Uchida Yoshiaki, Ferdousi Farhana, Zheng Yun-Wen, Oda Tatsuya, Isoda Hiroko
School of Integrative and Global Majors (SIGMA), University of Tsukuba, Tsukuba, Japan.
Alliance for Research on the Mediterranean and North Africa (ARENA), University of Tsukuba, Tsukuba, Japan.
Front Cell Dev Biol. 2020 Nov 5;8:578036. doi: 10.3389/fcell.2020.578036. eCollection 2020.
Human amnion epithelial cells (hAECs), derived from discarded term placenta, is anticipated as a new stem cell resource because of their advantages over embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), such as no risk of tumorigenicity and minimal ethical issue. hAECs have been reported to differentiate into hepatic-like cells (HLCs) with variable functionalities suitable for cell-based therapy of end-stage liver diseases, drug screening, and drug toxicity tests. On the other hand, a new research stream has been evolving to use natural compounds as stimulants of stem cell differentiation because of their high availability and minimum side effects. Isorhamnetin is a naturally occurring flavonoid commonly found in fruits and vegetables and has been reported to improve hepatic fibrosis and steatosis. In this present study, we have screened the differentiation potential of isorhamnetin in hAECs. The cells were grown on 3D cell culture and were treated with 20 μM of synthesized isorhamnetin for 10 days without adding any additional growth factors. DNA microarray global gene expression analysis was conducted for differentially expressed genes between isorhamnetin-treated and untreated control cells, gene expression validation was carried out using RT-qPCR method, and finally, several hepatic functions were assessed. Microarray analysis showed that isorhamnetin could activate essential biological processes, molecular functions, and signaling pathways for hepatic differentiation. Hepatic progenitor markers, and , were upregulated in the isorhamnetin-treated hAECs. was downregulated, while was upregulated on Day 10. Furthermore, isorhamnetin-treated cells could show increased CYP enzyme mRNA levels, ICG uptake and release, glycogen storage activity, and urea secretion. Additionally, isorhamnetin-treated cells did not show any trace of transdifferentiation evident by significant downregulation of several colon- and cholangiocyte-specific markers. However, longer treatment with isorhamnetin did not promote hepatic maturation. Altogether, our findings indicate that isorhamnetin has a promising effect on directing the hepatic-lineage specific differentiation in hAECs.
人羊膜上皮细胞（hAECs）源自废弃的足月胎盘，因其相对于胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）具有优势，如无致瘤风险且伦理问题最小，有望成为一种新的干细胞资源。据报道，hAECs可分化为具有多种功能的肝样细胞（HLCs），适用于终末期肝病的细胞治疗、药物筛选和药物毒性测试。另一方面，由于天然化合物具有高可用性和最小副作用，一种利用天然化合物作为干细胞分化刺激剂的新研究方向正在不断发展。异鼠李素是一种常见于水果和蔬菜中的天然黄酮类化合物，据报道它可改善肝纤维化和脂肪变性。在本研究中，我们筛选了异鼠李素在hAECs中的分化潜能。细胞在三维细胞培养中生长，并用20μM合成异鼠李素处理10天，不添加任何额外的生长因子。对异鼠李素处理组和未处理的对照组细胞之间的差异表达基因进行DNA微阵列全局基因表达分析，使用RT-qPCR方法进行基因表达验证，并最终评估几种肝功能。微阵列分析表明，异鼠李素可激活肝分化的关键生物学过程、分子功能和信号通路。在异鼠李素处理的hAECs中，肝祖细胞标志物 和 上调。 在第10天下调，而 上调。此外，异鼠李素处理的细胞可显示CYP酶mRNA水平升高、吲哚氰绿摄取和释放增加、糖原储存活性增加以及尿素分泌增加。此外，异鼠李素处理的细胞未显示出任何转分化迹象，这可通过几种结肠和胆管细胞特异性标志物的显著下调来证明。然而，用异鼠李素进行更长时间的处理并未促进肝成熟。总之，我们的研究结果表明，异鼠李素在指导hAECs向肝系特异性分化方面具有显著作用。
