Figure 1

Enrichment of α-MHC⁺ cells isolated from the α-MHC⁺ ES cell lineage after puromycin treatment. (a) Progressive purification of α-myosin heavy chain (MHC)⁺ cardiac cell aggregates after treatment of the 8-day-old embryoid bodies (EBs) with 4 μg/ml puromycin for 7 days. Puromycin containing medium was refreshed every second day. (b) Reverse transcription (RT)-polymerase chain reaction (PCR) analysis of α-MHC expression during EB differentiation (for RT-PCR conditions and primers, see Additional data file 3). (c,d) Cells from 15-day-old EBs and 15-day-old puromycin purified α-MHC⁺ aggregates were dissociated by trypsinization and the purity of the α-MHC⁺ cells in the 15-day-old EBs (panel c) and in the 15-day-old α-MHC⁺ aggregates (panel d) was examined by fluorescence-activate cell sorting analysis. (e) Characterization of ES cell derived cardiomyocytes by immunocytochemistry. α-MHC⁺ cardiomyocytes were dissociated with collagenase B and plated on fibronectin coated coverslips. (e) Enhanced green fluorescent protein (EGFP) expression of single α-MHC⁺ cells with different morphologies (subpanels a, c, and e). Detection of α-cardiac actinin (subpanels b, d, and f) and connexion-43 (subpanels g and h) was performed using anti-cardiac actinin (1:400) and anti-connexin-43 (1:400). Secondary detection was performed with anti-mouse-IgG₁-AlexaFluor555 and anti-rabbit-Ig-AlexaFluor647. Hoechst dye was used to stain nuclei. Bars in panel e (subpanels a to f) are 50 μm; bar in panel e (subpanel g) is 20 μm; and bar in panel (subpanel h) is 7.5 μm.