In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green
In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green
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DOI：
10.1634/stemcells.20-2-146
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发表时间：
2002-01-01
期刊：
影响因子：
5.2
通讯作者：
Tsunoda, Y
中科院分区：
文献类型：
--
作者：
Yamada, T;Yoshikawa, M;Tsunoda, Y
背景和目标。胚胎干细胞（ES细胞）具有在体外分化为多种细胞谱系的多能性。我们最近发现出现的细胞簇，显示细胞摄取的吲哚菁绿色（ICG）在分化的ES细胞培养。ICG在临床上用作评价肝功能的供试品，因为它仅由肝细胞消除。本研究的目的是探讨ICG染色细胞的肝脏特性。通过ES细胞的5天悬滴培养形成的胚状体（EBs）被允许在放置的培养物中生长。通过逆转录-聚合酶链反应分析肝细胞标志物的基因表达，并通过化学方法检测白蛋白的产生。形态学和细胞成分进行了研究，通过电子显微镜。将ICG染色的细胞进一步移植到小鼠门静脉中。ICG染色的细胞在EB培养的14天左右出现，并形成独特的三维结构。它们对白蛋白具有免疫反应性，并表达白蛋白、甲胎蛋白、甲状腺素运载蛋白、肝细胞核因子3 β、α-1-抗胰蛋白酶、色氨酸-2，3-双加氧酶、尿素循环酶、促凝血酶和肝特异性有机阴离子转运蛋白-1等mRNA。超微结构分析显示，一个发达的系统的细胞器，如线粒体，溶酶体，高尔基体，粗面内质网和光面内质网。将ICG阳性细胞移植到门静脉导致其并入小鼠肝脏，在那里它们与邻近的肝细胞在形态上无法区分。胚胎干细胞来源的ICG阳性细胞具有肝细胞的特征，ICG染色是体外鉴定胚胎干细胞分化为肝细胞的有用标记。
Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found the emergence of cell clusters that show the cellular uptake of indocyanine green (ICG) in the culture of differentiated ES cells. ICG is clinically used as a test substance to evaluate liver function because it is eliminated exclusively by hepatocytes. The aim of the present study was to investigate the hepatic characteristics of ICG-stained cells.Methods. Embryoid bodies (EBs), formed by a 5-day hanging drop culture of ES cells, were allowed to outgrow in the placed culture. Gene expression of hepatocyte markers was analyzed by reverse transcriptase-polymerase chain reaction, and albumin production was examined immunohistochemically. Morphology and cellular components were investigated by electron microscopy. ICG-stained cells were further transplanted into the portal vein of mice.Results. ICG-stained cells appeared around 14 days of the EB culture and formed distinct three-dimensional structures. They were inummoreactive to albumin and expressed mRNAs such as albumin, alpha-fetoprotein, transthyretin, hepatocyte nuclear factor 3 beta, alpha-1-antitrypsin, tryptophan-2,3-dioxygenase, urea cycle enzyme, gluconeogenic enzyme, and liver-specific organic anion transporter-1. An ultrastructural analysis revealed a well-developed system of organelles such as mitochondria, lysosomes, Golgi apparatus, and rough and smooth endoplasmic reticulum. The transplantation of ICG-positive cells into the portal vein resulted in the incorporation into mice livers, where they were morphologically indistinguishable from neighboring hepatocytes.Conclusions. ES cell-derived ICG-positive cells possess characteristics of hepatocytes, and ICG-staining is a useful marker to identify differentiated hepatocytes from EBs in vitro.