An in vitro strategy using multiple human induced pluripotent stem cell-derived models to assess the toxicity of chemicals: A case study on paraquat

Nunes, Carolina; Singh, Pranika; Mazidi, Zahra; Murphy, Cormac D.; Bourguignon, Aurore; Wellens, Sara; Chandrasekaran, Vidya; Ghosh, Syamal K.; Zana, Melinda; Pamies, David; Thomas, Aurélien; Verfaillie, Cathérine; Culot, Maxime; Dinnyés, András; Hardy, Barry; Wilmes, Anja; Jennings, Paul; Grillari, Regina; Grillari, Johannes; Zurich, Marie-Gabrielle; Exner, Thomas E.

doi:10.1016/j.tiv.2022.105333

Cited by 14 publications

(11 citation statements)

References 52 publications

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“…BMT was calculated using BMDExpress software from the best fit-ted model; log2FoldChange was calculated in R using the weighted 5 − P logistic regression/progression model using transcriptomics. Other iPSC-derived systems for different cell types had been successfully used in TempO-Seq transcriptomic studies, including iPSCderived hepatocytes (ter Braak et al 2021;Ghosh et al 2021) and neurons (Dreser et al 2020), and recently, we have reported a study employing iPSCderived systems that were differentiated into kidney (podocyte-like and PTL cells), liver (hepatocyte-like cells), vasculature (endothelial-like cells), and brain (blood-brain-barrier-like cells, neuronal-like cells, and brain spheres) using TempO-Seq transcriptomics to predict mechanisms of toxicity in response to paraquat (Nunes et al, 2022). Paraquat is a well-described herbicide toxin that induces oxidative stress in the neurons, liver, and kidney (McCarthy et al 2004;Onur et al 2022) that was also picked up by iPSCderived models in that study (Nunes et al 2022).…”

Section: Sodium Arsenite Sodium Arsenite Tunicamycinmentioning

confidence: 99%

“…Other iPSC-derived systems for different cell types had been successfully used in TempO-Seq transcriptomic studies, including iPSCderived hepatocytes (ter Braak et al 2021;Ghosh et al 2021) and neurons (Dreser et al 2020), and recently, we have reported a study employing iPSCderived systems that were differentiated into kidney (podocyte-like and PTL cells), liver (hepatocyte-like cells), vasculature (endothelial-like cells), and brain (blood-brain-barrier-like cells, neuronal-like cells, and brain spheres) using TempO-Seq transcriptomics to predict mechanisms of toxicity in response to paraquat (Nunes et al, 2022). Paraquat is a well-described herbicide toxin that induces oxidative stress in the neurons, liver, and kidney (McCarthy et al 2004;Onur et al 2022) that was also picked up by iPSCderived models in that study (Nunes et al 2022). In addition to paraquat exposure, PTL cells had been previously reported to activate the Nrf2 oxidative stress response and metal stress response after treatment with the heavy metal cadmium chloride (Singh et al 2021) at concentrations that had been previously reported to activate these pathways in the human proximal tubular cell lines HK2 (Wilmes et al 2011) and RPTEC/TERT1 (Aschauer et al 2015).…”

Section: Sodium Arsenite Sodium Arsenite Tunicamycinmentioning

confidence: 99%

“…Therefore, they may also be useful for detecting individual and disease-specific responses after drug exposure. Several studies have demonstrated this, including studies in iPSC-derived cardiomyocytes and hepatocytes (Takayama et al 2014;Blau et al 2016;Grimm et al 2018;Nunes et al 2022). iPSC models also allow the possibility to test several tissues from the same donors.…”

Section: Introductionmentioning

confidence: 99%

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Capturing time-dependent activation of genes and stress-response pathways using transcriptomics in iPSC-derived renal proximal tubule cells

