Department of Parasitology, Nara Medical University, 840 Shijo-cho, Kashihara 634-8521, Japan.
J Biosci Bioeng. 2011 Jan;111(1):71-7. doi: 10.1016/j.jbiosc.2010.08.005.
We investigated the effects of coculture with hepatic stellate cells (HSCs) on the differentiation of embryonic stem (ES) cells and embryoid bodies (EBs). Rat HSCs were incubated until becoming semi-confluent and adherent to the dish. Undifferentiated mouse ES cells and 4-day EBs were cultured in gelatin-coated or HSC-feeder dishes, then induced hepatocyte-like cells and the remaining undifferentiated ES cells were examined using immunocytochemical and RT-PCR methods. HSCs promoted the differentiation of EBs into hepatocyte-like cells, whereas they inhibited the differentiation of undifferentiated ES cells. Among EB outgrowths cocultured with HSCs, albumin-immunopositive cells were clearly and abundantly observed, while they were faintly and scarcely seen in EB outgrowths without HSCs. mRNA expressions of the hepatocyte-related markers such as albumin, transthyretin, alpha-1-antitrypsin, tryptophan-2,3-dioxygenase, phosphoenolpyruvate carboxykinase, hepatocyte nuclear factor 4α and cytochrome P4507a1 were clearly detected in EB outgrowths cocultured with HSCs, while they were only weakly detected or undetected in spontaneous EB outgrowths without HSCs. In contrast to the promoted hepatic differentiation of EBs by HSCs, undifferentiated ES cells formed cellular colonies in HSC-feeder dishes that were similar to the colonies of undifferentiated ES cells kept in maintenance medium containing leukemia inhibitory factor. In addition, ES cell colonies were immunopositive for Oct-3/4, markers of an undifferentiated state, and there were few ALB-immunopositive cells in the colonies. Thus, HSCs have contrasting effects on EBs undergoing differentiation and undifferentiated ES cells, i.e., positive and negative modulation, respectively.
我们研究了与肝星状细胞（HSCs）共培养对胚胎干细胞（ES 细胞）和类胚体（EBs）分化的影响。将大鼠 HSCs 孵育至半融合并贴壁于培养皿。将未分化的小鼠 ES 细胞和 4 天的 EBs 培养在涂有明胶或 HSC 饲养细胞的培养皿中，然后使用免疫细胞化学和 RT-PCR 方法检测诱导的肝细胞样细胞和剩余的未分化 ES 细胞。HSCs 促进 EBs 分化为肝细胞样细胞，而抑制未分化的 ES 细胞分化。在与 HSCs 共培养的 EB 外植体中，白蛋白免疫阳性细胞清晰且丰富，而在没有 HSCs 的 EB 外植体中则微弱且稀少。在与 HSCs 共培养的 EB 外植体中，明显检测到与肝细胞相关的标志物如白蛋白、转甲状腺素蛋白、α-1-抗胰蛋白酶、色氨酸-2,3-双加氧酶、磷酸烯醇丙酮酸羧激酶、肝核因子 4α 和细胞色素 P4507a1 的 mRNA 表达，而在没有 HSCs 的自发 EB 外植体中则仅微弱检测到或未检测到。与 HSCs 促进 EBs 的肝分化相反，未分化的 ES 细胞在 HSC 饲养细胞中形成类似于保持在含有白血病抑制因子的维持培养基中的未分化 ES 细胞的细胞集落。此外，ES 细胞集落对 Oct-3/4 呈免疫阳性，这是未分化状态的标志物，并且在集落中很少有 ALB 免疫阳性细胞。因此，HSCs 对正在分化的 EBs 和未分化的 ES 细胞具有相反的作用，即分别为正向和负向调节。
