Chronic airway diseases are a common medical condition worldwide and include diseases such as cystic fibrosis, asthma or chronic obstructive pulmonary disease (COPD). COPD is the third leading cause of death in the world, and there is still no curative treatment. Air liquid interface cultures (ALI) mimic the lung development and disease. We and others have recently generated a functional airway multiciliated epithelium in ALI conditions by using human pluripotent stem cells, called “iALI” (Ahmed et al., BioRxiv, 2020). During this differentiation process, a mesenchymal stromal component is consistently described but poorly studied.

To comprehensively define the cell types, mechanisms driving iALI model and understand the crosstalk between epithelial and mesenchymal components, we performed single-cell mRNA sequencing. RT-qPCR and immunofluorescence assays were used to confirm the sequencing results.

In addition to the epithelial cell subtypes, the iALI cultures also contain a previously poorly documented EPCAM-COL1A1+DCN+ mesenchymal stromal compartment. Several populations, that partially overlap can be identified : 1/ACTA2+ myofibroblasts including fibromyocytes characterized by high expression of contractile genes such as CNN1 and TAGLN, 2/a transition population closer to epithelial cells expressing MAF and the long non coding mRNA EPB41L4A-AS1 and 3/a highly proliferating PCNA+CDK1+ cells. Moreover, stromal cells expressed growth factors such as FGF10 while its receptor FGFR2 is expressed on the epithelial cells, suggesting that stromal cells could support iALI development.

These data provide high-resolution insights into the complexity and plasticity of the iALI and suggest that a crosstalk between epithelium and mesenchyme is essential to iALI development. The functional role of the relevant pathways will be assessed in our iALI model in control condition but also in a pathological context.