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

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.

Generation of functional human pancreatic organoids by transplants of embryonic stem cell derivatives in a 3D-printed tissue trapper.

Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards ß cells. Here, we devised a strategy to enhance maturation of PP by assembly of three-dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell derivatives including ES-derived pancreatic duodenal homeobox 1 (PDX1) + early PP, mesenchymal stem cells, and endothelial cells at an optimized cell ratio, on Matrigel. The PO was placed in a 3D-printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicated that, in contrast to corresponding early PP transplants, 3D PO developed more vascularization as indicated by greater area and number of vessels, a higher number of insulin-positive cells and improvement of human C-peptide secretions. Based on our findings, PO-derived ß cells could be considered a novel strategy to study human ß-cell development, novel therapeutics, and regenerative medicine for diabetes.

Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

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

Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health.

A Novel Insight into Endothelial and Cardiac Cells Phenotype in Systemic Sclerosis Using Patient-Derived Induced Pluripotent Stem Cell.

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage and multi organ fibrotic changes with unknown pathogenesis. Most SSc patients suffer from defective angiogenesis/vasculogenesis and cardiac conditions leading to high mortality rates. We aimed to investigate the cardiovascular phenotype of SSc by cardiogenic differentiation of SSc induced pluripotent stem cells (iPSC). MATERIALS AND METHODS: In this experimental study, we generated iPSC from two diffuse SSc patients, followed by successful differentiation into endothelial cells (ECs) and cardiomyocytes (CMs). RESULTS: SSc-derived EC (SSc-EC) expressed KDR, a nearly EC marker, similar to healthy control-EC (C1-EC). After sorting and culturing KDR+ cells, the resulting EC expressed CD31, a late endothelial marker, but vascular endothelial (VE)-cadherin expression markedly dropped resulting in a functional defect as reflected in tube formation failure of SSc-EC. Interestingly, upregulation of SNAI1 (snail family transcriptional repressor 1) was observed in SSc-EC which might underlie VE-cadherin downregulation. Furthermore, SSc-derived CM (SSc-CM) successfully expressed cardiacspecific markers including ion channels, resulting in normal physiological behavior and responsiveness to cardioactive drugs. CONCLUSION: This study provides an insight into impaired angiogenesis observed in SSc patients by evaluating in vitro cardiovascular differentiation of SSc iPSC.

Improving the biological function of decellularized heart valves through integration of protein tethering and three-dimensional cell seeding in a bioreactor.

Decellularized xenogeneic heart valves (DHVs) are promising products for valve replacement. However, the widespread clinical application of such products is limited due to the risk of immune reaction, progressive degeneration, inflammation, and calcification. Here, we have developed an optimized decellularization protocol for a xenogeneic heart valve. We improved the biological function of DHVs by protein tethering onto DHV and three-dimensional (3D) cell seeding in a bioreactor. Our results showed that heart valves treated with a Triton X-100 and sodium deoxycholate-based protocol were completely cell-free, with preserved biochemical and biomechanical properties. The immobilization of stromal derived factor-1α (SDF-1α) and basic fibroblast growth factor on DHV significantly improved recellularization with endothelial progenitor cells under the 3D culture condition in the bioreactor compared to static culture conditions. Cell phenotype analysis showed higher fibroblast-like cells and less myofibroblast-like cells in both protein-tethered DHVs. However, SDF-DHV significantly enhanced recellularization both in vitro and in vivo compared to basic fibroblast growth factor DHV and demonstrated less inflammatory cell infiltration. SDF-DHV had less calcification and platelet adhesion. Altogether, integration of SDF-1α immobilization and 3D cell seeding in a bioreactor might provide a novel, promising approach for production of functional heart valves.