Pathophysiological studies in neonatal tracheal aspirate derived mesenchymal stromal cells and animal models to determine the role of Pbx1 and other candidate genes in congenital diaphragmatic hernia.

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Our goal with this proposal is to understand the role of the transcription factor Pbx1 in the pathophysiology of congenital diaphragmatic hernia (CDH) using cell-based and animal models. CDH is characterized by a diaphragmatic defect that occurs during embryonic development and is associated with pulmonary hypoplasia and persistent pulmonary hypertension. The high mortality (30-50%) of affected neonates depends largely on the degree of pulmonary hypoplasia, of which the underlying pathobiology is not sufficiently understood. We know from genome wide association studies, that transcription factor Pbx1 is a candidate gene in CDH and knockdown of Pbx1 in mice results in pulmonary hypoplasia and hypertension as seen in CDH patients. Pbx1 is expressed until the postnatal period and direct stimulation of Pbx1 downstream targets can rescue compromised lung development in affected lungs. Therefore, it is potentially relevant as a therapeutic target for postnatal interventions. Here, we aim to describe the role of Pbx1 in normal and abnormal lung development due to CDH. We will use mesenchymal stromal cells isolated from tracheal aspirates from intubated neonates with CDH and “lung healthy” controls and perform different mechanistic studies. We will leverage CRISPR/Cas9 mediated knockdown of Pbx1 in these mesenchymal stromal cells and test how this affects cell morphology and determine changes in epigenetic signature of these cells. Moreover, we will obtain proteomic profiles of the respective cells at different developmental timepoints to analyze the changes on protein level. As they emerge other candidate genes will be tested accordingly with the established work pipeline. Finally, CDH tracheal aspirate cells will be subjected to gene replacement or treatment with drugs designed to regulate or perturb pathways identified by protein interaction analyses.