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1 EOH, GSPH Univ Pittsburgh, PGH, Pennsylvania, United States
2 Radiation Oncology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
3 First Department of Medicine, Hokkaido University School of Medicine, Kita-ku, Japan
4 Division Pulm Allergy Crit Care Medicine, University Pittsburgh Sch Med, Pittsburgh, Pennsylvania, United States
5 Department of Environmental and Occupational Health, Univeristy of Pittsburgh; Department of Environmental and Occupational Health, Univeristy of Pittsburgh, United States
* To whom correspondence should be addressed. E-mail: sdr1{at}pitt.edu.
Previous analysis of lung injury and repair has provided evidence for region-specific stem cells that maintain proximal and distal epithelial compartments. However, redundant expression of lineage markers by cells at several levels of the stem cell hierarchy has complicated phenotypic and functional characterization of clonogenic airway cells. Based on the demonstration that rapid efflux of the DNA dye Hoechst 33342 can be used to prospectively purify long-term repopulating hematopoietic stem cells, we hypothesized that lung cells with similar biochemical properties would be enriched for clonogenic progenitors. We demonstrate that Hoechst-dim side population (SP) cells isolated from proximal and distal compartments of the mouse lung were relatively small and agranular, exhibited low red and green autofluorescence, and that the SP fraction was highly enriched in clonogenic cells. Quantitative RT-PCR indicated that vimentin mRNA was enriched and that epithelial markers were depleted in these preparations of SP cells. Bleomycin exposure was associated with decreased clonogenicity among alveolar SP and suggested that SP cell function was compromised under pro-fibrotic conditions. We conclude that the SP phenotype is common to clonogenic cells at multiple airway locations and suggest that Hoechst efflux is a property of cells expressing a wound-repair phenotype.
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