Endothelial cells synergize with fibroblasts to form a niche for engineering alveolar epithelium

We perform single-cell RNA sequencing of whole engineered lungs generated either by alveolar epithelial type 2 cell (AEC2)/fibroblast (FB) co-culture or by AEC2/FB/endothelial cell (EC) tri-culture, to investigate AEC2 and FB phenotype in the presence and absence of endothelial cells. Primary postnatal day 7 (P7) AEC2s, P7 lung FBs, and primary rat lung microvascular ECs (from 4-6 week-old rats) used for engineered cultures, as well as native P7 whole rat lung, were sequenced for comparison. We find that native-like AEC2 phenotype, alveolar structure, and cellular signaling patterns are better recapitulated via epithelial-mesenchymal-endothelial tri-culture rather than by a more simplified system. FBs cultured in the presence of endothelial cells demonstrate reduced expression of contractile markers and upregulate features of an AEC2 niche-supportive signature. Overall design: Single-cell RNA sequencing data of AEC2/fibroblast and AEC2/fibroblast/endothelial (tri-culture) d7 engineered lungs, with AEC2 and fibroblast isolates, primary ECs, as well as P7 native rat lung (pooled from 6 rats) for comparison