Immunophenotypic subsetting of functionally distinct lung epithelial progenitor cells

Epithelial injury followed by defective repair is an important component of several lung diseases. The repair process largely depends on proliferation and differentiation of endogenous epithelial stem cells. 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. We determined that Epithelial Cell Adhesion Molecule (EpCAM) and Integrin alpha 6 are expressed on the cell surface of alveolar and bronchiolar epithelial cells, and that low levels of Sca-1 expression characterize the bronchiolar epithelium. Within the Sca-1 super(low) EpCAM super(pos) Integrin alpha 6 super(pos) population of bronchiolar epithelial cells, autofluorescence (AF) levels distinguish the facultative transit-amplifying population (AF super(hi)) from bronchiolar stem cells (AF super(low)). Use of transgenic animal models allowing expansion or depletion of the stem cell pool and lineage tracing allowed us to determine the identity of cells isolated based on their cell surface phenotype and autofluorescence characteristics. Injury models 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 enriched 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 features of the bronchiolar stem cell compartment.