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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.
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.
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....
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)
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.......
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.......
This work has been supported by NIH grants HL148068-04 and R44ES027703-02 (TJH).
....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 1640Â | 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 | MAB1691Â | |
Versene (ethylenediaminetetraacetic acid - EDTA solution) | Gibco | 15040-066 | |
Y-27632 dihydrochloride | Tocris | 1254 | |
β-mercaptoethanol | Gibco | 21985023 |
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