title: Molecular profiling of single Sca-1+/CD34+,- cells--the putative murine lung stem cells.
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 10(6) 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.
authors.affiliation: Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Hittinger
authors.affiliation: Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany.
authors.forename: Zbigniew T
authors.initials: ZT
authors.lastname: Czyz
authors.affiliation: Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany.
authors.forename: Yves
authors.initials: Y
authors.lastname: Huesemann
authors.affiliation: Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany ; Institute of Functional Genomics, University of Regensburg, Regensburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Maneck
authors.affiliation: Project Group "Personalized Tumor Therapy", Fraunhofer Institute for Experimental Medicine and Toxicology, Regensburg, Germany.
authors.forename: Catherine
authors.initials: C
authors.lastname: Botteron
authors.affiliation: Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kaeufl
authors.affiliation: Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany ; Project Group "Personalized Tumor Therapy", Fraunhofer Institute for Experimental Medicine and Toxicology, Regensburg, Germany.
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Klein
authors.affiliation: Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany ; Project Group "Personalized Tumor Therapy", Fraunhofer Institute for Experimental Medicine and Toxicology, Regensburg, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Polzer
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism*; D000950:Antigens, Ly / Q000235:genetics / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism*; D002469:Cell Separation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D018952:Antigens, CD34; D000950:Antigens, Ly; D015415:Biomarkers; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D012333:RNA, Messenger