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  2. Establishment of drug screening in human embryonic stem cells based on a high-content screening system

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

  • J Pharmacol Toxicol Methods. 2020 Nov-Dec;106:106913. doi: 10.1016/j.vascn.2020.106913.
Yunfen Hua 1 Yongqin Wu 1 Xing Chun 2 Huarong Huang 2 Junyan Yan 3 Baowei Hu 3 Ming Zhang 4 Lifang Jin 5
Affiliations

Affiliations

  • 1 College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, China.
  • 2 College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036, China.
  • 3 School of Life Science, Shaoxing University, Shaoxing 312000, China.
  • 4 Hangzhou Precision Medicine Research Center, Hangzhou 310019, China.
  • 5 School of Life Science, Shaoxing University, Shaoxing 312000, China; Hangzhou Precision Medicine Research Center, Hangzhou 310019, China. Electronic address: lifangj@usx.edu.cn.
Abstract

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, respectively. The Oct-4-EGFP-H9 transgenic hESCs were cultured in mTeSR medium with different concentrations of 5-Fluorouracil (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.

Keywords

Colony area; Drug screening; High-content screening system; Human embryonic stem cells.

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