Expression of epithelial and mesenchymal markers in <i>Sca-1⁺/CD34^+,⁻</i> cells.

<p><b>Panel A:</b> The expression of epithelial and mesenchymal transcripts was tested by analytical PCR and is illustrated in a hierarchical cluster heatmap. The analysis shows that the majority of <i>Sca-1⁺/CD34⁺</i> cells (light grey) show similar marker expression as <i>Sca-1^−/CD34⁺</i> cells (white), while all <i>Sca-1⁺/CD34</i>⁻ cells (dark grey) are located in the second branch. Red squares indicate specific bands in analytical PCR, black squares indicate negative PCR results. <b>Panel B:</b> FACS analysis reveals EpCAM⁺/Pdgfrα⁺ subpopulation within Sca-1⁺/CD34^−/CD31^−/CD45⁻ cells. For each of 5 mice, 5×10⁶ murine lung cells were isolated from lung explants and stained with antibodies directed against Sca-1, CD34, CD31, CD45, Pdgfrα and Epcam. While Sca-1⁺/CD34⁻ cells consistently showed Epcam expression, Pdgfrα expression was predominantly found in Sca-1⁺/CD34⁺ cells. However, Sca-1⁺/CD34^−/Epcam⁺ cells could be divided in two major subpopulations defined by Pdgfrα expression. Relative quantification is given for corresponding selected subpopulation as indicated by arrows. <b>Panel C:</b> Scatter plots of the detected cell populations for mouse 5, only.</p

PCR results of corresponding transcripts in Sca-1+/CD31- and cells CD34+/CD45- cells.

<p>PCR results of corresponding transcripts in Sca-1+/CD31- and cells CD34+/CD45- cells.</p

Gene expression microarray analysis of isolated single cells.

<p><b>Panel A:</b> The comparison of microarray expression profiles of <i>Sca-1⁺/CD34⁺</i> cells and pulmonary reference (Sca-1^−/CD34^−/CD31^−/CD45⁻) cells showed 107 differentially expressed genes. <b>Panel B:</b> Pools of analyzed cells from <i>Sca-1⁺/CD34⁺</i> subpopulation (red), <i>Sca-1⁺/CD34</i>⁻ subpopulation (blue) and pulmonary reference cells (green) were analyzed by quantitative PCR against differentially expressed genes <i>Dcn</i>, <i>Esd</i> and <i>Gsn</i>. The selected genes not only show significant differences regarding to their expression, but also represent different subpopulations of proteins. Error bars indicate standard deviation of the mean calculated for analyzed triplicates. Expression values are calculated by relative quantification against housekeeping gene <i>Actb</i> and illustrated in comparison to pulmonary reference cells (expression value = 1.0) on a logarithmic scale. All comparisons between different groups, as determined by quantitative PCR, showed significantly different expression levels (student’s t-test, * indicating p<0.05).</p

Distribution of PCR-based <i>Sca-1/CD34</i> expression in isolated putative BASCs.

<p>Distribution of PCR-based <i>Sca-1/CD34</i> expression in isolated putative BASCs.</p

Non-erythrocytic cells, Sca-1⁺ and CD34⁺ cells per lung preparation.

<p>Non-erythrocytic cells, Sca-1⁺ and CD34⁺ cells per lung preparation.</p

Flowchart of the experimental setup.

<p>Simplified schematic overview on the workflow of single cell isolation and immunofluorescent staining strategy: Cells are divided into two groups after separation and stained with antibodies directed against CD31 and Sca-1 or CD34 and CD45, respectively. In order to gain appropriate reference cells for comparative gene expression analysis, additional lung cells are stained with antibodies directed against CD31 and CD45, and only CD31^−/CD45⁻ cells are isolated. Propidium iodide (PI) and GFP-Annexine (GFP-A) are applied to exclude apoptotic cells. Genes recently introduced in literature in order to differentiate between epithelial and mesenchymal cells are tested by specific PCR. PCR results enable further subdivision of analyzed cells (Sca-1⁺/CD34⁺ cells, Sca-1⁺/CD34⁻ cells, Sca-1^−/CD34⁺ cells). In a final step, selected Sca-1⁺/CD34⁺ cells, Sca-1⁺/CD34⁻ cells and Sca-1^−/CD34⁻ reference cells are subjected to comparative gene expression analysis. Results are validated by qPCR of pooled samples.</p