Distal Lung Epithelial Regeneration is Driven by Notch Pathway Inhibition and Enhanced by Ex Vivo Native Lung Scaffold Biomimetic Culture

Lung tissue engineering on native extracellular matrix involves combining essential cell populations with corresponding scaffolds. Functional regeneration will require both airway and alveolar epithelial repopulation, along a proximal-to-distal axis. Basal epithelial cells (BECs) can be isolated from donor lung tissue and used for airway recellularization. Yet, a robust source of distal alveolar epithelial cells remains a challenge. Inhibiting the Notch pathway by targeting γ-secretase can direct patient-derived BECs toward a distal Type-2 pneumocyte (AT2)-like fate in vitro, as demonstrated by increased surfactant protein-C (SP-C, +22.06-fold), ABCA3 (+2.79-fold), and LAMP1 (+11.48-fold) gene expression. Notch inhibition was confirmed by decreased HES1 (-2.69-fold) and by a Notch-responsive luciferase reporter. Notch inhibition following delivery of BECs to the airways of lung scaffolds and ex vivo biomimetic culture enabled greater induction of distal AT2-like fate (+27.61-fold SP-C, -3.73-fold HES1 vs in vitro). Regenerated lungs displayed alveolar recellularization with organized architecture and morphology. Revascularization of the scaffold with pulmonary artery endothelial cells (PAECs) further aided BEC distalization during ex vivo regeneration (+2.38-fold SP-C, -6.17-fold HES1, vs no PAECs). Addition of air ventilation during ex vivo lung culture further enhanced this response (+2.54 SP-C vs no ventilation). These results demonstrate an exciting potential of BECs for lung tissue regeneration and endogenous repair. The microenvironment and reparative niche are also important drivers of epithelial cell fate during regeneration.