When cultured as aggregates in suspension, embryonic stem cells form three-dimensional, multicellular complexes called embryoid bodies. These spherical structures resemble the egg cylinders of embryos 6 to 8 days post-fertilization.
After approximately 4 to 8 days, these embryoid bodies typically contain skeletal structures between the inner mesenchymal cells and outer epithelial cells, which begin to contract spontaneously. These skeletal structures express key cardiac proteins and exhibit contractile properties, action potentials, and chronotropic responses similar to those described for embryoid bodies.
Early embryonic stem cells derived from embryonic Purkinje cells develop. stem cells that form in epithelial cells resemble pacemaker cells and primary cardiomyocytes. Later, atrial, ventricular, and ganglion cells, histaminergic cells, and Cardiac transcription factors Nkx2.5, GATA-4, and MEF2C are expressed early during differentiation, while more mature stem cells express atrial natriuretic factor (ANF), sodium-calcium exchanger, and phospholamban, as well as structural proteins such as myosin light chain (MLC)-2v, myosin heavy chain α and β, titin (Z and M domains), α-actinin, cardiac troponin T, α-actin, and M protein.
