Impact of cell culture on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells

The alveolar epithelial type 2 cell (AEC2) is the facultative progenitor of lung alveoli tasked to maintain distal lung homeostasis. AEC2 dysfunction has been implicated in the pathogenesis of a number of pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), highlighting the importance of human in vitro models of the alveolar epithelium. However, AEC2-like cells captured in cell culture have yet to be directly compared to their in vivo counterparts at single cell resolution. Here, we apply single cell RNA sequencing to perform head-to-head comparisons between the global transcriptomes of freshly isolated primary (1{degrees}) adult human AEC2s, their isogenic cultured progeny, and human iPSC-derived AEC2s (iAEC2s) cultured in identical conditions. We find each population occupies a distinct transcriptomic space with both types of cultured AEC2s (1{degrees} and iAEC2s) exhibiting similarities to and differences from freshly purified 1{degrees} cells. Across each cell type, we find an inverse relationship between proliferative states and AEC2 maturation states, with uncultured 1{degrees} AEC2s being most quiescent and mature, their cultured progeny being more proliferative/less mature, and cultured iAEC2s being most proliferative/least mature. iAEC2s also express significantly lower levels of major histocompatibility complex (MHC) genes compared to 1{degrees} cells, suggesting immunological immaturity. Cultures of either type of human AEC2 (1{degrees} or iAEC2) do not generate detectable type 1 alveolar cells in these defined conditions; however, iAEC2s after co-culture with fibroblasts can give rise to a subset of cells expressing "transitional cell markers" recently described in fibrotic lung tissue of patients with pulmonary fibrosis or in mouse models of pulmonary fibrosis. Hence, we provide direct comparisons of the transcriptomic programs of 1{degrees} and engineered AEC2s, two in vitro model systems that can be harnessed for studies of human lung health and disease.