Functional Maturation of hiPSC-CMs
Utility of hiPSC-CMs for Truly Predictive Assays
Human iPSC-CM’s inherent morphology, structural hallmarks, calcium handling mechanism, electrophysiology, and gene expression profile resemble neonatal cardiomyocytes which limits its application for safety/toxicity assessment, drug discovery and even disease modeling.
Long-term electrical pacing is quickly becoming the preferred method of choice to improve the maturation status of hiPSC-CMs in a consistent and scalable manner. Recent discoveries using an Integrated Pacing Function to pace hiPSC-CM indicate that the functionally mature hiPSC-CM provide a significantly improved model that can be used for various applications including safety/tox assessment, drug discovery, and cardiac disease modeling. The xCELLigence CardioECR system utilizes planar impedance electrodes to electrically pace hiPSC-CM and achieve reliable pacing of hiPSC-CM.
Long-term Electrical Pacing Improves Maturation of hiPSC-CM
Human iPSC-CM were electrically paced with the xCELLigence CardioECR system using escalating frequency of pacing over 3 weeks and then assessed for gene expression, protein expression, and functional assays.
Improve Gene Expression by Electrical Pacing
|The expression of key maturation gene markers in non-paced (CTRL) and paced cardiomyocytes was determined by RT-PCR. The human fetal heart (FH) and adult left ventricle (ALV) were included as benchmarks of known level of maturation. The amount of RNA was normalized to the expression of GAPDH. Gene expression level was presented as a fold change to the human fetal heart. The data are presented as mean± SD, N=4. (* P< 0.05).|
Improve Protein Expression by Electrical Pacing
|Protein expression level of sarcomeric α-actinin, myosin heavy chain, cTNT, SERCA2a and PLN in paced and non-paced cells were determined by western blot analysis, which was quantified using protein to GAPDH ratio and further normalized to the non-paced cardiomyocytes. The data are presented as mean ± SD, N=4. (* P< 0.05).|
|Isoproterenol treatment in CTRL and paced cardiomyocytes (a-d). Non-paced and paced cells were treated with ISO at 10, 100 and 1000 nM. The averaged percentage change of beating amplitude (a) and beating rate (b) to the time-matched DMSO control were calculated in non-paced/CTRL (in blue) and paced cells (in orange) at 30 min after ISO exposure. The data are presented as mean ± SD, N=3. The IMP waveforms were continuously measured for 30 s before and after drug addition, which were further calculated to averaged single waveforms (C). Representative averaged IMP waveforms obtained before (in black) and after ISO addition (paced cells, orange trace). (See Drug Discovery)|
Human iPSC-CMs possess an inherent negative impedance amplitude-frequency relationship which is reversed after electrical pacing.
Overcome cardiomyocyte neonatal phenotype expression to obtain truly predictive assays
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