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

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.

Bardoxolone Methyl Ameliorates Compression-Induced Oxidative Stress Damage of Nucleus Pulposus Cells and Intervertebral Disc Degeneration Ex Vivo

“…Oxidative stress can accelerate the process of IDD in many ways, including through apoptosis, ECM degeneration, senescence, and autophagy (Feng et al, 2017). Many reports have shown that BARD has a strong antioxidative effect (Khurana et al, 2020;Pang et al, 2021;Snijders et al, 2021). However, whether BARD can alleviate IVD degeneration caused by compression has not been studied.…”

Specific attenuation of purinergic signaling during bortezomib-induced peripheral neuropathy

“…We used the human iPSC lines mciPS (model no. SC301A-1; System Biosciences, Palo Alto, CA, USA), SBAD2 [75], Sigma iPSC0028 (Si28) (EPITHELIAL-1, #IPSC0028, Merck, Darmstadt, Germany) and the transgenic iPSC line Si28-NGN1. IPSC cultures were maintained under xeno-free conditions (see supplementary methods) [76].…”

Current Status and Challenges of Human Induced Pluripotent Stem Cell-Derived Liver Models in Drug Discovery

“…As hPSCs are highly proliferative, editing their genome is more readily feasible compared to primary human liver cells that have limited expansion capacity. Therefore, they are amenable to the creation of fluorescent stress pathway reporters [26][27][28][48][49][50][51]. These reporters can be generated by introducing an exogenous promoter and reporter gene in 'safe harbor' loci, such as the adeno-associated virus integration site 1 (AAVS1) locus [28,52], the citrate lyase beta-like gene locus (CLYBL) [53] or the ROSA26 locus [54] in iPSCs.…”

“…This allows monitoring of not only the presence of a specific protein but also the sub-cellular localization of the protein of interest. As an alternative for the linker sequence, an internal ribosomal entry site (IRES) or a 2A peptide sequence can be used, which will lead to the translation of two separate proteins [48,49,51,56]. These fluorescent reporter lines allow for the monitoring of cellular pathways, studying the mode of action (MOA) of drug toxicity or to investigate in vivo transplanted PSC-derived progeny [26].…”

“…Moreover, like for PHHs, hPSC-HLCs can also be used for the identification of drug-mediated steatosis, apoptosis and cholestasis [128]. Due to the relatively easy genome engineering of iPSC, mode of action (MOA) of drugs can be evaluated in real time using HLCs derived from PSCs wherein stress pathway reporter genes have been incorporated, which cannot be achieved using PHHs [51]. 2D hPSC-HLC culture systems exploiting patient-derived iPSC, including genetically corrected isogenic cells, can also be used to model genetic liver diseases and identify new drug candidates for diseases, such as familial hypercholesterolemia, glycogen storage disease, Wilson's disease, Alpers syndrome, α1AT deficiency, Crigler-Najjar Type 1, defective mitochondrial respiratory chain complex disorder and hereditary tyrosinemia [41,42,44,45,[128][129][130][131][132][133][134].…”