et al. 2022

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Transcriptomic analysis is a powerful method in the utilization of New Approach Methods (NAMs) for identifying mechanisms of toxicity and application to hazard characterization. With this regard, mapping toxicological events to time of exposure would be helpful to characterize early events. Here, we investigated time-dependent changes in gene expression levels in iPSC-derived renal proximal tubular-like cells (PTL) treated with five diverse compounds using TempO-Seq transcriptomics with the aims to evaluate the application of PTL for toxicity prediction and to report on temporal effects for the activation of cellular stress response pathways. PTL were treated with either 50 μM amiodarone, 10 μM sodium arsenate, 5 nM rotenone, or 300 nM tunicamycin over a temporal time course between 1 and 24 h. The TGFβ-type I receptor kinase inhibitor GW788388 (1 μM) was used as a negative control. Pathway analysis revealed the induction of key stress-response pathways, including Nrf2 oxidative stress response, unfolding protein response, and metal stress response. Early response genes per pathway were identified much earlier than 24 h and included HMOX1, ATF3, DDIT3, and several MT1 isotypes. GW788388 did not induce any genes within the stress response pathways above, but showed deregulation of genes involved in TGFβ inhibition, including downregulation of CYP24A1 and SERPINE1 and upregulation of WT1. This study highlights the application of iPSC-derived renal cells for prediction of cellular toxicity and sheds new light on the temporal and early effects of key genes that are involved in cellular stress response pathways.

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Section: Sodium Arsenite Sodium Arsenite Tunicamycinmentioning

confidence: 99%

Section: Sodium Arsenite Sodium Arsenite Tunicamycinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Capturing time-dependent activation of genes and stress-response pathways using transcriptomics in iPSC-derived renal proximal tubule cells

et al. 2022

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“…Additionally, specific cell markers/enzymes potentially relevant for TCE biotransformation were investigated across cell models from the mRNA raw counts. Differential expression analysis (pair-wise comparison, condition vs medium control) per cell type were acquired through DESeq2 Bioconductor package (Online Resources 2-Table S1) (Love et al 2014;Nunes et al 2022). For differently expressed genes (DEGs) cut-off, the following filters were applied: padj < 0.01, basemean > 35 and log2 fold change >| 0.58 |.…”

Section: Sample Quality Check and Statistical Analysis Of Tempo-seq Datamentioning

confidence: 99%

“…mpg. de) with a minimum overlap of 2 probes and p-value cut-off of 0.01, as previously described (Nguyen et al 2021;Nunes et al 2022). Over-representation analysis was performed for two conditions: low concentrations and high concentrations of 1,2-DCVC tested.…”

Section: Sample Quality Check and Statistical Analysis Of Tempo-seq Datamentioning

confidence: 99%

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models

Capinha

Zhang²,

Holzer³

et al. 2022

Arch Toxicol

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Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

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Differentiation and Subculturing of Renal Proximal Tubular‐like Cells Derived from Human iPSC

Meijer,

Naderlinger,

Jennings

et al. 2023

Current Protocols

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Recently, we have developed a protocol to differentiate human induced pluripotent stem cells (iPSC) into proximal tubular‐like cells (PTL) (Chandrasekaran et al., 2021). These cells express proximal tubular‐specific markers, including megalin, and form a polarized monolayer expressing tight junction proteins, including ZO‐3 and occludin. Furthermore, PTL display functional properties, including megalin‐facilitated endocytosis, P‐glycoprotein (ABCB1) efflux, and respond to parathyroid hormone. Here, we report step‐by‐step protocols to culture iPSC prior to differentiation (Basic Protocol 1), to differentiate PTL from iPSC (Basic Protocol 2), and to passage and freeze‐thaw PTL (Basic Protocol 3). Additionally, we provide a protocol (Basic Protocol 4) to culture PTL on microporous growth supports (transwells). Immunofluorescence stainings for characteristic markers, including megalin, are shown for unpassaged (Basic Protocol 2) and passaged (Basic Protocol 3) PTL. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: iPSC cultureBasic Protocol 2: iPSC‐derived PTL differentiationBasic Protocol 3: PTL passaging, culturing, and freezingBasic Protocol 4: PTL culturing on transwellsSupport Protocol 1: Preparation of Geltrex‐coated cell culture platesSupport Protocol 2: Preparation of RPTEC/TERT1 or fHDF/TERT166‐ECM‐coated cell culture platesSupport Protocol 3: Preparation of human collagen IV‐coated cell culture platesSupport Protocol 4: Immunofluorescence staining

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An in vitro strategy using multiple human induced pluripotent stem cell-derived models to assess the toxicity of chemicals: A case study on paraquat

Cited by 14 publications

References 52 publications

Capturing time-dependent activation of genes and stress-response pathways using transcriptomics in iPSC-derived renal proximal tubule cells

Capturing time-dependent activation of genes and stress-response pathways using transcriptomics in iPSC-derived renal proximal tubule cells

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models

Differentiation and Subculturing of Renal Proximal Tubular‐like Cells Derived from Human iPSC

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