Biology and Ontogeny of Human Small Airway Epithelium Club Cells Identified with Single Cell Transcriptome and Principle Component Gradient Analysis

Club (“Clara”) cells, dome shaped cells with disease cytoplasmic granules and microvilli, are the major secretory cell of the human small (>6 generation) airways. Little is known regarding the biology of club cells in the human airway, nor of the ontogeny of this cell type. Taking advantage of the SCGB1A1 as the marker for club cells and our ability to sample the normal small airway epithelium by bronchoscopy and brushing healthy volunteers, we defined the transcriptome of the normal human airway club cell using single cell transcriptome sequencing. Analysis of the human small airway epithelial single cell transcriptome together with in vitro validation provides novel insights into the molecular phenotype and biological functions of the human club cell population and identifies the basal cell as the human progenitor cells for club cells. Overall design: Three healthy nonsmokers, with no history of exposure to any tobacco products, were assessed using approved clinical protocols by Weill Cornell Medical College Institutional Review Board and the small airway epithelium (10th-12th generations of bronchi) were collected by bronchoscopic brushing. Single cell capture and RNA-sequencing was performed for 157 single-cell samples. To assess the ontogeny of human club cells using a computational strategy, semi-supervised, multivariate analysis was carried out using Principal Component Gradient Analysis (PCGA) performed with several pairs of cell-type marker genes including KRT5/SCGB1A1, DNAI1/SCGB1A1, and MUC5AC/SCGB1A1. To assess whether the pure population of human small airway epithelium KRT5+ BC could differentiate into SCGB1A1+ KRT5? club cells, the BC were cultured on air-liquid interface.