Side population cells and Bcrp1 expression in lung.
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-3beta, 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.
Summer
Ross
R
The Pulmonary Center, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, USA. rsummer@lung.bumc.bu.edu
Kotton
Darrell N
DN
Sun
Xi
X
Ma
Bei
B
Fitzsimmons
Kathleen
K
Fine
Alan
A
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000235:genetics*; D000367:Age Factors; D000818:Animals; D015415:Biomarkers; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D051542:Hepatocyte Nuclear Factor 3-beta; D008168:Lung / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009130:Muscle, Smooth / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D009363:Neoplasm Proteins; D009687:Nuclear Proteins / Q000235:genetics; D010641:Phenotype; D013234:Stem Cells / Q000737:chemistry* / Q000502:physiology*; D014157:Transcription Factors
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D004268:DNA-Binding Proteins; C496503:Foxa2 protein, mouse; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta