Yamada Takatsugu, Yoshikawa Masahide, Kanda Seiji, Kato Yoko, Nakajima Yoshiyuki, Ishizaka Shigeaki, Tsunoda Yukio
Division of Developmental Biology, Department of Parasitology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan.
Stem Cells. 2002;20(2):146-54. doi: 10.1634/stemcells.20-2-146.
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.
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.
ICG-stained cells appeared around 14 days of the EB culture and formed distinct three-dimensional structures. They were immunoreactive 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.
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.
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。我们最近发现在分化的ES细胞培养物中出现了摄取吲哚菁绿（ICG）的细胞簇。ICG在临床上用作评估肝功能的测试物质，因为它仅由肝细胞清除。本研究的目的是研究ICG染色细胞的肝脏特征。
通过ES细胞的5天悬滴培养形成的胚状体（EBs）在培养皿中生长。通过逆转录聚合酶链反应分析肝细胞标志物的基因表达，并通过免疫组织化学检查白蛋白的产生。通过电子显微镜研究形态和细胞成分。将ICG染色的细胞进一步移植到小鼠的门静脉中。
ICG染色的细胞在EB培养约14天时出现，并形成明显的三维结构。它们对白蛋白具有免疫反应性，并表达诸如白蛋白、甲胎蛋白、转甲状腺素蛋白、肝细胞核因子3β、α1抗胰蛋白酶、色氨酸-2,3-双加氧酶、尿素循环酶、糖异生酶和肝脏特异性有机阴离子转运蛋白-1等mRNA。超微结构分析显示线粒体、溶酶体、高尔基体以及粗面和滑面内质网等细胞器系统发育良好。将ICG阳性细胞移植到门静脉中导致其融入小鼠肝脏，在那里它们在形态上与相邻的肝细胞无法区分。
ES细胞来源的ICG阳性细胞具有肝细胞的特征，ICG染色是在体外从EB中鉴定分化肝细胞的有用标志物。
