Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.
Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.
Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. xiaoyangwang@gmail.com
Xiao-Yang
XY
Wang
Kathleen M
KM
Keefe
Sandra M
SM
Jensen-Taubman
Danlei
D
Yang
Kai
K
Yan
R Ilona
RI
Linnoila
D000818:Animals; D001980:Bronchi / Q000166:cytology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry; D007150:Immunohistochemistry; D008168:Lung / Q000166:cytology; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D014598:Uteroglobin / Q000378:metabolism*
D014598:Uteroglobin