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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to β-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Introduction

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an attractive human-based platform to model a large variety of cardiac diseases in vitro1,2,3,4,5,6,7,8. Moreover, iPSC-CMs can be used for the prediction of patient responses to novel or existing drugs, to screen hit compounds, and develop new personalized drugs9,10. Howev....

Protocol

The experiments using adult rat cardiomyocytes in this study were conducted with approved Institutional Animal Care and Use Committee (IACUC) protocols of Icahn School of Medicine at Mount Sinai. The adult rat cardiomyocytes were isolated from Sprague Dawley rat hearts by the Langendorff-based method as previously described16.

1. Preparation of Media

  1. Prepare hiPSC media.
    1. Equilibrate the supplement and the basal medium to room temperature (RT). En.......

Representative Results

The protocol described in Figure 1A generated highly pure cardiomyocytes that acquire a ventricular/adult-like phenotype with time in culture. As assessed by immunofluorescence staining for the atrial and ventricular myosin regulatory light chain 2 isoforms (MLC2A and MLC2V, respectively), the majority of the cells generated by this protocol were MLC2A-positive at day 30 after induction of cardiac differentiation, while MLC2V was expressed in much lower amounts at the same t.......

Discussion

Critical steps for using human iPSC-CMs as experimental models are: 1) generating high-quality cardiomyocytes (CMs) that can ensure the consistent performance and reproducible results; 2) allowing the cells to mature in culture for at least 90 days to adequately assess their phenotype; 3) performing electrophysiological studies, e.g. calcium (Ca2+) transient measurements, to provide a physiologically relevant functional characterization of human iPSC-CMs. We developed a monolayer-based differentiation method t.......

Acknowledgements

This research was supported by AHA Scientist Development Grant 17SDG33700093 (F.S.); Mount Sinai KL2 Scholars Award for Clinical and Translational Research Career Development KL2TR001435 (F.S.); NIH R00 HL116645 and AHA 18TPA34170460 (C.K.).

....

Materials

NameCompanyCatalog NumberComments
Anti-Actin, α-Smooth Muscle antibody, Mouse monoclonalSigma AldrichA5228
Alexa Fluor 488 goat anti mouseInvitrogenA11001
Alexa Fluor 555 goat anti rabbitInvitrogenA21428
B27 SupplementGibco17504-044
B27(-) insulin SupplementGibcoA18956-01
CHIR-99021SelleckchemS2924
DAPI nuclear stainThermoFisherD1306
DMEM/F12 (1:1) (1X) + L- Glutamine + 15mM HepesGibco11330-032
Double Ended Cell lifter, Flat blade and J-HookCelltreat229306
Falcon Multiwell Tissue Culture Plate, 6 wellCorning353046
Fluidic inline heaterLive Cell InstrumentIL-H-10
Fura-2, AMInvitrogenF1221
hESC-qualified matrixCorning354277Matrigel Matrix
hPSC mediaGibcoA33493-01StemFlex Basal Medium
IWR-1Sigma AldrichI0161
Live cell imaging chamberLive Cell InstrumentEC-B25
MLC-2A, Monoclonal Mouse AntibodySynaptic Systems311011
Myocyte calcium and contractility systemIonoptixISW-400
Myosin Light Chain 2 Antibody, Rabbit Polyclonal (MLC2V)Proteintech10906-1-AP
Nalgene Rapid Flow Sterile Disposable Filter units with PES MembraneThermoFisher124-0045
PBS with Calcium and MagnesiumCorning21-030-CV
PBS without Calcium and MagensiumCorning21-031-CV
Premium Glass Cover SlipsLab Scientific7807
RPMI medium 1640 (-) D-glucose (1X)Gibco11879-020
RPMI medium 1640 (1X)Gibco11875-093
Sodium L-lactateSigma AldrichL7022
StemFlex SupplementGibcoA33492-01
ThiazovivinTocris3845
Trypsin-EDTA (0.25%)ThermoFisher25200056
Tyrode's solutionBoston BioproductsBSS-355wAdjust pH at 7.2. Add 1.2mM Calcium Chloride

References

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