High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

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 heart^2,3. 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.......

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.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin EDTA	Gibco	25200-056
0.5 mg/mL BSA (7.5 µmol/L)	Millipore Sigma	A3294
2.9788 g/500 mL HEPES (25 mmol/L)	Millipore Sigma	H4034
AdGCaMP6m	Vector biolabs	1909
Albumin human	Sigma	A9731-1G
alpha actinin antibody	ThermoFisher	MA1-22863
B27	Gibco	17504-044
Blebbistatin	Sigma	B0560
CalBryte 520AM	AAT Bioquest	20650
CELLvo MatrixPlus 96wp	StemBiosys	N/A	https://www.stembiosys.com/products/cellvo-matrix-plus
CHIR99021	LC Laboratories	c-6556
Clear Assay medium (fluorobrite)	ThermoFisher	A1896701	For adenovirus transduction
DAPI	ThermoFisher	62248
DMEM:F12	Gibco	11330-032
FBS (Fetal Bovine Serum)	Sigma	F4135-500ML
FluoVolt	ThermoFisher	F10488
HBSS	Gibco	14025-092
iCell CM maintenance media	FUJIFILM/Cellular Dynamics	M1003
iCell2 CMs	FUJIFILM	1434
Incucyte Zoom	Sartorius
iPS DF19-9-11T.H	WiCell
Isoproterenol	MilliporeSigma	CAS-51-30-9
IWP4	Tocris	5214
L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate	Sigma	A8960-5g
L-glutamine	Gibco	A2916801
LS columns	Miltenyii Biotec	130-042-401
MACS Buffer (autoMACS Running Buffer)	Miltenyii Biotec	130-091-221
Matrigel	Corning	354234
MitoTracker Red	ThermoFisher	M7512
Nautilus HTS Optical Mapping	CuriBio		https://www.curibio.com/products-overview
Nikon A1R Confocal Microscope	Nikon
nonessential amino acids	Gibco	11140-050
pre-separation filter	Miltenyii Biotec	130-041-407
PSC-Derived Cardiomyocyte Isolation Kit, human	Miltenyii Biotec	130-110-188
Pulse	CuriBio		https://www.curibio.com/products-overview
Quadro MACS separator (Magnet)	Miltenyii Biotec	130-091-051
Retinoic acid	Sigma	R2625
RPMI 1640	Gibco	11875-093
RPMI 1640 (+HEPES, +L-Glutamine)	Gibco	22400-089
StemMACS iPS-Brew XF	Miltenyii Biotec	130-107-086
TnI antibody (pan TnI)	Millipore Sigma	MAB1691
Versene (ethylenediaminetetraacetic acid - EDTA solution)	Gibco	15040-066
Y-27632 dihydrochloride	Tocris	1254
β-mercaptoethanol	Gibco	21985023