The human small airways have a unique position, morphology and cellular composition within the lung resulting in regional susceptibility to toxicant induced injury. Current in vitro airway models generally lack regional organotypic differentiation. Therefore, this work aimed to optimize in vitro models relevant for small airway toxicity testing and to determine the cellular specificity of small airway toxicants with the overall aim to improve in vitro testing capabilities.

Primary small airway epithelial cell (SAEC) models were optimized for regional specific differentiation. DCI differentiated primary SAEC showed higher levels of club cell markers compared to basal and PneumaCultTM-ALI differentiated SAEC and were thus identified as most applicable to small airway regional specific toxicity testing. iPSC derived SAEC models did not show sufficient expression of mature airway markers within this thesis and were not further explored. Macrophages and dendritic cells play an important role in small airway responses to toxicants. Therefore, CD34+ and iPSC derived macrophage and dendritic cell models were established and thoroughly characterized using single cell sequencing. Although these models likely represent inflammatory phenotypes, they are relevant for airway toxicity testing.

Chemical exposures were used to further characterize the in vitro models and identify cellular specificity of exposures. Paraquat (PQ) exposure showed cellular specific effects, as demonstrated by specific gene activation in differentiated primary SAEC and CD34+ derived dendritic cells. Both Basal SAEC and iPSC derived macrophages and dendritic cells did not show a profound effect upon PQ exposure, indicating adequate differentiation is required to detect PQ effects and further efforts are needed for the iPSC models developed within this thesis to be applicable to toxicity testing.

Overall, this demonstrates the importance and feasibility of organotypic differentiation to establish in vitro models that are applicable to in vitro small airway toxicity testing.