Isolation and functional characterization of early Mesp1-GFP–expressing cells. (A–C) Expression of cardiovascular markers after 8 d of differentiation of the indicated cell populations isolated at D3. Cardiac and endothelial differentiation were quantified by FACS using a cardiac-specific isoform of the troponin T (cTNT; A) and the endothelial marker CD31 (B). SMC differentiation was assessed by counting the percentage of cells expressing smooth muscle actin (SMA) on cytospin slides (C; also see Fig. S1 A). n = 4. (D) Relative mRNA expression of cardiovascular markers in Mesp1-GFP positive–derived cells (black bars) and in all sorted cells (gray bars) assessed by real-time RT-PCR 8 d after replating. Results are normalized to the expression of the different transcripts in the Mesp1-GFP negative (Neg)–derived cells (white bars). n = 4. (E) Immunostaining for cTNT (CMs), VE-cadherin (VE-cadh; ECs), and SMA (SMCs) in individual colonies obtained after the replating at the clonal density of isolated Mesp1-GFP cells at D3 and cultured for 13 d. Bars, 50 µm. (F) Quantification of colonies expressing cardiovascular (cTNT and VE-cadherin), cardiac (cTNT), and endothelial (VE-cadherin) markers as obtained in E. n = 3. (G) RT-PCR analysis of cardiovascular markers in colonies derived from a single Mesp1-GFP isolated cell in 96 wells after 13 d of differentiation. Only clones positive for β-actin are shown, with dividing lines indicating the removal of intervening lanes from the gels. Samples tested in different experiments are shown as distinct panels with their respective positive (+) and negative (−) control samples. (H) Cardiovascular potential of Mesp1-GFP isolated cells at D3 of ESC differentiation, which were transplanted under the kidney capsule of nonobese diabetic/severe combined immunodeficient mice. Cardiovascular differentiation was assessed after 4 wk by immunostaining for cTNT, VE-cadherin, and SMA. n = 3. Bars, 100 µm. Error bars indicate means ± SEM.