Embryonic Stem Cell-Derived Cardiomyocyte Heterogeneity and the Isolation of Immature and Committed Cells for Cardiac Remodeling and Regeneration
Figure 1
Characterization of syNP4 ES cell derived-CMs. (a) Traits of ESC-CM generated using the hanging drop technique. Following differentiation and plating, puromycin-resistant cells aggregate in clusters and show spontaneous contractions. Day 8-9 CMs (one or two days after plating) readily incorporate BrdU (green) and dividing cells can be readily identified by the use of an antibody against a phosphorylated form of histone H3. (b) As a function of differentiation time, syNP4 derived-CMs begin to express p21CIP and p27KIP1 markers of contact inhibition and cell cycle checkpoint control, but the cells also continue to express transcripts to α-smooth muscle actin (α-SMA), a marker of immature cardiomyocytes. (c) Using the mass culture system, plated puromycin-resistant syNP4-derived CMs cluster similarly to that described in 1A above (see inset). Cells were immunostained with an antibody against cardiac TnT (cTnT), demonstrating that these clusters consist of CMs. (d) By flow cytometry, the purity of the cells has been determined to range from 75 to 92% (), depending on the cultivation and selection conditions. LC: loading control. Size markers = 50 μm.
