Mapping fetal myeloid differentiation in airway samples from premature neonates with single-cell profiling

Single-cell genomic technologies hold great potential to advance our understanding of development and disease. A major limitation lies in isolating intact cells from primary tissues for profiling. Sampling methods compatible with current clinical interventions could enable longitudinal studies, the enrollment of large cohorts, and even the development of novel diagnostics. To explore single-cell RNA-seq (scRNA-seq) profiling of the cell types present at birth in the airway lumen of extremely premature (<28 weeks gestation) neonates, we isolated cells from endotracheal aspirates collected from intubated neonates within the first hour after birth. We generated data on 10 subjects, providing a rich view of airway luminal biology at a critical developmental period. Our results show that cells present in the airways of premature neonates primarily represent a continuum of myeloid differentiation, including fetal monocytes (25% of all cells), intermediate myeloid populations (48% of cells), and macrophages (2.6% of cells). To our knowledge, this is the first single-cell transcriptomic characterization of human monocytes in the neonatal airway isolated within an hour of birth. Applying trajectory analysis to the premature neonate myeloid populations, we identified two trajectories consistent with the developmental stages of interstitial and alveolar macrophages, as well as a third trajectory presenting a potential alternative pathway bridging these terminal macrophage states. While the three trajectories share many dynamic genes (5,451), they also have distinct transcriptional changes (259 alveolar-specific genes, 666 interstitial-specific genes, and 285 bridging-specific genes). Overall, our results define high quality single-cell data from cells isolated within the so-called "golden hour of birth" in extremely premature neonate airways representing complex lung biology and can be utilized in studies of human development and disease.