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In This Article

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

Summary

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Abstract

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating.

Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols.

High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Introduction

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been validated on an international scale, and are available for in vitro cardiotoxicity screening1. Highly pure hiPSC-CMs can be generated in virtually unlimited numbers, cryopreserved, and thawed. Upon replating, they also reanimate and begin contracting with a rhythm reminiscent of the human heart2,3. Remarkably, individual hiPSC-CMs couple to each other and form functional syncytia that beat as a single tissue. Nowadays, hiPSCs are routinely derived from patient blood samples, so any person can be re....

Protocol

hiPSC usage in this protocol was approved by the University of Michigan HPSCRO Committee (Human Pluripotent Stem Cell Oversight Committee). See the Table of Materials for a list of materials and equipment. See Table 1 for media and their compositions.

1. Thawing and plating commercially available cryopreserved hiPSC-CMs for maturation on a maturation-inducing extracellular matrix (MECM)

  1. Warm all the reagents to room temperature and.......

Representative Results

hiPSC-CM maturation characterized by phase contrast and immunofluorescent confocal imaging
The timeline for ECM-mediated maturation of commercially available hiPSC-CMs using MECM coated 96-well plates is presented in Figure 1A. These data are collected using commercially available cardiomyocytes that arrive in the laboratory as cryopreserved vials of cells. Each vial contains >5 × 106 viable cardiomyocytes. The cells are ~98% pure and rigorously tes.......

Discussion

There are several different approaches to in vitro cardiotoxicity screening using hiPSC-CMs. A recent "Best Practices" paper on the use of hiPSC-CMs presented the various in vitro assays, their primary readouts, and importantly, each assay's granularity to quantify human cardiac electrophysiological function20. In addition to using membrane-piercing electrodes, the most direct measure of human cardiac electrophysiological function is provided by VSDs. VSD assay readou.......

Acknowledgements

This work has been supported by NIH grants HL148068-04 and R44ES027703-02 (TJH).

....

Materials

NameCompanyCatalog NumberComments
0.25% Trypsin EDTAGibco25200-056
0.5 mg/mL BSA (7.5 µmol/L)Millipore SigmaA3294
2.9788 g/500 mL HEPES (25 mmol/L)Millipore SigmaH4034
AdGCaMP6mVector biolabs1909
Albumin humanSigmaA9731-1G
alpha actinin antibodyThermoFisherMA1-22863
B27Gibco17504-044
BlebbistatinSigmaB0560
CalBryte 520AMAAT Bioquest20650
CELLvo MatrixPlus 96wpStemBiosysN/Ahttps://www.stembiosys.com/products/cellvo-matrix-plus
CHIR99021LC Laboratoriesc-6556
Clear Assay medium (fluorobrite)ThermoFisherA1896701For adenovirus transduction
DAPIThermoFisher62248
DMEM:F12Gibco11330-032
FBS (Fetal Bovine Serum)SigmaF4135-500ML
FluoVoltThermoFisherF10488
HBSSGibco14025-092
iCell CM maintenance mediaFUJIFILM/Cellular DynamicsM1003
iCell2 CMsFUJIFILM1434
Incucyte Zoom Sartorius
iPS DF19-9-11T.HWiCell
IsoproterenolMilliporeSigmaCAS-51-30-9
IWP4Tocris5214
L-ascorbic acid 2-phosphate sesquimagnesium salt hydrateSigmaA8960-5g
L-glutamineGibcoA2916801
LS columnsMiltenyii Biotec130-042-401
MACS Buffer (autoMACS Running Buffer)Miltenyii Biotec130-091-221
MatrigelCorning354234
MitoTracker RedThermoFisherM7512
Nautilus HTS Optical Mapping CuriBiohttps://www.curibio.com/products-overview
Nikon A1R Confocal MicroscopeNikon
nonessential amino acidsGibco11140-050
pre-separation filterMiltenyii Biotec130-041-407
PSC-Derived Cardiomyocyte Isolation Kit, humanMiltenyii Biotec130-110-188
PulseCuriBiohttps://www.curibio.com/products-overview
Quadro MACS separator (Magnet)Miltenyii Biotec130-091-051
Retinoic acidSigmaR2625
RPMI 1640 Gibco11875-093
RPMI 1640 (+HEPES, +L-Glutamine)Gibco22400-089
StemMACS iPS-Brew XFMiltenyii Biotec130-107-086
TnI antibody (pan TnI)Millipore SigmaMAB1691 
Versene (ethylenediaminetetraacetic acid - EDTA solution)Gibco15040-066
Y-27632 dihydrochlorideTocris1254
β-mercaptoethanolGibco21985023

References

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HiPSC CMCardiotoxicity ScreeningHigh throughputMature CardiomyocytesExtracellular MatrixEP AssayMACS

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