467 - Progressive Increases in Mesenchymal Cell Diversity Modulate Lung Development and are Attenuated by Hyperoxia

Poster Number: 467
Publication Number: 467.432

Fabio Zanini, UNSW Sydney, Sydney, New South Wales, Australia; Xibing Che, Stanford University School of Medicine, Palo Alto, CA, United States; Carsten Knutsen, Lucile Packard Children's Hospital Stanford, Palo Alto, CA, United States; Min Liu, Stanford University School of Medicine, Stanford, CA, United States; Nina E. Suresh, Stanford University School of Medicine, Redwood City, CA, United States; Cristina M. Alvira, Stanford University School of Medicine, Stanford, CA, United States; David N. Cornfield, Stanford University, Stanford, CA, United States

Background: Mesenchymal cells (MC) are central to lung development. We sought to interrogate the perinatal lung MC relative to cellular diversity, dynamic gene expression changes during alveolarization, and in hyperoxia-induced neonatal lung injury.

Objective: To test the hypothesis that single cell RNA-sequencing would identify developmentally distinct lung MC that modulate physiologic and pathophysiologic postnatal lung development.

Design/Methods: Murine pulmonary MC (5493 cells) were isolated at early saccular (E18.5), late saccular (P1), early alveolar (P7) and late alveolar (P21) stages of development, and from P7 mice exposed to hyperoxia (FiO2=0.8 x 7d), a lung injury model. Single MC were sequenced on Illumina NovaSeq at a depth of ~107 reads/cell. MC populations and genes of interest were validated via in situ hybridization.

Results: Unsupervised clustering identified 14 transcriptionally distinct MC clusters, with dynamic and developmental changes in diversity. Harmonization with adult mouse data demonstrated a distinction between perinatal (E18.5, P1, P7) and adult lung MC subtypes. Fibroblast precursors, present at E18.5, disappeared by P1 and were replaced by two distinct fibroblast subtypes expressing either Col13a1 (alveolar) or Col14a1 (adventitial). A separate embryonic MC present at E18.5 disappeared by P1 and was replaced by two distinct clusters, myofibroblasts (MyoF) expressing Pdgfra and airway smooth muscle (ASM) expressing Hhip. ASM/MyoF precursors (E18.5) exclusively expressed corticotropin releasing hormone (Crh), a signal for glucocorticoid production. Hyperoxia slowed the developmental transcriptomic changes of the lung such that hyperoxic MC at P7 more closely resembled healthy MC from P1 than P7 mice (P=0.001). Hyperoxia decreased the abundance of MyoF, total pericytes (P=0.003, KS test) and proliferating pericytes (P=0.01). Two novel cell clusters, seen predominately from male mice, emerged in hyperoxia. One cluster expressed genes shared by alveolar fibroblasts (Wnt2, Col13a1), but also novel genes including neuron-specific tubulin (Tubb3), pro-contractile genes (e.g. Acta1 and Actc1), and Lgals3, a gene up-regulated in fibrosis. Conclusion(s): In the lung, the neonatal MC transcriptome demonstrates progressive increases in cellular diversity and dynamic gene expression. Hyperoxia decreased pericyte abundance and prevented developmental evolution of the mesenchymal transcriptome, mirroring the structural arrest in lung development that characterizes BPD. Two novel cell clusters in hyperoxia-exposed male mouse may be clinically significant given the sexual dimorphism of BPD.