文献类型：期刊文献

英文题名：Establishment of drug screening in human embryonic stem cells based on a high-content screening system

作者：Hua, Yunfen[1];Wu, Yongqin[1];Chun, Xing[2];Huang, Huarong[2];Yan, Junyan[3];Hu, Baowei[3];Zhang, Ming[4];Jin, Lifang[3,4]

机构：[1]Zhejiang Univ Technol, Coll Pharmaceut Sci, Hangzhou 310014, Peoples R China;[2]Hangzhou Normal Univ, Coll Life & Environm Sci, Hangzhou 310036, Peoples R China;[3]Shaoxing Univ, Sch Life Sci, Shaoxing 312000, Peoples R China;[4]Hangzhou Precis Med Res Ctr, Hangzhou 310019, Peoples R China

年份：2020

卷号：106

外文期刊名：JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS

收录：SCI-EXPANDED(收录号：WOS:000593745700008)、、Scopus(收录号：2-s2.0-85090593680)、WOS

基金：This work was supported by the Zhejiang Province New Talent Program (grant number: 2019R426059).

语种：英文

外文关键词：High-content screening system; Human embryonic stem cells; Colony area; Drug screening

外文摘要：High-content screening (HCS) systems can be used for high-throughput screening of drugs in human embryonic stem cells (hESCs). However, hESCs require immunofluorescence staining with stemness markers (e.g., Oct-4) prior to HCS, which can be time consuming and labor intensive. In this study, we employed transgenic hESCs with enhanced green fluorescent protein driven by stemness gene Oct-4 promoter (Oct-4-EGFP-H9), in which the colony area and relative green fluorescence area inferred a state of hESC proliferation and stemness, respec-tively. The Oct-4-EGFP-H9 transgenic hESCs were cultured in mTeSR medium with different concentrations of 5Fluorouracil (5-FU), vitamin C (VC), or retinoic acid (RA) for 5-7 days, followed by repeated imaging using the HCS system. Finally, the hESC colony area and green fluorescence area were calculated. Results showed that 5 FU treatment markedly reduced colony area in a dose-dependent manner, whereas VC and RA treatments did not. MTT assay and flow cytometry indicated that 5-FU inhibited the proliferation of hESCs significantly, verifying reliability of the data from the HCS system based on colony area analysis. The green fluorescence to total colony area ratio decreased with RA treatment, suggesting that RA significantly promoted differentiation, whereas 5-FU and VC had almost no effect, as verified by quantitative real-time polymerase chain reaction and western blot analysis. In conclusion, our study established a rapid and efficient drug screening system without the requirement of staining based on HCS.