Computational protocol: Induced Pluripotent Stem Cell–Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function

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Protocol publication

[…] Action potentials were recorded from spontaneously contracting small cell clusters or isolated cells generated by dissociating iPSC-CMs after plating on top of gelatin or fibronectin-coated glass coverslips. Specifically, the EBs generated by our differentiation protocol were inspected under microscope to identify contracting areas. In general, ≈25% of the EBs demonstrated spontaneous contractions. To obtain cardiomyocytes for the electrophysiological experiments, we surgically isolated small contracting areas from the whole contracting EB. These areas were then enzymatically treated to yield spontaneously contracting small clusters (containing ≤5 cells) and single cardiomyocytes, used for action potential recordings. The pacemaker current If was recorded only from single cardiomyocytes. The patch pipette solution contained (mmol/L) 120 KCl, 1 MgCl2, 3 Mg-ATP, 10 HEPES, and 10 EGTA (pH, 7.3). The bath solution contained (mmol/L) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH, 7.4; all materials were purchased from Sigma-Aldrich, St. Louis, MO). Axopatch 200B, Digidata 1322, and pClamp10 (Molecular Devices, Sunnyvale, CA) were used for data amplification, acquisition, and analysis. [...] Normally distributed data are presented as mean±SEM. The effects of Ivabradine on mean heart beat rate were analyzed using a 1-way ANOVA followed by the Holm–Sidak method. P<0.05 per Holm–Sidak method was considered statistically significant. In analyzing the percentage of matching beats for in vivo experiments, statistical significance was examined by a 1-way ANOVA for repeated measures. Post hoc analysis was performed with the Bonferroni correction. Offline data analysis and statistical evaluations were performed with SigmaPlot (Systat Software Inc, San Jose, CA) and Prism (GraphPad Software, San Diego, CA). In all tests, P<0.05 was considered significant.The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the article as written. […]

Pipeline specifications

Software tools pCLAMP, SigmaPlot
Applications Miscellaneous, Patch-clamp
Organisms Canis lupus familiaris, Homo sapiens
Chemicals Epinephrine