Identification, isolation, and clonal culture of stem cells is essential for understanding their proliferative and differentiation potential, and the cellular and molecular mechanisms that regulate their fate. Akin to development in vivo, the in vitro growth of adult lung epithelial stem cells requires support of mesenchymal-derived growth factors. In the adult mouse lung, epithelial stem/progenitor cells are defined by the phenotype CD45neg CD31neg EpCAMpos CD104pos CD24low, and mesenchymal cells are defined by the phenotype CD45neg CD31neg EpCAMneg Sca-1hi. Here we describe a method for primary cell isolation from the adult mouse lung, a flow cytometry strategy for fractionation of epithelial stem/progenitor cells and mesenchymal cells, and a three-dimensional epithelial colony-forming assay.
Keywords: Colony-forming assay; Flow cytometry; Lung epithelium; Stem cells.