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.
Highlights
Generated pharmaceuticals and chemicals need to be assessed for their potential toxic effects in humans
We report the endogenous tagging of Heme oxygenase-1 (HMOX1) in human induced pluripotent stem cell (hiPSC) by CRISPR/Cas9 genome editing and present the complete characterization and functional validation of the generated cell line, which can be coupled with cell lineagespecific differentiations in combination with high content imaging (HCI) platforms to serve as a precious multi-organ oxidative stress reporter test system in toxicology studies
For imaging and compound exposures, hiPSCs were dissociated into single cells using 1X TrypLE Select, 62,500 cells/cm2 were seeded into Matrigel-coated 96-well microplates (Greiner Bio-One) and the culture medium was supplemented with 1X RevitaCell for 24 h. 48 h post-seeding hiPSCs were ready for exposure. hiPSCs underwent routine mycoplasma screening and karyotyping
Summary
Generated pharmaceuticals and chemicals need to be assessed for their potential toxic effects in humans. Archives of Toxicology (2021) 95:3285–3302 developed as alternatives of animal models for the prediction of chemical-induced toxicities (Scrivens and Bhogal 2007), including systems based on transformed cell lines and primary cell cultures (Collet et al 2019; Michael 2017; Sonneveld et al 2005). IPSCs are characterized by their capacity of long-term self-renewal and differentiation potential to all lineages, representing an unlimited source of organ-specific cells (Takahashi et al 2007; Yu et al 2007). They provide a unique advantage for testing organ-specific sensitivities from cells carrying the same genetic background, offering great potential to refine organspecific toxicology models
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