Manipal Institute of Regenerative Medicine, Manipal University Branch Campus, Bangalore, India.
J Cell Physiol. 2011 Jun;226(6):1583-95. doi: 10.1002/jcp.22484.
In vitro models based on embryonic stem cells (ESC) are highly promising for improvement of predictive toxicology screening in humans. After the successful validation of embryonic stem cell test (EST) in 2001; concerns have been raised on the usage of mouse ESC and also the morphological evaluation of beating cell clusters. This requires specialized skill-sets and is highly prone to misjudgement and false positive results. To overcome these limitations, we undertook the present study incorporating improvisations over the conventional EST. Here, we explored the potential of a human ESC (hESC)-based assay to evaluate the potential toxicity of penicillin-G, caffeine, and hydroxyurea. Drug treatment inhibited hESC adhesion and substantially altered the morphology and viability (∼ 50%) of embryoid bodies (EBs). Flow cytometry analysis not only showed a significant increase of apoptotic cells in the highest doses but also induced a diverse pattern in DNA content and cell cycle distribution relative to control. Both semi-quantitative and quantitative RT-PCR studies revealed a selective down regulation of markers associated with stemness (Nanog, Rex1, SOX-2, and hTERT); cardiac mesoderm (Cripto1, MEF-2C, and Brachyury); hepatic endoderm (AFP, HNF-3β, HNF-4α, GATA-4, and SOX-17); and neuroectoderm (Nestin, SOX-1, NURR1, NEFH, Synaptophysin, TH, and Olig2) in a drug as well as dose dependent manner indicating abnormal differentiation. Furthermore, a decrease in the expression of AFP and GFAP proteins followed by a dose-dependent reduction in the levels of hCG-β, progesterone-II, and estradiol hormones was demonstrated by immunocytochemistry and ECLIA, respectively. This new and unique approach comprising of DNA cell cycle analysis, germ layer-specific marker expression and hormone levels as endpoints might offer a clinically relevant and commercially viable alternative for predicting in vivo developmental toxicity.
基于胚胎干细胞 (ESC) 的体外模型在提高人类预测性毒理学筛选方面具有很高的应用前景。2001 年，胚胎干细胞测试 (EST) 成功验证后；人们对使用小鼠 ESC 以及对搏动细胞簇的形态评估提出了担忧。这需要专门的技能，并且非常容易出现误判和假阳性结果。为了克服这些限制，我们在常规 EST 的基础上进行了改进，开展了本研究。在这里，我们探索了基于人 ESC (hESC) 的测定法在评估青霉素-G、咖啡因和羟基脲潜在毒性方面的潜力。药物处理抑制 hESC 黏附，并显著改变胚状体 (EB) 的形态和活力（约 50%）。流式细胞术分析不仅显示在最高剂量下凋亡细胞显著增加，而且与对照相比，还诱导了 DNA 含量和细胞周期分布的不同模式。半定量和定量 RT-PCR 研究均揭示了与干性（Nanog、Rex1、SOX-2 和 hTERT）；心脏中胚层（Cripto1、MEF-2C 和 Brachyury）；肝内胚层（AFP、HNF-3β、HNF-4α、GATA-4 和 SOX-17）；神经外胚层（Nestin、SOX-1、NURR1、NEFH、Synaptophysin、TH 和 Olig2）相关标志物的选择性下调，呈药物依赖性。此外，通过免疫细胞化学和 ECLIA 分别证明了 AFP 和 GFAP 蛋白表达减少以及 hCG-β、孕激素-II 和雌二醇激素水平呈剂量依赖性降低。这种新的、独特的方法包括 DNA 细胞周期分析、胚层特异性标志物表达和激素水平作为终点，可能为预测体内发育毒性提供一种具有临床相关性和商业可行性的替代方法。
