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.

Direct transcriptomic comparison of xenobiotic metabolism and toxicity pathway induction of airway epithelium models at an air-liquid interface generated from induced pluripotent stem cells and primary bronchial epithelial cells.

The airway epithelium represents the main barrier between inhaled air and the tissues of the respiratory tract and is therefore an important point of contact with xenobiotic substances into the human body. Several studies have recently shown that in vitro models of the airway grown at an air-liquid interface (ALI) can be particularly useful to obtain mechanistic information about the toxicity of chemical compounds. However, such methods are not very amenable to high throughput since the primary cells cannot be expanded indefinitely in culture to obtain a sustainable number of cells. Induced pluripotent stem cells (iPSCs) have become a popular option in the recent years for modelling the airways of the lung, but despite progress in the field, such models have so far not been assessed for their ability to metabolise xenobiotic compounds and how they compare to the primary bronchial airway model (pBAE). Here, we report a comparative analysis by TempoSeq (oligo-directed sequencing) of an iPSC-derived airway model (iBAE) with a primary bronchial airway model (pBAE). The iBAE and pBAE were differentiated at an ALI and then evaluated in a 5-compound screen with exposure to a sub-lethal concentration of each compound for 24 h. We found that despite lower expression of xenobiotic metabolism genes, the iBAE similarly predicted the toxic pathways when compared to the pBAE model. Our results show that iPSC airway models at ALI show promise for inhalation toxicity assessments with further development.

SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells.

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs.

Co-expression of SARS-CoV-2 entry genes in the superficial adult human conjunctival, limbal and corneal epithelium suggests an additional route of entry via the ocular surface.

PURPOSE: The high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear. METHODS: Using single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface. RESULTS: The co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKß and IFNG as upstream regulators of the transcriptional profile of ACE2+TMPRSS2+ cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface. CONCLUSIONS: Together our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection.