Neonatal airway development and injury are poorly understood, in part due to challenges of studying extremes of phenotype in human pathological samples and difficulties obtaining relevant comparator samples. Ex vivo model systems are needed to improve understanding of airway development, injury, and repair in the neonatal lung. We optimized a protocol for organotypic culture of primary murine neonatal tracheal epithelial cells (MNTECs). We compared expansion and differentiation properties of MNTECs in five different media conditions, ranging from previously published "lab-made" media to commercial sources of media. We measured the success of our organotypic cultures by quantifying the relative proportions of ciliated epithelium, TP63+ basal stem cells, and stromal cell contamination, as well as total cell numbers and air-liquid interface (ALI) thickness. Commercially available media performed better than standard lab-made media, with nearly 100% success and 20% success, respectively. Proliferation in commercial media improves expansion of TP63+ basal cells, inhibits growth of contaminating stromal cells, and improves differentiation to a polarized, ciliated pseudostratified airway epithelium, when compared with lab-made LP media. These results provide a reliable technique for studying neonatal airway epithelial cells in wild-type and genetically mutant mice.NEW & NOTEWORTHY In this study, we optimized murine neonatal tracheal epithelial cell (MNTEC) growth and differentiation for mechanistic studies of disrupted airway epithelial cell development, injury, and repair. Our protocol removes significant complexity and historical variability of murine air-liquid interface (ALI) cultures and is specifically designed for neonatal airway epithelial cell cultures with limited cell numbers. We also compared the transcriptomes of well-differentiated and poorly differentiated organotypic airway epithelial cultures to identify key genes for epithelial growth and polarization.

RATIONALE:Bronchopulmonary dysplasia (BPD) arises from disrupted lung development after preterm birth and produces structural deficits at every level of the respiratory tree. Lower airway disease is emerging as a clinically significant BPD phenotype with increased mortality, yet the molecular mechanisms whereby preterm birth disrupts airway development remain poorly defined. OBJECTIVES:To develop a human model of lower airway disease following preterm birth and to define a molecular endotype of evolving BPD (eBPD) at baseline and in response to injury. METHODS:An ex vivo organotypic airway epithelial cell (AEC) model was combined with well-characterized pathologic and transcriptomic patient samples for quantitative immunohistochemistry and RNA-sequencing analyses. MEASUREMENTS AND MAIN RESULTS:Compared to AECs from healthy controls, eBPD-derived AECs exhibited reduced proliferation, impaired differentiation to ciliated epithelium, and expansion of a vimentin-positive population with a transcriptional profile associated with impaired AEC differentiation. Following hyperoxia exposure, eBPD-derived AECs mounted a robust vimentin response ex vivo, paralleling increased vimentin expression observed in airway cells from lung tissue of human infants with BPD. CONCLUSIONS:Using an organotypic model of neonatal airway differentiation, we demonstrate eBPD is associated with impaired AEC differentiation, increased vimentin-expression and concomitant loss of ciliated cells, and an exaggerated vimentin response to hyperoxic injury. These findings mimic the effects of prematurity in airway cells in human patients. These data support a mechanism whereby hyperoxia leads to impaired epithelial differentiation and associated lower airway dysfunction in BPD and inform future mechanistic studies interrogating the role of intermediate filaments in maladaptive epithelial repair.

Objectives An estimated 23% of children with grade 3 bronchopulmonary dysplasia (BPD) require tracheostomy for long term positive pressure ventilation. Transition from an intensive care unit (ICU) ventilator to a portable home ventilator (PHV) is one of the key steps in the process of discharging a patient home. The objective of this study was to identify clinical factors and ventilator settings associated with successful transition to a PHV in infants with grade 3 BPD.Study Design We performed a retrospective record review of 74 ventilator-dependent infants with grade 3 BPD hospitalized at 15 centers within the BPD Collaborative who transitioned to PHV between March 2021 and March 2023. Hierarchical cluster analysis of patient demographics and ICU ventilator settings at the time of transition was identify similar groups and compare factors associated with successful transitions.Results Patients with higher mean airway pressure (MAP) on the PHV compared with the MAP on their ICU ventilator were more likely to successfully transition on the first attempt, had fewer days from their first attempt until successful transition, and transitioned at an earlier postmenstrual age than patients with a lower MAP on their PHV. Cluster analysis of baseline ICU ventilator settings did not predict successful transition to PHV.Conclusions We conclude that higher PHV pressures may enable patients with severe BPD to transition to a PHV. Premature weaning of ventilator pressures may be associated with prolonged need for an ICU ventilator.

Introduction: The role of the developing airway epithelium in bronchopulmonary dysplasia pathogenesis is poorly understood, in part due to lack of model systems. Organotypic culture of primary murine neonatal tracheal epithelial cells (MNTEC) allows for studies of epithelial-specific genes in genetically mutant mice. Successful MNTEC culture is challenging due to limited cell numbers and required pre-plating to remove fibroblasts. Objective: To characterize expansion and differentiation properties of organotypic MNTEC in different growth media and conditions. Methods: Primary MNTEC were isolated from wild-type PN2-3 C57BL/6 mice (n=5) as previously described. Four culture conditions (Fig 1A-B) were established with lab-prepared proliferation (LP) and differentiation (LD) media with BPE-containing DMEM/F12 media and commercial proliferation (CP) and differentiation (CD) media (PneumaCultTM EX Plus and ALI media). CP3dLP cells were cultured in CP media for 3 days before culture in LP media. Without pre-plating, cells were seeded at a density of 200,000 cells/24-well transwell and grown in LP or CP media for 7 days before staining with anti-TP63 and anti-Vimentin. After 21 days at ALI in LP or CP, differentiation was assessed with phalloidin, anti-Tuba4A (cilia), and anti-Vimentin staining. Quantitative confocal microscopy was performed and analyzed with Fiji and Prism (Mann-Whitney, p<0.05 significance). Bulk RNA-Sequencing was performed on differentiated cultures from the four conditions. Data analysis was performed with R, using Limma and edgeR packages and GSEA analysis for hallmark genes. Results: MNTEC grown in LP media for 7d had a disorganized combination of polarized and unpolarized regions (Fig 1D). More vimentin-positive cells were present (5.6%) in LP compared with CP media for 3d (0%) or CP for 7d (0.09%), and the increased vimentin-expressing cells persisted into differentiated cultures (Fig 1C and 1E). Conversely, 1.8% MTECs in LP media for 7d were positive for the epithelial stem cell marker TP63, compared with 5.7% in CP media and 4.8% in CP3dLP. Only 17.4% of MTEC cultures in LP-LD successfully differentiated at ALI, compared with 100% of the 3 other systems. Finally, MNTECs grown in LP had increased expression of genes in EMT pathways compared with CP conditions. Conclusions: CP media improves expansion and differentiation of MNTECs with decreased stromal cell contamination when compared with LP media. MNTECs grown in CP media were well differentiated and generated a functional ciliary epithelium without failure. These findings offer a feasible strategy for efficiently culturing mouse airway epithelial cells, reducing animal numbers, and decreasing in vitro experimental costs.

