A tracheal aspirate-derived airway basal cell model reveals a proinflammatory epithelial defect in congenital diaphragmatic hernia

Abstract

Rationale: Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. Objectives: To establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. Methods: We developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from CDH patients and preterm and term, non-CDH controls were derived and analyzed by bulk RNA-sequencing, ATAC-sequencing, and air-liquid-interface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main Results: Transcriptomic and epigenetic profiling of CDH and non-CDH basal stem cells reveals a disease-specific, proinflammatory signature independent of severity or hernia size. In addition, CDH basal stem cells exhibit defective epithelial differentiation in vitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, steroid treatment normalizes epithelial differentiation phenotypes of human CDH basal stem cells in vitro and in nitrofen rat tracheas in vivo. Conclusions: Our findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

Administration of amniotic fluid stem cell extracellular vesicles promotes development of fetal hypoplastic lungs by immunomodulating lung macrophages

“…Using immunofluorescence on autopsy samples, we confirmed that human fetuses with CDH also have an inflammatory status with macrophage enrichment and increased TNFα and pNF-κB expression. A similar observation was made in neonates with CDH postnatally, who were found to have upregulation of pNF-κB in the proximal lung (basal cells) and TNFα in the distal lung ( 57, 58 ). Other studies reported that neonates with CDH had high levels of proinflammatory cytokines in the blood postnatally ( 59–64 ).…”

Single cell-guided prenatal derivation of primary epithelial organoids from the human amniotic and tracheal fluids

“…With these techniques, we validated the presence of amniotic fluid epithelial cells (AFEC), highlighting that these shed from a multiplicity of developing tissues, and demonstrating that this population contains progenitors capable of forming tissue-specific primary fetal organoids. Finally, with the adoption of fetal surgery procedures such as Fetal Endoluminal Tracheal Occlusion (FETO) to treat CDH, we can now sample and expand viable epithelial cells from tracheal fluid (TF) at various stages in the therapy [39][40][41] .…”