Isolation of hepatoblasts based on the expression of Dlk/Pref-1
Isolation of hepatoblasts based on the expression of Dlk/Pref-1
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DOI：
10.1242/jcs.00388
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发表时间：
2003-05-01
影响因子：
4
通讯作者：
Miyajima, A
中科院分区：
文献类型：
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作者：
Tanimizu, N;Nishikawa, M;Miyajima, A
成肝细胞是肝细胞和胆管上皮细胞的共同祖细胞，尽管它们的性质在很大程度上仍然未知。为了分离和表征成肝细胞，我们通过信号序列捕获方法寻找在小鼠胎肝细胞中表达的细胞表面抗原，发现Dlk，也称为Pref-1，在胎肝中强烈表达。免疫组化和北方分析表明Dlk在E10.5肝芽中高表达。这种强表达持续到E16.5期，此后显著下调。使用针对Dlk的单克隆抗体，我们通过荧光激活细胞分选仪或自动磁性细胞分选仪分离Dlk（+）细胞。从胎肝分离的Dlk（+）细胞表达白蛋白，并在低密度下与HGF和EGF培养5天时形成集落。来自E14.5 Dlk（+）细胞的集落中，超过60%同时含有白蛋白（+）和细胞角蛋白19（+）细胞，表明大多数集落形成Dlk（+）细胞能够分化为肝细胞和胆管上皮细胞谱系。电镜下观察到大部分培养细胞有大量的微绒毛，也表明Dlk（+）细胞在此条件下分化。此外，7%的集落形成Dlk（+）细胞不仅是双能的，而且是高度增殖的，在5天的培养期间形成包含超过100个细胞的大集落。通过将Dlk（+）细胞移植到脾脏中，在受体肝脏中发现供体来源的肝细胞，表明Dlk（+）细胞在体内分化为肝细胞。这些结果表明Dlk（+）细胞是成肝细胞，并且Dlk是从胎肝富集高度增殖的成肝细胞的有用标记。
Hepatoblasts are common progenitors for hepatocytes and biliary epithelial cells, although their nature remains largely unknown. In order to isolate and to characterize hepatoblasts, we searched for cell surface antigens expressed in mouse fetal hepatic cells by the signal sequence trap method and found that Dlk, also known as Pref-1, was strongly expressed in fetal liver. Immunohistochemical as well as northern analysis indicated that Dlk was highly expressed in the E10.5 liver bud. The strong expression continued until the E16.5 stage and was significantly downregulated thereafter. Using a monoclonal antibody against Dlk, we isolated Dlk(+) cells either by a fluorescence-activated cell sorter or by an automatic magnetic cell sorter. Dlk(+) cells isolated from fetal livers expressed albumin and formed colonies when cultured at low density with HGF and EGF for 5 days. Over 60% of colonies derived from E14.5 Dlk(+) cells contained both albumin(+) and cytokeratin 19(+) cells, indicating that a majority of colony-forming Dlk(+) cells are able to differentiate into both hepatocyte and biliary epithelial cell lineages. In addition, numerous microvilli were observed by electronmicroscopic analysis in most of those cultured cells, also indicating differentiation of Dlk(+) cells under this condition. Furthermore, 7% of the colony-forming Dlk(+) cells were not only bipotential but also highly proliferative, forming a large colony containing more than 100 cells during 5 days of culture. By transplantation of Dlk(+) cells into the spleen, donor-derived hepatocytes were found in the recipient liver, indicating that Dlk(+) cells differentiated into hepatocytes in vivo. These results indicate that Dlk(+) cells are hepatoblasts and that Dlk is a useful marker to enrich highly proliferative hepatoblasts from fetal liver.