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.

Induced pluripotent and CD34+ stem cell derived myeloid cells display differential responses to particle and dust mite exposure.

Myeloid cells form an essential component of initial responses to environmental hazards and toxic exposures. The ability to model these responses in vitro is central to efforts tasked with identifying hazardous materials and understanding mechanisms of injury and disease. Induced pluripotent stem cell (iPSC) derived cells have been suggested as alternatives to more established primary cell testing systems for these purposes. iPSC derived macrophage and dendritic like cells were compared to CD34+ haematopoietic stem cell derived populations using transcriptomic analysis. Using single cell sequencing-based characterisation of iPSC derived myeloid cells, we identified transitional, mature and M2 like macrophages as well as dendritic like antigen presenting cells and fibrocytes. Direct transcriptomic comparisons between iPSC and CD34+ cell derived populations revealed higher expression of myeloid differentiation genes such as MNDA, CSF1R and CSF2RB in CD34+ cells, while iPSC populations had higher fibroblastic and proliferative markers. Exposure of differentiated macrophage populations to nanoparticle alone or in combination with dust mite, resulted in differential gene expression on combination only, with responses markedly absent in iPSC compared to CD34+ derived cells. The lack of responsiveness in iPSC derived cells may be attributable to lower levels of dust mite component receptors CD14, TLR4, CLEC7A and CD36. In summary, iPSC derived myeloid cells display typical characteristics of immune cells but may lack a fully mature phenotype to adequately respond to environmental exposures.

Biodiesel exhaust particle airway toxicity and the role of polycyclic aromatic hydrocarbons.

Renewable alternatives to fossil diesel (FD) including fatty acid methyl ester (FAME) biodiesel have become more prevalent. However, toxicity of exhaust material from their combustion, relative to the fuels they are displacing has not been fully characterised. This study was carried out to examine particle toxicity within the lung epithelium and the role for polycyclic aromatic hydrocarbons (PAHs). Exhaust particles from a 20% (v/v) blend of FAME biodiesel had little impact on primary airway epithelial toxicity compared to FD derived particles but did result in an altered profile of PAHs, including an increase in particle bound carcinogenic B[a]P. Higher blends of biodiesel had significantly increased levels of more carcinogenic PAHs, which was associated with a higher level of stress response gene expression including CYP1A1, NQO1 and IL1B. Removal of semi-volatile material from particulates abolished effects on airway cells. Particle size difference and toxic metals were discounted as causative for biological effects. Finally, combustion of a single component fuel (Methyl decanoate) containing the methyl ester molecular structure found in FAME mixtures, also produced more carcinogenic PAHs at the higher fuel blend levels. These results indicate the use of FAME biodiesel at higher blends may be associated with an increased particle associated carcinogenic and toxicity risk.

The relationship between epigenetic age and the hallmarks of aging in human cells.

Epigenetic clocks are mathematically derived age estimators that are based on combinations of methylation values that change with age at specific CpGs in the genome. These clocks are widely used to measure the age of tissues and cells1,2. The discrepancy between epigenetic age (EpiAge), as estimated by these clocks, and chronological age is referred to as EpiAge acceleration. Epidemiological studies have linked EpiAge acceleration to a wide variety of pathologies, health states, lifestyle, mental state and environmental factors2, indicating that epigenetic clocks tap into critical biological processes that are involved in aging. Despite the importance of this inference, the mechanisms underpinning these clocks remained largely uncharacterized and unelucidated. Here, using primary human cells, we set out to investigate whether epigenetic aging is the manifestation of one or more of the aging hallmarks previously identified3. We show that although epigenetic aging is distinct from cellular senescence, telomere attrition and genomic instability, it is associated with nutrient sensing, mitochondrial activity and stem cell composition.

Small Airway Susceptibility to Chemical and Particle Injury.

Small airways (SA) in humans are commonly defined as those conducting airways <2 mm in diameter. They are susceptible to particle- and chemical-induced injury and play a major role in the development of airway disease such as COPD and asthma. Susceptibility to injury can be attributed in part to structural features including airflow dynamics and tissue architecture, but recent evidence may indicate a more prominent role for cellular composition in directing toxicological responses. Animal studies support the hypothesis that inherent cellular differences across the tracheobronchial tree, including metabolic CYP450 expression in the distal conducting airways, can influence SA susceptibility to injury. Currently, there is insufficient information in humans to make similar conclusions, prompting further necessary work in this area. An understanding of why the SA are more susceptible to certain chemical and particle exposures than other airway regions is fundamental to our ability to identify hazardous materials, their properties, and accompanying exposure scenarios that compromise lung function. It is also important for the ability to develop appropriate models for toxicity testing. Moreover, it is central to our understanding of SA disease aetiology and how interventional strategies for treatment may be developed. In this review, we will document the structural and cellular airway regional differences that are likely to influence airway susceptibility to injury, including the role of secretory club cells. We will also describe recent advances in single-cell sequencing of human airways, which have provided unprecedented details of cell phenotype, likely to impact airway chemical and particle injury.

CD34+ derived macrophage and dendritic cells display differential responses to paraquat.

Paraquat (PQ) is a redox cycling herbicide known for its acute toxicity in humans. Airway parenchymal cells have been identified as primary sites for PQ accumulation, tissue inflammation and cellular injury. However, the role of immune cells in PQ induced tissue injury is largely unknown. To explore this further, primary cultures of human CD34+ stem cell derived macrophages (MCcd34) and dendritic cells (DCcd34) were established and characterised using RNA-Seq profiling. The impact of PQ on DCcd34 and MCcd34 cytotoxicity revealed increased effect within DCcd34 cultures. PQ toxicity mechanisms were examined using sub-cytotoxic concentrations and TempO-seq transcriptomic assays. Comparable increases for several stress response pathway (NFE2L2, NF-kB and HSF) dependent genes were observed across both cell types. Interestingly, PQ induced unfolded protein response (UPR), p53, Irf and DC maturation genes in DCcd34 but not in MCcd34. Further exploration of the immune modifying potential of PQ was performed using the common allergen house dust mite (HD). Co-treatment of PQ and HD resulted in enhanced inflammatory responses within MCcd34 but not DCcd34. These results demonstrate immune cell type differential responses to PQ, that may underlie aspects of acute toxicity and susceptibility to inflammatory disease.