| Objective To investigate the effects of electrical stimulation on thedifferentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytein vitro.Methods1.Thawed iPSCs derived from mice and plated them on theMEF cell feeder layer which were pretreated by Mitomycin C. Themorphological changes under optical microscope,the expression of greenfluorescent protein(GFP) promoted by stem cell totipotential genes Oct-4under fluorescence microscopy and the alkaline phosphatase(AP) stainingwere used to identify the undifferentiation iPSCs.2.Hanging-drop method was applied to induce iPSCs to formembryoid bodies(E Bs)and vitamin C was used for inducing theirdifferentiation. Electrical stimulation was used or not in the whole processof induction, iPSCs were divided into electrical stimulation group andnon-stimulation group. Growth situations were observed, the time point thatbeating EBs in each group appeared was recorded and their numbers were counted. The differentiation rate of cardiomyocyte was calculated.Furthermore, expression of cardiac troponin T(cTnT) was observed byconfocal laser scanning microscope (CLSM) and mRNA expression levelsof the related genes Oct-4, GATA-4and α-MHC were analyzed by RT-PCR.Results1. The iPSCs clones plated on the MEF cell feeder layerwere adherent and scattered like clone groups.The cells in the center ofiPSCs clones were arranged tightly. The refractivity at the edge of theclones was strong,which made the boundaries between the iPSCs and theMEF cell feeder layer clear. The iPSCs clones emitted green fluorescenceunder fluorescence microscopy (Oct-4-GFP positive).AP staining showedthat the iPSCs clones were dyed deep purple.2. Spontaneously beating EBs could be observed in bothgroups in the course of induction. Compared with the non-stimulationgroup, beating EBs in electrical stimulation group differentiatedfaster,spontaneous beating appeared earlier[3rdday vs5thday] and thedifferentiation rate of cardiomyocytes was higher [(68.89±5.09)%vs.(52.22±3.85)%, P<0.05].3. Cells in the beating areas of both groups expressedcardiac-specific troponin c-TnT, whereas the electrical stimulation groupshowed a clearer cytoskeleton.4. The mRNA level of Oct-4decreased as the induction timewent by. Furtheremore, it decreased faster in the electrial stimulated group (P<0.05). In addition, mRNA expressions of GATA4and α-MHC could bedetected in both groups,while ecletrical stimulation group had higherexpressions than non-stimulated group at the same point-in-time (P<0.05).Conclusion In the surroundings with LIF and the MEF cell feederlayer which were pretreated by Mitomycin C, iPSCs maintainedundifferentiated; The electrical stimulation which could simulated cardiacelectrical microenvironment to some extent facilitated the differentiation ofiPSCs induced by vitamin C into cells with myocardial cell phenotypiccharateristics in vitro. |
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