View Abstract

A2458 - Distal Differentiation of Basal Epithelial Stem Cells by Notch Pathway Inhibition Is Enhanced by Ex Vivo Lung Scaffold and Endothelial Cell Co-Culture
Author Block: S. E. Gilpin, D. E. Gorman, T. Wu, H. C. Ott; Surgery, Massachusetts General Hospital, Boston, MA, United States.
The aim of engineering lung tissue on native extracellular matrix scaffolds involves combining essential cell populations with corresponding matrices to form living, functional grafts. Epithelial tissue regeneration will require the efficient repopulation of both airway and alveolar epithelium, along a proximal-to-distal axis. Basal epithelial stem cells (BESCs), identified by p63 and Keratin5 (Krt5) expression, can be isolated from donor lung tissue and utilized to recellularize lung scaffolds. Yet, a robust source of distal alveolar epithelial cells remains a current challenge toward functional lung regeneration. Inhibition of the Notch signal pathway using small molecules targeting gamma-secretase can direct BESCs toward a distal Type2 pneumocyte-like fate in vitro. This was verified by an increase in surfactant protein-C (SP-C, +22.06±0.29-fold), ATP binding cassette subfamily A-3 (ABCA3, essential for surfactant production, +2.79±0.25-fold), and Lysosomal-associated membrane protein-1 (LAMP-1, +11.48±0.06-fold) gene expression, after 5 days of treatment in vitro. Notch pathway inhibition was confirmed by decreased HES1 expression (-2.69±0.62-fold). No significant changes in BESC-associated gene (p63, Krt5) were found. All qPCR analysis is expressed relative to untreated BESCs (n=3 per group) and confirmed by immunofluorescent cell staining. Greater induction of a distal Type2 pneumocyte-like fate was confirmed following delivery of BESCs to the airways of acellular rat lung scaffolds and subsequent ex vivo biomimetic culture, with delivery of the inhibitors through the vascular perfusate for 7 days (+27.61±8.39-fold SP-C, -3.73±0.98-fold HES1 compared to in vitro treatment). As a control, BESCs cultured on rat or human acellular lung tissue slices in vitro did not significantly increase SP-C gene expression, compared to BESCs treated without matrix. Additional recellularization of the lung scaffold vasculature with primary pulmonary artery endothelial cells (PAECs) further aided Notch pathway inhibition and greater increase in SP-C expression during ex vivo regeneration (+2.38±0.68-fold SP-C, -6.17±2.83-fold HES1, compared to ex vivo culture without PAECs). Analysis of the regenerated lung tissue confirmed extensive alveolar recellularization with organized tissue architecture and morphology. Addition of cyclic air ventilation (6 breaths/min, 5ml/breath) to the regenerating lungs during ex vivo culture further increased SP-C expression with Notch inhibition (+2.54±0.45-fold SP-C, -2.11±0.97-fold HES1, compared to perfusion-only ex vivo culture). Together, these results highlight the potential of endogenous tissue-derived epithelial stem cells for lung tissue regeneration and further contribute to the evolving understanding of epithelial cell aptitude for tissue repair. The addition of multiple cell types and biomechanical stimuli during ex vivo regeneration is an important driver of epithelial cell fate and function.