Titration of Activin A and BMP4 improves cardiomyocyte yield.

EBs were formed from E14.Nkx2–5-GFP mES cells as above. On day 4 EBs were stimulated with Activin A 4 ng/ml and a range of BMP4 concentrations or unstimulated (0 ng/ml of both Activin A and BMP4). 28 hours (day 5+4 hours) after induction of cardiac mesoderm, EBs were dissociated with TrypLE. A subset of cells was analyzed for the expression of Nkx2.5 (GFP) using flow cytometry. The remaining cells were plated for continued differentiation into cardiomyocytes. On day 8 cells were stained for cardiac troponin (cTroponin) and analyzed for the percentage of cells expressing Nkx2.5 (GFP) and cTroponin using flow cytometry. Data are from a representative experiment that was performed at least 10 times utilizing 5 different lots of Activin A and 5 different lots of BMP4 for titration.