Department of Pharmacology, Faculty of Health Sciences, School of Medicine, University of Pretoria, Pretoria 0007, South Africa.
Wellcome Trust-Medical Research Council Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, University of Cambridge, Cambridge CB2 0SZ, UK.
Toxicol Sci. 2018 Jul 1;164(1):229-239. doi: 10.1093/toxsci/kfy084.
Experimental drugs need to be screened for safety within time constraints. Hepatotoxicity is one concerning contributor to the failure of investigational new drugs and a major rationale for postmarketing withdrawal decisions. Ethical considerations in preclinical research force the requirement for highly predictive in vitro assays using human tissue which retains functionality reflective of primary tissue. Here, the proteome of cells commonly used to assess preclinical hepatotoxicity was compared. Primary human hepatocytes (PHHs), hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells, HepG2 cell monolayers and HepG2 cell 3D spheroids were cultured and collected as whole cell lysates. Over 6000 proteins were identified and quantified in terms of relative abundance in replicate proteomic experiments using isobaric tagging methods. Comparison of these quantitative data provides biological insight into the feasibility of using HLCs, HepG2 monolayers, and HepG2 3D spheroids for hepatotoxicity testing. Collectively these data reveal how HLCs differentiated for 35 days and HepG2 cells proteomes differ from one another and that of PHHs. HepG2 cells possess a strong cancer cell signature and do not adequately express key metabolic proteins which mark the hepatic phenotype, this was not substantially altered by culturing as 3D spheroids. These data suggest that while no single hepatic model reflects the diverse array of outcomes required to mimic the in vivo liver functions, that HLCs are the most suitable investigational avenue for replacing PHHs in vitro.
实验药物需要在时间限制内进行安全性筛选。肝毒性是导致新药研究失败的一个令人担忧的因素，也是药物上市后撤回的主要原因之一。临床前研究中的伦理考虑因素迫使我们需要使用保留了与原组织相似功能的人类组织进行高度预测性的体外检测。在这里，我们比较了常用于评估临床前肝毒性的细胞的蛋白质组。原代人肝细胞（PHH）、人多能干细胞分化的肝细胞样细胞（HLC）、HepG2 细胞单层和 HepG2 细胞 3D 球体被培养并作为全细胞裂解物收集。使用等重标记方法在重复的蛋白质组学实验中鉴定和定量了超过 6000 种蛋白质。对这些定量数据的比较提供了关于使用 HLC、HepG2 单层和 HepG2 3D 球体进行肝毒性测试的可行性的生物学见解。这些数据共同表明，分化 35 天的 HLC 和 HepG2 细胞的蛋白质组彼此之间以及与 PHH 的蛋白质组不同。HepG2 细胞具有强烈的癌细胞特征，并且不能充分表达标记肝表型的关键代谢蛋白，而将其培养成 3D 球体并没有显著改变这一点。这些数据表明，虽然没有单一的肝模型能够反映出模拟体内肝脏功能所需的多样化结果，但 HLC 是替代 PHH 进行体外研究的最合适途径。
