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A2464 - Clonal Analysis of Airway Stem Cell Heterogeneity in Cystic Fibrosis
Author Block: S. Niroula1, W. Rao2, K. Goller1, R. Mahalingam1, S. Wang1, J. Xie1, M. Duleba1, M. L. Metersky3, J. F. Engelhardt4, K. R. Parekh5, F. McKeon1, W. Xian2; 1Biology and Biochemistry, University of Houston, Houston, TX, United States, 2Institute of Molecular Medicine, McGovern Medical School of The University of Texas Health Science Center, Houston, TX, United States, 3Center for Bronchiectasis Care Pulmonary, Critical Care and Sleep Medicine, University of Connecticut Health Center, Farmington, CT, United States, 4Department of Anatomy and Cell Biology and Gene Therapy Center for Cystic Fibrosis and Other Genetic Diseases, University of Iowa Carver College of Medicine, Iowa City, IA, United States, 5Division of Thoracic Surgery, University of Iowa Carver College of Medicine, Iowa City, IA, United States.
Despite congenital CFTR mutations and the severity of advanced cystic fibrosis (CF), the onset and progression of lung disease in these patients is not uniform. This phenomenon raises the possibility that symptomatic CF involves contributions to the disease process of an epigenetic nature beyond those conferred by CFTR mutations themselves. As a first test of this hypothesis, we asked how airway stem cells from patients with end-stage CF undergoing lung transplant therapy differ from those of normal patients without CFTR mutations. Using technology that enables single stem cell cloning, we confirmed that normal proximal airway stem cells are p63+/Krt5+ and can be differentiated to yield 90% ciliated cells and 10% goblet cells. Likewise, distal airway stem cells (DASCs), which are also p63+Krt5+, differentiate to Type I and Type II pneumocytes. In contrast, clones from end-stage CF yielded three distinct types of p63+Krt5+ stem cells that respectively differentiated to normal upper airway epithelia, goblet cell metaplasia (GCM), and squamous cell metaplasia (SCM). Long-term passaging of these three clonal types from the proximal airways revealed that these differentiation fates were absolute and likely epigenetically committed. In addition, both the GCM and SCM clones from the proximal airways of CF patients showed distinct yet robustly hyperinflammatory gene expression profiles that were also stably maintained despite months of in vitro cultivation. The combination of secreted chemokines and cytokines from both GCM and SCM were sufficient to trigger endothelial cell activation in vitro including the presentation and expression of E-selectin and ICAM-1, confirming the enhanced expression of leukocyte extravasation pathways in these stem cell variants. And while the DASCs from CF patients retained their ability to differentiate to Type I and Type II pneumocytes in vitro, the gene expression profiles of these cells were distinguished from normal DASCs by their stable expression of multiple inflammatory genes. Efforts to characterize the epigenetic modifications and the role of CFTR mutations in this stem cell heterogeneity are ongoing. Our provisional conclusions from these studies are that end-stage CF patients display a heterogeneity of proximal airway stem cells that is epigenetic in origin and includes variants that conceivably contribute to the progression of CF disease presentation.