Kugler Josephine, Kemler Rolf, Luch Andreas, Oelgeschläger Michael
*Department of Chemical & Product Safety, German Federal Institute for Risk Assessment (BfR), Berlin, Germany
Emeritus Laboratory, Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
Toxicol Sci. 2016 Aug;152(2):382-94. doi: 10.1093/toxsci/kfw094. Epub 2016 May 20.
Embryonic stem cells (ESCs) are commonly used for the analysis of gene function in embryonic development and provide valuable models for human diseases. In recent years, ESCs have also become an attractive tool for toxicological testing, in particular for the identification of teratogenic compounds. We have recently described a Bmp-reporter ESC line as a new tool to identify teratogenic compounds and to characterize the molecular mechanisms mediating embryonic toxicity. Here we describe the use of a Wnt/β-Catenin-reporter ESC line isolated from a previously described mouse line that carries the LacZ reporter gene under the control of a β-Catenin responsive promoter. The reporter ESC line stably differentiates into cardiomyocytes within 12 days. The reporter was endogenously induced between day 3-5 of differentiation reminiscent of its expression in vivo, in which strong LacZ activity is detected around gastrulation. Subsequently its expression becomes restricted to mesodermal cells and cells undergoing an epithelial to mesenchymal transition. The Wnt/β-Catenin-dependent expression of the reporter protein allowed quantification of dose- and time-dependent effects of teratogenic chemicals. In particular, valproic acid reduced reporter activity on day 7 whereas retinoic acid induced reporter activity on day 5 at concentrations comparable to the ones inhibiting the formation of functional cardiomyocytes, the classical read-out of the embryonic stem cell test (EST). In addition, we were also able to show distinct effects of teratogenic chemicals on the Wnt/β-Catenin-reporter compared with the previously described Bmp-reporter ESCs. Thus, different reporter cell lines provide complementary tools for the identification and analysis of potentially teratogenic compounds.
胚胎干细胞(ESCs)常用于胚胎发育中基因功能的分析,并为人类疾病提供有价值的模型。近年来,ESCs也成为毒理学测试的一种有吸引力的工具,特别是用于鉴定致畸化合物。我们最近描述了一种Bmp报告基因ESC系,作为鉴定致畸化合物和表征介导胚胎毒性分子机制的新工具。在此,我们描述了一种Wnt/β-连环蛋白报告基因ESC系的应用,该系是从先前描述的小鼠品系中分离出来的,该品系在β-连环蛋白反应性启动子的控制下携带LacZ报告基因。该报告基因ESC系在12天内稳定分化为心肌细胞。在分化的第3至5天内源性诱导报告基因,这让人想起其在体内的表达,在体内,原肠胚形成期可检测到强烈的LacZ活性。随后,其表达局限于中胚层细胞和经历上皮-间充质转化的细胞。报告蛋白的Wnt/β-连环蛋白依赖性表达允许对致畸化学物质的剂量和时间依赖性效应进行定量。特别是,丙戊酸在第7天降低了报告基因活性,而视黄酸在第5天诱导了报告基因活性,其浓度与抑制功能性心肌细胞形成的浓度相当,功能性心肌细胞形成是胚胎干细胞试验(EST)的经典检测指标。此外,与先前描述的Bmp报告基因ESCs相比,我们还能够显示致畸化学物质对Wnt/β-连环蛋白报告基因的不同影响。因此,不同的报告细胞系为鉴定和分析潜在致畸化合物提供了互补工具。