构的诸多特征。培养基的关键组分为Wnt激动剂Rspo，其为新近发现的Lgr5配体。本研究发现，健康成年肝脏中无Lgr5-LacZ的表达，但当肝脏遭受损伤时，胆管附近会出现少量Lgr5-LacZ+细胞，这与Wnt信号通路的强烈激活相伴随。通过构建新型Lgr5-ires-CreERT2敲入（knock-in）等位基因开展谱系示踪（lineage tracing）实验，结果显示损伤诱导产生的Lgr5+细胞在体内可分化为肝细胞与胆管细胞。从损伤肝脏中分离的单个Lgr5+细胞，可在基于Rspo1的培养基中进行长达数月的克隆扩增，形成类器官。这类克隆类器官可在体外诱导分化，当移植到FAH-/-小鼠体内后，可产生具有功能的肝细胞。上述研究结果表明，此前关于活跃自我更新组织中Lgr5+干细胞的研究结论，可拓展至自发更新速率较低的组织中由损伤诱导产生的干细胞。本研究首先从多种野生型组织中制备基因表达芯片，包括肌肉、白色脂肪组织、棕色脂肪组织、肝脏与胰腺；随后从不同培养条件下维持的肝脏来源培养物中制备基因表达芯片，并将其表达谱与对应的亲本组织及其他无关组织进行对比。