RationaleThe lung mesenchyme gives rise to multiple distinct lineages of cells in the mature respiratory system, including smooth muscle cells (SMCs) of the airway and vasculature, vascular endothelial cells and parenchymal fibroblasts. However, a thorough understanding of the specification and inter‐relationship among mesenchymally derived cell diversity in the human lung is lacking.MethodsWe completed single cell RNA sequencing analysis of human pseudoglandular and early canalicular stage human lung cells. Canonical correlation analysis, clustering, cluster marker gene identification and tSNE representation was performed in Seurat. Cell populations were annotated using Toppfun. Immunohistochemistry (IHC) and in situ hybridization (ISH) of human lung tissues from multiple developmental stages validated spatial gene expression patterns for key marker genes.Main ResultsWe identified molecularly distinct populations representing “committed” fetal human lung endothelial cells, pericytes, and smooth muscle cells (SMCs). Early endothelial lineages expressed “classic” endothelial cell markers (PECAM, CDH5, VWF) while pericytes expressed PDGFRB, THY1 and basement membrane molecules (COL4, laminin, proteoglycans). We also observed a large population of “nonspecific” human lung mesenchymal progenitor (HLMP) cells characterized by expression of COL1, PDGFRA and multiple elastin fiber genes (ELN, MFAP4, FBLN1). We further characterized diversity of mesenchymal lineages defined by ACTA2 expression. Two mesenchymal cell populations, with the highest levels of ACTA2, expressed unique sets of markers associated with airway‐ or vascular‐associated SMCs. ISH for unique SMC cluster marker genes confirmed early and persistent spatial specification of airway (HHIP, MYLK, IGF1) and vascular (NTRK3, MEF2C) SMCs in the developing human lung.ConclusionThese data add significantly to our understanding of transcriptional phenotypes of human lung cells and provide the first ever description of molecular heterogeneity at the single cell level in human fetal lung tissue. From these data, we can identify a rich and heterogeneous set of mesenchymal cell populations, and define distinct molecular profiles for fetal human lung endothelial cells, fibroblasts, pericytes and smooth muscle cells. These data also facilitated the identification of markers for molecularly distinct populations of airway‐ and vascular smooth muscle cells, which were spatially validated across fetal development.Support or Funding InformationThis work is funded by NIH/NHLBI R01HL (to DA), and the Hastings center for pulmonary research (to SD).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.