Figure 8: Precise co-segregation of the cardiogenic signature to PDGFRα⁺/CD31⁻ SP and non-SP cells.

(a) Left, FACS density plots illustrating the four-way strategy to flow-sort Lin⁻/Sca1⁺ cells by SP staining plus PDGFRα and CD31. Right, bar graph of the subpopulations’ prevalence. Mean±s.e.m.; n=5; *P<0.0001. (b) Bar graph indicating the % of single cells generating clones. Mean±s.e.m.; n=4–5; *P<0.0001. (c) Single-cell qRT–PCR profiles, showing co-expression of selected genes in Pdgfrα⁺/CD31⁻ versus Pdgfrα⁻/CD31⁺ cells from the SP and non-SP fractions. The heatmap shows expression as −ΔCt values (blue, low; red, high). Genes are ordered based on the variance of mean expression levels of the four groups. Highlighted genes: green, Cdh5 and Kdr, enriched in PDGFRα⁻/CD31⁺ cells; light red, Pdgfra, Tcf21, Tbx20, Gata4 and Hand2, in PDGFRα⁺/CD31⁻ cells; yellow: Ly6a, in all four subpopulations. n=60–70 for each. (d) Left, gating strategy to identify mutually exclusive PDGFRα⁺ and CD31⁺ cells within the Sca1⁺ population. Right, bar graph showing the % of cells from the indicated populations. Mean±s.e.m.; n=5; *P<0.0001. (e) Density plots show −ΔCt values of the indicated genes in single PDGFRα⁺/CD31⁻ (light red) and PDGFRα⁻/CD31⁺ (light blue) cells from the three sample groups (SP, non-SP and Sca1⁺). Genes are ranked based on the loadings extracted from PC2, which reflect the across-sample variability. (f) PCA of the single-cell expression profiles from the six populations in panel e. (Top) PC1 (43% of variability) captures within-group variance. PC2 (20% of variability) corresponds to between-group variance, mainly separating cells according to PDGFRα/CD31 status, irrespective of their isolation as SP, non-SP or total Sca1⁺ cells. (Bottom) Gene loadings associated with PC1 and PC2. Genes associated with PC1 are unrelated to cell class: consistently absent/low expression across all samples at the left (Nkx2-5) and high expression at the right (Ly6a). PC2 resolves the genes expressed in different cell populations: Cdh5 and Kdr, prevalent in PDGFRα⁻/CD31⁺ cells, at the top, with Pdgfra, Tcf21 and cardiac transcription factors, prevalent in PDGFRα⁺/CD31⁻ cells, at the bottom.