DESCRIPTION (Applicant's abstract): Identification of pulmonary epithelial stem cells with pluripotential differentiation capacity will significantly impact development of gene therapeutic modalities, and development of new approaches for the treatment or prevention of chronic and neoplastic lung disease. Our preliminary studies have defined the neuroepithelial body (NEB) microenvironment as a critical reservoir of proliferative cells following acute airway injury and suggest that these may have stem cell-like character. Proliferation and hyperplasia of NEB-associated cells, which is common to many types of chronic lung disease in humans, is only observed among mouse models when progenitor (Clara) cells are among those injured. Goals of this application are to test the hypothesis that injury of a progenitor cell population, such as the mature Clara cell, is a stimulus for recruitment of NEB-associated stem cells for epithelial renewal. Aims will define contributions made by proliferative cells originating from the NEB towards repair from ozone or naphthalene-induced airway injury; agents preferentially targeting terminally differentiated (ciliated) or progenitor (Clara) cells, respectively. Classification of NEB-associated cells with regenerated capacity as "stem cells" will be made by fulfilling three criteria: 1) absence of certain differentiated functions, 2) pluripotential differentiation capacity and 3) relatively slow cycling time in the quiescent lung. Preliminary data demonstrate a requirement for Clara cell secretory protein (CCSP)-expressing (CE) cells in airway repair. Aim 1 will build upon these studies by further exploring variability in functional and molecular phenotype of CE cells in the quiescent and injured adult mouse lung. Aim 2 will expand upon preliminary studies demonstrating that both CE and pulmonary neuroendocrine (PNE) cells proliferate following acute naphthalene-induced airway injury. Through use of transgenic mice allowing cell type-specific conditional ablation of either CE or PNE cells, we will identify the contribution of each group to airway homeostasis and repair. Finally, studies proposed in Aim 3 will classify NEB-associated cells with proliferative potential as either progenitor cells or stem cells through analysis of cycling time and differentiation capacity. Completion of these aims will define pulmonary stem cell populations, their contributions to airway repair, and how this may be modified by injury to either progenitor or non-progenitor epithelial cell populations. These finding will significantly impact the understanding of mechanisms of normal epithelial renewal and neoplasia, and will identify critical cells to be targeted for stable maintenance of therapeutic genes.