Tsukada Hiroaki, Takada Tatsuyuki, Shiomi Hisanori, Torii Ryuzo, Tani Tohru
Department of Surgery, Shiga University of Medical Science, Otsu-city, Shiga, Japan 520-2192.
In Vitro Cell Dev Biol Anim. 2006 Mar-Apr;42(3-4):83-8. doi: 10.1290/0506039.1.
Embryonic stem (ES) cells can replicate indefinitely and differentiate into all cell types, including hepatocytes. Research using primate ES cells is considered to be important for studies of potential cell therapies. Recently, we established cynomolgus monkey ES cells designated as CMK6. The CMK6 cell line is a useful tool for investigating the mechanism of differentiation in primate ES cells and developing cell therapies, because of its biological similarity to human ES cells. To examine whether cynomolgus monkey ES cells differentiate into hepatocytes, CMK6 cells were cultured with or without acidic fibroblast growth factor (aFGF). Evaluation of the hepatic differentiation was performed by analysis of the mRNA expression in early hepatic marker genes using the reverse transcriptase-polymerase chain reaction (RT-PCR). The protein expression of albumin (ALB) was also studied by immunocytochemistry. RT-PCR analyses revealed mRNA expressions of alpha-fetoprotein, transthyretin, and ALB in the presence of aFGF at 3 wk of differentiation, whereas no mRNA expression of these genes was detected in cells without aFGF. The protein expression of ALB in the presence of aFGF at 3 wk of differentiation was also confirmed by immunocytochemistry. However, tyrosine aminotransferase, which is a mature hepatic marker, was not detected in the presence or absence of aFGF at any stage of differentiation. These results suggested that aFGF successfully promoted in vitro differentiation of cynomolgus monkey ES cells to an early hepatic lineage.
胚胎干细胞（ES细胞）能够无限增殖并分化为包括肝细胞在内的所有细胞类型。使用灵长类ES细胞进行的研究被认为对潜在细胞疗法的研究具有重要意义。最近，我们建立了名为CMK6的食蟹猴ES细胞系。由于CMK6细胞系与人类ES细胞具有生物学相似性，因此它是研究灵长类ES细胞分化机制和开发细胞疗法的有用工具。为了检测食蟹猴ES细胞是否能分化为肝细胞，将CMK6细胞在有或没有酸性成纤维细胞生长因子（aFGF）的情况下进行培养。通过逆转录聚合酶链反应（RT-PCR）分析早期肝标志物基因中的mRNA表达来评估肝分化情况。还通过免疫细胞化学研究了白蛋白（ALB）的蛋白表达。RT-PCR分析显示，在分化3周时，存在aFGF的情况下，甲胎蛋白、甲状腺素转运蛋白和ALB的mRNA有表达，而在没有aFGF的细胞中未检测到这些基因的mRNA表达。免疫细胞化学也证实了在分化3周时存在aFGF的情况下ALB的蛋白表达。然而，在分化的任何阶段，无论有无aFGF，均未检测到作为成熟肝标志物的酪氨酸转氨酶。这些结果表明，aFGF成功促进了食蟹猴ES细胞在体外向早期肝谱系的分化。
