Abstract
Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker-positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD34 − and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD34 − cells (p = 0.03), whereas mRNA of the mesenchymal marker Pdgfrα (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p = 0.04). FACS analysis confirmed the existence of a Pdgfrα positive subpopulation within Epcam+/Sca-1+/CD34− epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD34 − cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,− cells in the biology of the murine lung.
Highlights
The murine lung contains at least 40 morphologically distinct cell types of mesodermal or endodermal origin [1]
All isolated cells were subjected to whole transcriptome amplification (WTA) and WTA quality controlled by expression of Actb and Gapdh
In this study we investigated the gene expression profile of single stem cell antigen 1 (Sca-1)+/CD312/PI2 and cluster of differentiation 34 (CD34)+/CD452/GFP-A2 cells that were detected by immunofluorescence and subsequently sub-divided into groups based on the protein expression of Sca-1 and CD34, as well as mRNA expression of subgroup-specific markers
Summary
The murine lung contains at least 40 morphologically distinct cell types of mesodermal or endodermal origin [1]. It can be subdivided anatomically in three different regions: large airways, bronchioles, and alveoli. Considerable efforts have been undertaken to further characterize a specific subgroup of Sca-1+ cells. These cells were named bronchioalveolar stem cells (BASCs) based on their location at the bronchioalveolar duct junction [4] where they proved to play a key role in distal airway repair [4,5,6,7]
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