Introduction: Severe bronchopulmonary dysplasia (sBPD) is a consequence of extreme prematurity and results in abnormal lung development. While the importance of alveolar and vascular hypoplasia is recognized in BPD pathogenesis, the impact of airway maldevelopment is a knowledge gap. We previously reported that tracheal airway epithelial cells (AEC) from infants with evolving BPD have dysregulated differentiation in an ex vivo organotypic AEC model. Here, we explore the role of vimentin, an intermediate filament protein important for epithelial proliferation, differentiation, and repair, in the developing airway epithelium in BPD. Methods: Organotypic AEC cultures were generated from bronchial brushings (healthy pediatric patients with elective procedures) or tracheal aspirates (intubated premature infants <28wk GA) after expansion and differentiation at air-liquid interface, and bulk RNA Sequencing was performed. We compared airway tissue from autopsy specimens from infants born at less <26 weeks GA and under 1 month of age stratified by the severity of their lung disease. Quantitative immunostaining for tubulin, EPCAM, vimentin (VIM) was performed on AEC cultures and tissue samples. We further interrogated a recently released scRNA Seq atlas of human infant lung injury from infants who died with BPD, acute preterm lung injury, and term infant controls, to look for differential expression of vimentin. Results: AEC from patients with evolving sBPD had increased vimentin expression, by bulk RNA Sequencing and by increased VIM+ immunostaining, compared with AEC isolated from controls. In well-polarized cultures from patients with evolving sBPD, there was a subset of VIM+EPCAM+ AECs and in less differentiated cultures, there were VIM+EPCAM-cells with fibroblast morphology. Tracheal AECs in pathological samples from infants with evolving BPD have decreased differentiation to ciliated cells with decreased apical cilia (TUBA+), increased vimentin staining (VIM+), and decreased expression of EPCAM, compared with premature controls ([asterisk]p<0.05). Finally, in lungs of premature infants who develop sBPD, nonciliated airway epithelial cells expressed more vimentin compared with term controls (p=0.002). Conclusions: Increased vimentin expression is a notable feature in AEC derived from patients with sBPD and in non-ciliated airway cells of sBPD patients by single-cell transcriptomics. The role of vimentin as an intermediate filament in mediating adult chronic lung disease and cancer is well-described, and this discovery of an association between airway epithelial vimentin expression in sBPD provides a foundation for future research to explore the function of vimentin in these cells its mechanistic role in the response to hyperoxic injury in sBPD.

A molecular understanding of lung organogenesis requires delineation of the timing and regulation of the cellular transitions that ultimately form and support a surface capable of gas exchange. Although the advent of single-cell transcriptomics has allowed for the discovery and identification of transcriptionally distinct cell populations present during lung development, the spatiotemporal dynamics of these transcriptional shifts remain undefined. With imaging-based spatial transcriptomics, we analyzed the gene expression patterns in 17 human infant lungs at varying stages of development and injury, creating a spatial transcriptomic atlas of approximately 1.2 million cells. We applied computational clustering approaches to identify shared molecular patterns among this cohort, informing how tissue architecture and molecular spatial relationships are coordinated during development and disrupted in disease. Recognizing that all preterm birth represents an injury to the developing lung, we created a simplified classification scheme that relies upon the routinely collected objective measures of gestational age and lifespan. Within this framework, we have identified cell type patterns across gestational age and life span variables that would likely be overlooked when using the conventional "disease versus control" binary comparison. Together, these data represent an open resource for the lung research community, supporting discovery-based inquiry and identification of targetable molecular mechanisms in both normal and arrested human lung development.NEW & NOTEWORTHY Mapping the spatial and temporal transcriptional relationships during lung development is fundamental to understanding regeneration and chronic lung disease; however, the classification of samples as control or disease is especially challenging in the setting of preterm birth (itself a lung injury). Here, we report the largest neonatal lung transcriptomic atlas to date and an analysis framework based only on gestational age and lifespan, providing a new resource for hypothesis generation to the lung community.

Lung injury in preterm infants leads to structural and functional respiratory deficits, with a risk for bronchopulmonary dysplasia (BPD) that in its most severe form is accompanied by pulmonary hypertension (PH). To examine cellular and molecular dynamics driving evolving BPD in humans, we performed single-cell RNA sequencing of preterm infant lungs in early stages of BPD and BPD+PH compared to term infants. Analysis of the endothelium revealed a unique aberrant capillary cell-state primarily in BPD+PH marked by ANKRD1 expression. Predictive signaling analysis identified deficits in the semaphorin guidance-cue signaling pathway and decreased expression of pro-angiogenic transcription factor FOXF1 within the alveolar parenchyma in neonatal lung samples with BPD/BPD+PH. Loss of semaphorin signaling was replicated in a murine BPD model and in humans with alveolar capillary dysplasia (ACDMPV), suggesting a mechanistic link between the developmental programs underlying BPD and ACDMPV and a critical role for semaphorin signaling in normal lung development.

