In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes
In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes
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DOI：
10.1016/j.yexcr.2005.05.028
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发表时间：
2005-09-10
影响因子：
3.7
通讯作者：
Ikai, I
中科院分区：
文献类型：
--
作者：
Ishii, T;Yasuchika, K;Ikai, I
体外诱导胚胎干（ES）细胞成熟为肝细胞是很困难的。我们之前报道过，来自小鼠胎肝的Thy1阳性间充质细胞可促进肝祖细胞的成熟。在这里，我们从小鼠 ES 细胞中分离出产生甲胎蛋白 (AFP) 的细胞，随后通过与 Thy1 阳性细胞共培养在体外分化为肝细胞。在 AFP 启动子控制下表达绿色荧光蛋白 (GFP) 的 ES 细胞在无血清和饲养层的培养条件下培养。到第 7 天，GFP 阳性细胞的比例稳定在 41.6 +/- 12.2%（平均值 +/- SD）。通过流式细胞术分离的 GFP 阳性细胞在存在或不存在 Thy1 阳性细胞作为饲养层的情况下进行培养。对分离的 GFP 阳性细胞进行 AFP、Foxa2 和白蛋白染色。仅在与 Thy1 阳性细胞共培养后才检测到编码酪氨酸氨基转移酶、色氨酸 2,3-双加氧酶和葡萄糖-6-磷酸酶的 mRNA 表达。与 Thy1 阳性细胞共培养后，分离的细胞产生并储存糖原。共培养后氨清除活性也得到增强。电镜分析表明共培养的细胞表现出成熟肝细胞的形态特征。总之，体外与 Thy1 阳性细胞共培养可诱导从 ES 细胞培养物中分离出的 AFP 产生细胞成熟为肝细胞。 (c) 2005 Elsevier Inc. 保留所有权利。
It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 +/- 12.2% (means +/- SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes. (c) 2005 Elsevier Inc. All rights reserved.