Single-Cell Profiling of Premature Neonate Airways Reveals a Continuum of Myeloid Differentiation

Single-cell genomic technologies hold great potential to advance our understanding of lung development and disease. A major limitation lies in accessing intact cells from primary lung tissues for profiling human airway health. Sampling methods such as endotracheal aspiration that are compatible with clinical interventions could enable longitudinal studies, the enrollment of large cohorts, and the development of novel diagnostics. To explore single-cell RNA sequencing profiling of the cell types present at birth in the airway lumen of extremely premature neonates (<28 wk gestation), we isolated cells from endotracheal aspirates collected from intubated neonates within the first 1 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 total), intermediate myeloid populations (48%), and macrophages (2.6%). Applying trajectory analysis to the myeloid populations, we identified two trajectories consistent with the developmental stages of interstitial and alveolar macrophages, as well as a third trajectory presenting an alternative pathway bridging the distinct macrophage precursors. The three trajectories share many dynamic genes (N= 5,451), but also have distinct transcriptional changes (259 alveolar-specific, 666 interstitial-specific, and 285 bridging-specific). Overall, our results define cells isolated within the so-called “golden hour of birth” in extremely premature neonate airways, representing complex lung biology, and can be used in studies of human development and disease. scRNA-seq of cells isolated from 10 premature neonatal endotracheal aspirates upon initial intubation. Raw data is available from dbGaP (https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs003427.v1.p1 or search for "phs003427.v1.p1" at http://www.ncbi.nlm.nih.gov/gap).

单细胞基因组学技术在深化我们对肺发育与疾病的认知方面拥有巨大潜力。当前的主要局限在于，难以从人体原代肺组织中获取完整细胞以开展人类气道健康相关的组学分析。而适配临床操作的采样方法，例如气管吸引术（endotracheal aspiration），则可支持纵向研究、大型队列招募以及新型诊断技术的开发。为了对极早早产儿（妊娠时长<28周）出生时气道腔内的细胞类型开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析，我们从出生后1小时内的插管早产儿的气管吸引物中分离得到了细胞。我们为10名受试者构建了测序数据集，从而在这一关键发育阶段，详尽刻画了气道腔的生物学特征。研究结果显示，早产儿气道内的细胞主要呈现髓系分化的连续谱系，包括胎儿单核细胞（占总细胞数的25%）、中间髓系细胞群（占48%）以及巨噬细胞（占2.6%）。我们对髓系细胞群开展轨迹分析后，发现了两条分别对应间质巨噬细胞与肺泡巨噬细胞发育阶段的分化轨迹，同时还识别出第三条可衔接不同巨噬细胞前体的替代性分化通路。这三条分化轨迹共拥有5451个动态表达基因，但同时也存在各自独特的转录调控变化：其中肺泡巨噬细胞相关轨迹有259个特异性基因，间质巨噬细胞相关轨迹有666个特异性基因，桥接通路相关轨迹则有285个特异性基因。综上，本研究明确了极早早产儿气道内于所谓“出生黄金一小时”内分离得到的细胞特征，其反映了复杂的肺部生物学过程，可用于人类发育与疾病相关研究。本数据集为针对10名早产儿初始插管时采集的气管吸引物分离细胞所开展的单细胞RNA测序数据。原始数据可从dbGaP数据库获取（https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs003427.v1.p1，或在http://www.ncbi.nlm.nih.gov/gap 搜索"phs003427.v1.p1"）。