Journal: Nature Communications
Article Title: PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium
Figure Lengend Snippet: Single-cell profiles map cardiogenic gene expression to Sca1 + SP cells co-expressing Pdgfra. ( a ) Left, flow sorting of fresh cardiac Lin − Sca1 + cells after immunomagnetic enrichment for Sca1. Center, further purification of Lin − Sca1 + cells for the SP phenotype by Hoechst 33324 staining±ABC transporter inhibitors: FTC, fumitremorgin C; Res, reserpine; Ver, verapamil. Right, bar graph, mean±s.e.m.; n =4; * P ≤0.0001. ( b ) Single-cell qRT–PCR profiles of fresh total Sca1 + , SP and non-SP cells, compared with cardiomyocytes (CMC). The heatmap illustrates expression as −ΔCt values (blue, low or absent; red, high) and hierarchical clustering reveals the co-expression of functionally related genes in the populations indicated. Highlighted are: yellow, the Sca1 gene Ly6a ; blue, Kdr and Cdh5 , enriched in Sca1 + and non-SP cells; red, cardiogenic transcription factors, enriched in SP cells and CMC; green, Pdgfra and Tcf21 , enriched in SP cells; violet, CMC genes. Sca1 + , n =23; non-SP, n =44; SP, n =43; CMC, n =18. For the full set of 44 genes, see Supplementary Fig. 1 . ( c ) Density plots of expression (−ΔCt) for selected genes in SP (light red) versus non-SP (light blue) cell populations. Genes are ordered according to increasing P -values. Those with a significantly divergent prevalence of expression between SP and non-SP cells are indicated by an asterisk. ( d ) PCA of the single-cell expression profiles. (Top) PC2 (19% variability) separates SP from non-SP sample scores, whereas PC3 (9% variability) establishes a distinct separation between SP/non-SP/Sca1 + cells and CMC. Non-SP and unfractionated Sca1 + cells cluster together in the PC projection. (Bottom) Gene loadings contributing to each PC indicate that a small subset of genes explain the cross-group variability captured by PC2 and PC3. Cdh5 and Kdr are predominantly associated with non-SP and unfractionated Sca1 + cells, while Pdgfra and Tcf21 are correlated with SP cells (as given by PC2). Differences between CMCs and the remaining samples are strongly reflected in PC3, with cardiac structural genes ( Myh6 and Myl2 ) clustered consistently.
Article Snippet: Single-cell qRT–PCR Single cells were sorted directly (FACSAria II) into 96-well plates containing 10 μl of the reaction mixture for pre-amplification, using CellDirect One-Step qRT–PCR Kits (Invitrogen).
Techniques: Expressing, Flow Cytometry, Purification, Staining, Quantitative RT-PCR