Generation and characterization of human cardiomyocytes derived from pluripotent stem cells

In vitro development has been widely studied using murine ESCs (mESCs), whose differentiation procedure in culture implies the initial leukemia inhibitory factor (LIF) removal and the formation of cellular aggregates using the “hanging drop” method. These three dimensional (3D) structures, called embryoid bodies (EBs), replicate in vitro the different stages of murine embryonic development. Around differentiation day (dd) 8, clusters of spontaneously beating cells appear in culture; these cells express several transcriptional and structural cardiac markers and were therefore classified generically as cardiomyocytes. Since their isolation, human ESCs have shown different culturing needs from the murine counterpart, and their behavior revealed also minor differentiation plasticity. Cardiac differentiation is the most glaring example of this statement: only a modest proportion of EBs derived from either hESC or hiPSC contains contracting cells. This occurrence leads to the setup of different methods aimed to increase cardiac differentiation during in vitro development of pluripotent stem cells. While some of these procedures retain an initial 3D EB formation, others start from a confluent monolayer. However, CMs differentiated in vitro vary considerably from cells isolated from a mature human heart, because of the absence of humoral factors and organized mechanical and electrical stress. In general, many of the features of hPSC-CMs are reminiscent of normal fetal cells. hPSC-CMs are spontaneously beating cells co-expressing atrial-, ventricular-, and nodal- markers, with unorganized sarcomeres, immature mitochondria, and an expression profile different from adult CMs. The CMs that arise during early hESC or hiPSC in vitro differentiation exhibit spontaneous AP, with a relatively depolarized resting membrane potential, probably due to the temporary absence of the inward rectifier potassium current. The expression of the ion channels and, consequently, the ionic currents will undergo developmental maturation over time, as assessed by modifications in current density and property. hPSC-CMs immaturity is also reflected in their excitation–contraction machinery, lacking clear T-tubuli, disorganized sarcomeric striations, and immature Ca2+ handling. Then the possibility to obtain human CMs in a culture dish is a powerful technique that will allow identification of new drugs in pharmacological studies as well as the identification of new causative genes in modeling genetic pathologies. Nevertheless, newly differentiated human CMs show a low degree of maturation that must be considered and, eventually, overcome by physical, mechanical or cultural stimuli.