A High-Throughput Screen for Teratogens Using Human Pluripotent Stem Cells
A High-Throughput Screen for Teratogens Using Human Pluripotent Stem Cells
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DOI：
10.1093/toxsci/kft239
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发表时间：
2014-01-01
影响因子：
3.8
通讯作者：
Chiao, Eric
中科院分区：
文献类型：
--
作者：
Kameoka, Sei;Babiarz, Joshua;Chiao, Eric
在制药和化学工业中，需要高通量的基于人体细胞的分析来识别危险化学品，从而减少对预测人类毒性反应风险的动物研究的总体依赖。尽管有人类特有的致畸物质，如沙利度胺，但人类细胞致畸试验的使用才刚刚开始探索。在此，描述了一种用于鉴定致畸物质的人类多能干细胞测试(HPST)，将体外结果与传统的临床前毒理学致畸研究进行比较，并在可用于人类致畸结果的情况下进行比较。HPST方法采用3天单层定向分化人类胚胎干细胞。化合物的致畸风险是通过测量转录因子SOX17在中内胚层细胞中核转位的减少来衡量的。在hPST模型中，核SOX17的降低与体内的致畸作用密切相关。具体地说，在hPST中检查了71种已知的体内效应的类药物化合物，包括沙利度胺。以5m为阈值，诊断准确率为94%(敏感度97%，特异度92%)。此外，还检测了15种具有不同于小分子药物的物理化学性质的环境毒物，并观察到与体内研究的致畸结果具有同样强烈的一致性。最后，为了评估hPST在高通量筛选中的适用性，测试了一个包含300种激酶抑制剂的小文库，展示了hPST平台在询问致畸机制和药物安全性预测方面的实用价值。因此，hPST分析是一个强有力的致畸性预测指标，似乎是对现有体外模型的改进。
There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human pluripotent stem cell test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically, 71 drug-like compounds with known in vivo effects, including thalidomide, were examined in the hPST. A threshold of 5M demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore, 15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally, to assess the suitability of the hPST for high-throughput screens, a small library of 300 kinase inhibitors was tested, demonstrating the hPST platforms utility for interrogating teratogenic mechanisms and drug safety prediction. Thus, the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.