Developmental changes of cardiomyocytes differentiated from human embryonic stem cells: combined molecular and functional approaches

Background: Human embryonic stem cells (hESC) are emerging as a promising cell source for cardiac cell-based therapy. To this aim, it is mandatory to assess the functional and molecular properties of hESC-derived cardiac myocytes (CM) in vitro. Methods and results: hESC were differentiated in spontaneously beating CM and characterized over time of differentiation, using patch-clamp or intracellular recordings and RT-PCR to evaluate mRNA expression of channels. CM were pooled in two groups: early CM (eCM, from 15 to 40 days of differentiation) and late CM (lCM, from 50 to 110 days). The L-type Ca2+, the rapid delayed rectifier and the funny currents (ICaL, IKr, and If ) were recorded both in hESC and CM; the transient outward and inward rectifier currents (Ito and IK1) were present in CM only. Channel mRNAs were always present when currents were recorded. Interestingly, during cardiac differentiation, channel isoforms expression became similar to that shown by adult human heart and currents underwent specific modifications: maximal Ito and IK1 densities increased by 81% and 255%, respectively, from eCM to lCM; maximal ICaL density increased by 560% from hESC to CM. If activation rate slowed down, as time constant of activation increased by 7% and 258 % in eCM and lCM, respectively, as compared to hESC. Intracellular recordings in beating clusters of CM revealed changes in action potential parameters: maximum diastolic potential varied from -47±7 in eCM to -53±3 mV in lCM; Vmax increased from 4±0.6 in eCM to 6±0.4 V/s in lCM; action potential duration at 50% and 90% of repolarization lengthened from 167±10 to 265±39 ms and from 200±11 to 341±48 ms, in eCM and lCM; the slope of diastolic depolarization rate (DDR) was flattened from 23± 5.8 in eCM to 10±1.0 mV/s in lCM; the rate of spontaneous firing was reduced from 36±6 in eCM to 26±3 bpm in lCM . Application of specific blockers caused the following: E4031 revealed a key role of IKr in the regulation of excitability threshold both in eCM and lCM; blockade of IK1 by BaCl2 and of If by zatebradine exerted opposite effects on DDR and spontaneous activity, BaCl2 increasing and zatebradine decreasing them; isoprenaline produced a positive chronotropic effect both in eCM and lCM; ICa,L blockade by lacidipine reduced plateau duration and height. Conclusions: hESC-derived CM mature over time during in vitro differentiation and approach an adult phenotype.