Objective Bronchopulmonary dysplasia (BPD) remains the most common late morbidity for extremely premature infants. Care of infants with BPD requires a longitudinal approach from the neonatal intensive care unit to ambulatory care though interdisciplinary programs. Current approaches for the development of optimal programs vary among centers. Study Design We conducted a survey of 18 academic centers that are members of the BPD Collaborative, a consortium of institutions with an established interdisciplinary BPD program. We aimed to characterize the approach, composition, and current practices of the interdisciplinary teams in inpatient and outpatient domains. Results Variations exist among centers, including composition of the interdisciplinary team, whether the team is the primary or consult service, timing of the first team assessment of the patient, frequency and nature of rounds during the hospitalization, and the timing of ambulatory visits postdischarge. Conclusion Further studies to assess long-term outcomes are needed to optimize interdisciplinary care of infants with severe BPD. Key Points

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a significant contributor to morbidity and death in infants who are born premature. Male sex is an independent risk factor for the development of BPD. However, whether male sex is associated with adverse outcomes that occur after formal diagnosis of severe BPD prior to hospital discharge remains unclear. RESEARCH QUESTION: Is male sex associated with a higher risk of adverse outcomes in infants with established severe BPD? STUDY DESIGN AND METHODS: A retrospective, multicenter cohort study of infants enrolled in the BPD Collaborative Registry from January 1, 2015, to June 29, 2022, was performed. Demographics, clinical characteristics, and outcomes were stratified by sex (ie, male vs female). Regression modeling was used to estimate the association of sex with the primary composite outcome of death or tracheostomy at hospital discharge. RESULTS: We identified 1,156 infants with severe BPD, defined at 36 weeks postmenstrual age by the National Institutes of Health 2001 consensus definition. The cohort was predominantly male (59% male infants, 41% female infants). However, rates of mechanical ventilation at 36 weeks postmenstrual age (ie, type 2 severe BPD) did not differ by sex. Overall mortality rates within the cohort were low (male infants, 5.3%; female infants, 3.6%). The OR of death or tracheostomy for male -to -female infants was 1.0 (95% CI, 0.7-1.5). INTERPRETATION: Our results lead us to speculate that, although sex is an important variable that contributes to the development and pathogenesis of severe BPD, it does not appear to be associated with adverse outcomes in this cohort of infants with established disease. The surprising results raise important questions surrounding the temporal role of biological sex in the development of severe BPD and its progression during the neonatal ICU stay. As we explore the phenotypes and endotypes of BPD, it is imperative to consider how sex modulates the disease from birth through hospital discharge. CHEST 2024; 165(3):610-620

Objective Routine blood gas measurements are common in infants with severe bronchopulmonary dysplasia (sBPD) and are a noxious stimulus. We developed a guideline-driven approach to evaluate the care of infants with sBPD without routine blood gas sampling in the chronic phase of NICU care (after diagnosis at 36 weeks PMA).Study design We examined blood gas utilization and outcomes in our sBPD inpatient care unit using data collected between 2014 and 2020.Results 485 sBPD infants met inclusion criteria, and 303 (62%) never had a blood gas obtained after 36 weeks PMA. In infants who had blood gas measurements, the median number of total blood gases per patient was only 4 (IQR 1-10). We did not identify adverse effects on hospital outcomes in patients without routine blood gas measurements.Conclusions We found that patients with established BPD could be managed without routine blood gas analyses after 36 weeks PMA.

Respiratory disease is one of the most common complications of preterm birth. Survivors of prematurity have increased risks of morbidities and mortalities independent of prematurity, and frequently require multiple medications, home respiratory support, and subspecialty care to maintain health. Although advances in neonatal and pulmonary care have improved overall survival, earlier gestational age, lower birth weight, chorioamnionitis and late onset sepsis continue to be major factors in the development of bronchopulmonary dysplasia. These early life events associated with prematurity can have respiratory consequences that persist into adulthood. Furthermore, after initial hospital discharge, air pollution, respiratory tract infections and socioeconomic status may modify lung growth trajectories and influence respiratory outcomes in later life. Given that the incidence of respiratory disease associated with prematurity remains stable or increased, there is a need for pediatric and adult providers to be familiar with the natural history, manifestations, and common complications of disease.

