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The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118
Submitted 10 January 2003 ; accepted in final form 5 March 2003
Side population (SP) cells are a rare subset of cells found in various
tissues that are highly enriched for stem cell activity. SP cells can be
isolated by dual-wavelength flow cytometry because of their capacity to efflux
Hoechst dye, a process mediated by the ATP-binding cassette transporter breast
cancer resistance protein (Bcrp) 1. By performing flow cytometry of
enzymedigested mouse lung stained with Hoechst dye, we found that SP cells
comprise 0.03–0.07% of total lung cells and are evenly distributed in
proximal and distal lung regions. By RT-PCR, we found that lung SP cells
express hepatocyte nuclear factor-3
, but not thyroid transcription
factor-1. Surface marker analysis revealed lung SP cells to be stem cell
antigen 1 positive, Bcrp1 positive, lineage marker negative, and heterogeneous
at the CD45 locus. As expected, we did not detect lung SP cells in
Bcrp1-deficient animals. We, therefore, employed nonisotopic in situ
hybridization and immunostaining for Bcrp1 as a strategy to localize these
cells in vivo. Expression was observed in distinct lung cell types: bronchial
and vascular smooth muscle cells and round cells within the distal air space.
We confirmed the expression of Bcrp1 in primary bronchial smooth muscle cell
cultures (BSMC) and in lavaged distal airway cells, but neither possessed the
capacity to efflux Hoechst dye. In BSMC, Bcrp1 was localized to an
intracellular compartment, suggesting that the molecular site of Bcrp1
expression regulates SP phenotype.
Hoechst; stem cells; smooth muscle cells; breast cancer resistance protein 1
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