Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages

Kirsten E. Snijders, Anita Fehér, Zsuzsanna Táncos, István Bock, Annamária Téglási, Linda van den Berk, Marije Niemeijer, Peter Bouwman, Sylvia E. Le Dévédec, Martijn J. Moné, Rob Van Rossom, Manoj Kumar, Anja Wilmes, Paul Jennings, Catherine M. Verfaillie, Julianna Kobolák, Bas ter Braak, András Dinnyés, Bob van de Water*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

Original languageEnglish
JournalArchives of Toxicology
Early online date4 Sep 2021
DOIs
Publication statusE-pub ahead of print - 4 Sep 2021

Bibliographical note

Funding Information:
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 681002 (EU-ToxRisk).

Publisher Copyright:
© 2021, The Author(s).

Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.

Keywords

  • Endogenous gene tagging
  • High content imaging
  • In vitro toxicology
  • Induced pluripotent stem cells
  • Oxidative stress
  • Reporter cells

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