Isolation and functional characterization of early MCPs using a combination of monoclonal antibodies. (A) Cell surface marker expression in Mesp1-GFP–expressing cells as measured by real-time RT-PCR in isolated Mesp1-GFP–expressing cells at D3. Results are normalized for the mRNA expression in GFP-negative cells. n = 3. (B) Detection of CXCR4, PDGFRa, and Flk1 by FACS at D3 in all living cells (top) and in the Mesp1-GFP population (bottom). (C) Multicolor FACS analysis gated on Mesp1-GFP cells of CXCR4, PDGFRa, and Flk1 expression at D3 and D4. (D) Enrichment of Mesp1 expression in TP cells at D3 as measured by RT-PCR on FACS-isolated cells. Results are normalized for the relative transcript expression in all sorted cells. n = 3. (E) Temporal expression of CXCR4, PDGFRa, and Flk1 during ESC differentiation as detected by FACS. n = 2. (F) Combined detection of CXCR4, PDGFRa, and Flk1 expression at D3 and D4 in all living cells. (C and F) Percentages of cells in each quadrant are shown, and the percentage of CXCR4/PDGFRa/Flk1 TP cells are shown in parentheses. (G–I) Cardiac (G), endothelial (H), and SMC (I; also see Fig. S1 C) differentiation of TP cells as performed in Fig. 2 (A–C). n = 4. Error bars indicate means ± SEM.