Cellular and Molecular Biology of the Hepatic Stem Cell

在此，我们建立了一个从小鼠胚胎干细胞中分化、扩增和分离肝祖细胞的有效系统，并评估了其在损伤肝脏中的再生能力。利用转染了白蛋白（ALB）增强子/启动子调控的绿色荧光蛋白（GFP）报告基因的小鼠胚胎干细胞，我们发现无血清化学定义的培养基在缺乏外源生长因子或饲养细胞层的情况下支持胚状体（EBs）的形成和肝系细胞的分化。EB培养7天后，第一批表达ALB的GFP+细胞在“跳动”肌细胞附近被检测到。GFP+细胞数量增加，获得肝细胞样形态和肝细胞特异性标记物（即ALB， AAT， TO和G6P），并且在28天时占EB分离细胞的30%以上。在移植到MUP-uPA/SCID小鼠体内后，facs纯化的GFP+细胞发育成无细胞融合的功能性肝细胞，并参与了病变肝脏的修复。在ccl4诱导的肝损伤的刺激下，ES细胞来源的肝细胞对正常的生长调节有反应，并以与宿主肝细胞相同的速度增殖。移植的GFP+细胞也分化为胆道上皮细胞。综上所述，胚胎干细胞可以在体外产生高度富集的肝细胞前体，从而实现有效的细胞替代治疗。我们研究了c-Met信号在肝干细胞活化中的作用。为此，我们建立了含有卟啉和胆碱抑制剂3,5 -二乙基-1,4-二氢碰撞碱（DDC）的小鼠慢性肝损伤模型。先前，我们和其他研究人员证明，肝干细胞区室的激活对于ddc治疗小鼠的肝脏再生至关重要。ddc处理的MetLivKO小鼠没有表现出肝脏质量代偿性增加，这是由于肝细胞增殖和凋亡之间的不平衡。血清谷丙转氨酶、谷丙转氨酶、直接胆红素和胆汁酸水平升高证实了c- met缺乏症肝细胞的广泛损伤和功能受损。在DDC开始后6-8周，大多数MetLivKO小鼠出现严重的胆汁淤积并死于肝功能衰竭，而所有Cre-Ctl小鼠均存活。在这项工作中最引人注目的观察是在MetLivKO小鼠中几乎完全没有卵圆细胞增殖。相反，缺乏c- met的肝脏表现为典型的胆管增生，其局限于门静脉束的范围。相比之下，ddc处理的Cre-Ctl小鼠形成了一个广泛的导管卵形细胞网络，这些卵形细胞迁移到门脉周围实质，这是使用卵形细胞特异性抗体A6的共聚焦显微镜所证明的。只有在Cre-Ctl小鼠的肝脏中，才发现表达A6抗原的新形成的小肝细胞灶靠近卵圆细胞。这些结果表明，肝细胞中c-Met功能的丧失不仅会损害肝细胞的肝脏再生，还会阻碍干细胞介导的肝脏再生。

Here we established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we show that serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e. ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated with the same rate as host hepatocytes after an additional growth stimulus by CCl4-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy. We studied the involvement of c-Met signaling in the activation of liver stem cells. For this purpose we used a model of chronic liver injury induced in mice by diet containing the porphyrinogenic and cholestatic agent, 3, 5-diethocarbonyl-1,4-dihydrocollidine (DDC). Previously we and other investigators demonstrated that activation of the liver stem-cell compartment is essential for liver regeneration in DDC-treated mice. DDC-treated MetLivKO mice do not show a compensatory increase in liver mass is due to imbalance between hepatocyte proliferation and apoptosis. Extensive damage to and impaired functional capacity of c-Met-deficient hepatocytes are corroborated by elevated serum levels of AST, ALT, direct bilirubin, and bile acids. By 6-8 weeks after beginning DDC, most of the MetLivKO mice develop severe cholestasis and die from liver failure, whereas all Cre-Ctl mice survive this treatment. The most striking observation in this work is almost complete absence of oval cell proliferation in MetLivKO mice. Instead, c-Met-deficient livers show typical bile duct proliferation which is confined to the limits of portal tracts. In contrast, DDC-treated Cre-Ctl mice develop an extensive network of ductular oval cells which migrate into the periportal parenchyma, as is demonstrated by confocal microscopy using the oval cell-specific antibody, A6. Foci of small, newly formed hepatocytes that express A6 antigen are found in close proximity to oval cells only in livers of Cre-Ctl mice. These results show that loss of c-Met function in hepatocytes not only impairs regeneration of the liver from hepatocytes, but also blocks stem cell-mediated liver regeneration.