Multidisciplinary bronchopulmonary dysplasia (BPD) programs provide improved and consistent medical management, care of the developing infant, family support, and smoother transitions in care resulting in improved survival, pulmonary, and extra-pulmonary outcomes. This review summarizes the benefits of interdisciplinary BPD management, as well as strategies for initial programmatic development, program growth, and maintenance at centers across the United States factoring in institutional, provider, and parent reported goals that were derived from a consensus conference on BPD management.

Objective To describe our multidisciplinary bronchopulmonary dysplasia (BPD) consult team's systematic approach to BPD associated pulmonary hypertension (PH), to report our center outcomes, and to evaluate clinical associations with outcomes. Study design Retrospective cohort of 60 patients with BPD-PH who were referred to the Seattle Children's Hospital BPD team from 2018 to 2020. Patients with critical congenital heart disease were excluded. Demographics, comorbidities, treatments, closure of hemodynamically relevant intracardiac shunts, and clinical outcomes including time to BPD-PH resolution were reviewed. Results Median gestational age of the 60 patients was 25 weeks (IQR: 24–26). 20% were small for gestational age (SGA), 65% were male, and 25% received a tracheostomy. With aggressive cardiopulmonary management including respiratory support optimization, patent ductus arteriosus (PDA) and atrial septal defect (ASD) closure (40% PDA, 5% ASD, 3% both), and limited use of pulmonary vasodilators (8%), all infants demonstrated resolution of PH during the follow-up period, including three (5%) who later died from non-BPD-PH morbidities. Neither SGA status nor the timing of PH diagnosis (<36 vs. ≥36 weeks PMA) impacted the time to BPD-PH resolution in our cohort [median 72 days (IQR 30.5–166.5)]. Conclusion Our multidisciplinary, systematic approach to BPD-PH management was associated with complete resolution of PH with lower mortality despite less sildenafil use than reported in comparable cohorts. Unique features of our approach included aggressive PDA and ASD device closure and rare initiation of sildenafil only after lack of BPD-PH improvement with respiratory support optimization and diagnostic confirmation by cardiac catheterization.

INTRODUCTION:Evidence-based ventilation strategies for infants with severe bronchopulmonary dysplasia (BPD) remain unknown. Determining whether contemporary ventilation approaches cluster as specific BPD strategies may better characterize care and enhance the design of clinical trials. The objective of this study was to test the hypothesis that unsupervised, multifactorial clustering analysis of point prevalence ventilator setting data would classify a discrete number of physiology-based approaches to mechanical ventilation in a multicenter cohort of infants with severe BPD. METHODS:We performed a secondary analysis of a multicenter point prevalence study of infants with severe BPD treated with invasive mechanical ventilation. We clustered the cohort by mean airway pressure (MAP), positive end expiratory pressure (PEEP), set respiratory rate, and inspiratory time (Ti) using Ward's hierarchical clustering analysis (HCA). RESULTS:Seventy-eight patients with severe BPD were included from 14 centers. HCA classified three discrete clusters as determined by an agglomerative coefficient of 0.97. Cluster stability was relatively strong as determined by Jaccard coefficient means of 0.79, 0.85, and 0.77 for clusters 1, 2, and 3, respectively. The median PEEP, MAP, rate, Ti, and PIP differed significantly between clusters for each comparison by Kruskall-Wallis testing (p < 0.0001). CONCLUSIONS:In this study, unsupervised clustering analysis of ventilator setting data identified three discrete approaches to mechanical ventilation in a multicenter cohort of infants with severe BPD. Prospective trials are needed to determine whether these approaches to mechanical ventilation are associated with specific severe BPD clinical phenotypes and differentially modify respiratory outcomes.