Quantitative methods were established to measure changes in the gross morphology (i.e., shape and area), cytoskeletal organization, and intercalation of mitochondria between the myofibrils in human embryonic stem cell-ventricular cardiomyocytes (hESC-vCMs) that had been treated with different metabolic stimulants (i.e., oleic acid and isoproterenol (ISO)) or electrical stimulation during early differentiation. Cells were double-immunolabelled with α-actinin and mitochondria (COX IV) antibodies, after which images were acquired via confocal microscopy. Image analysis protocols were then designed using Image J software to measure the extent of differentiation of cells within the population. My data demonstrated that treatment with oleic acid or ISO alone or with a combination of the two i...[ Read more ]

Quantitative methods were established to measure changes in the gross morphology (i.e., shape and area), cytoskeletal organization, and intercalation of mitochondria between the myofibrils in human embryonic stem cell-ventricular cardiomyocytes (hESC-vCMs) that had been treated with different metabolic stimulants (i.e., oleic acid and isoproterenol (ISO)) or electrical stimulation during early differentiation. Cells were double-immunolabelled with α-actinin and mitochondria (COX IV) antibodies, after which images were acquired via confocal microscopy. Image analysis protocols were then designed using Image J software to measure the extent of differentiation of cells within the population. My data demonstrated that treatment with oleic acid or ISO alone or with a combination of the two induced a more elongated phenotype of hESC-vCMs than the untreated controls. In addition, cells treated with oleic acid alone or oleic acid and ISO exhibited a more organized (parallel) orientation of myofibrils, and the combined treatment also resulted in enhanced intercalation of mitochondria between the myofibrils. These data suggest that when used alone, these metabolic stimulants have a variable effect on hESC-vCMs but when they are applied together their effect on inducing a more mature phenotype in these cells is enhanced. The possible role of endogenous Ca²⁺ signaling during human embryonic stem cell (hESC) differentiation and maturation into ventricular cardiomyocytes (vCMs) was investigated using hES2 cells that were transduced with the jellyfish apo-aequorin gene using a lentiviral vector. Active holo-aequorin was reconstituted in the apo-aequorin expressed cells, by incubation with f-coelenterazine, the prosthetic co-factor of the holo-aequorin complex. Holo-aequorin is a Ca²⁺-sensitive bioluminescent complex that emits light at ~470 nm on binding with Ca²⁺. The temporal nature of the Ca²⁺ signals generated by aggregates of these cells (called cardiospheres) at the earliest stages of differentiation into hESC-vCMs was then investigated using a luminometer. My data indicated that during the earliest stages of vCM differentiation, no endogenous Ca²⁺ signals could be detected using the current aequorin method. However, as vCMs matured both IP₃Rs and RyRs were shown to be expressed via Ca²⁺ transients generated after the addition of exogenous ATP and caffeine, respective agonists of these SR-located Ca²⁺ release channels.