Isolation and functional analysis of cellular components of the bronchiolar stem cell hierarchy

Mouse bronchiolar stem cells have been identified in vivo based on functional characteristics including naphthalene resistance, long-term retention of labeled DNA precursors, and dual expression of markers for airway (CCSP) and alveolar (pro-SPC) epithelium. Further characterization would benefit from establishment of rigorous enrichment strategies allowing analysis of their behavior in vitro and following transplantation, and the establishment of a defining gene expression signature. We have determined that Epithelial Cell Adhesion Molecule (EpCAM) and Integrin α6 are expressed on the cell surface of both alveolar and bronchiolar epithelial cells and that low levels of Sca-1 expression characterize the bronchiolar epithelium. Within the Sca-1low EpCAMpos Integrinα6pos population of bronchiolar epithelial cells, autofluorescence (AF) levels distinguish the facultative transit-amplifying population which is AFhi from bronchiolar stem cells which are AFlow. Use of transgenic animal models allowing expansion or depletion of the stem cell compartment and use of lineage tracing strategies have allowed us to determine the identity of cells isolated based on their cell surface phenotype and autofluorescence characteristics. Injury models associated with depletion of terminally differentiated ciliated cells (ozone) or facultative transit amplifying population (naphthalene) were used to validate the functional characteristics of the two fractions of bronchiolar progenitors. In conclusion, we have developed and validated a fractionation approach for the generation of highly purified preparations of bronchiolar stem and Clara cells from the mouse lung. These data enable establishment of robust in vitro and transplantation assays to further validate the functional behavior of stem and facultative TA (Clara) cells and allows analysis of gene expression profile of the two populations towards a better understanding of unique characteristics of the bronchiolar stem cell compartment