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

被引:19
作者
Snijders, Kirsten E. [1 ]
Feher, Anita [2 ]
Tancos, Zsuzsanna [2 ]
Bock, Istvan [2 ]
Teglasi, Annamaria [2 ]
van den Berk, Linda [1 ]
Niemeijer, Marije [1 ]
Bouwman, Peter [1 ]
Le Devedec, Sylvia E. [1 ]
Mone, Martijn J. [1 ]
Van Rossom, Rob [4 ]
Kumar, Manoj [4 ]
Wilmes, Anja [5 ]
Jennings, Paul [5 ]
Verfaillie, Catherine M. [4 ]
Kobolak, Julianna [2 ]
ter Braak, Bas [1 ]
Dinnyes, Andras [2 ,3 ]
van de Water, Bob [1 ]
机构
[1] Leiden Univ, Leiden Acad Ctr Drug Res, Div Drug Discovery & Safety, Einsteinweg 55, NL-2333 CC Leiden, Netherlands
[2] BioTalentum Ltd, H-2100 Godollo, Hungary
[3] Hungarian Univ Agr & Life Sci, Inst Physiol & Anim Hlth, Dept Physiol & Anim Hlth, H-2100 Godollo, Hungary
[4] Katholieke Univ Leuven, Stem Cell Inst, Dept Dev & Regenerat, Leuven, Belgium
[5] Amsterdam Inst Mol Med & Syst, Div Mol & Computat Toxicol, Amsterdam, Netherlands
关键词
Oxidative stress; Reporter cells; Induced pluripotent stem cells; In vitro toxicology; Endogenous gene tagging; High content imaging; PROTEIN INSERTION; CARDIOMYOCYTES; TRANSITION; GENERATION; INDUCTION; TOXICITY; REPORTER; NEURONS;
D O I
10.1007/s00204-021-03127-8
中图分类号
R99 [毒物学(毒理学)];
学科分类号
100405 ;
摘要
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 mu M) 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.
引用
收藏
页码:3285 / 3302
页数:18
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