Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

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 vitro^{1,2,3,4,5,6,7,8}. 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 drugs^9,10. 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.......

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.......

Materials

Name	Company	Catalog Number	Comments
Anti-Actin, α-Smooth Muscle antibody, Mouse monoclonal	Sigma Aldrich	A5228
Alexa Fluor 488 goat anti mouse	Invitrogen	A11001
Alexa Fluor 555 goat anti rabbit	Invitrogen	A21428
B27 Supplement	Gibco	17504-044
B27(-) insulin Supplement	Gibco	A18956-01
CHIR-99021	Selleckchem	S2924
DAPI nuclear stain	ThermoFisher	D1306
DMEM/F12 (1:1) (1X) + L- Glutamine + 15mM Hepes	Gibco	11330-032
Double Ended Cell lifter, Flat blade and J-Hook	Celltreat	229306
Falcon Multiwell Tissue Culture Plate, 6 well	Corning	353046
Fluidic inline heater	Live Cell Instrument	IL-H-10
Fura-2, AM	Invitrogen	F1221
hESC-qualified matrix	Corning	354277	Matrigel Matrix
hPSC media	Gibco	A33493-01	StemFlex Basal Medium
IWR-1	Sigma Aldrich	I0161
Live cell imaging chamber	Live Cell Instrument	EC-B25
MLC-2A, Monoclonal Mouse Antibody	Synaptic Systems	311011
Myocyte calcium and contractility system	Ionoptix	ISW-400
Myosin Light Chain 2 Antibody, Rabbit Polyclonal (MLC2V)	Proteintech	10906-1-AP
Nalgene Rapid Flow Sterile Disposable Filter units with PES Membrane	ThermoFisher	124-0045
PBS with Calcium and Magnesium	Corning	21-030-CV
PBS without Calcium and Magensium	Corning	21-031-CV
Premium Glass Cover Slips	Lab Scientific	7807
RPMI medium 1640 (-) D-glucose (1X)	Gibco	11879-020
RPMI medium 1640 (1X)	Gibco	11875-093
Sodium L-lactate	Sigma Aldrich	L7022
StemFlex Supplement	Gibco	A33492-01
Thiazovivin	Tocris	3845
Trypsin-EDTA (0.25%)	ThermoFisher	25200056
Tyrode's solution	Boston Bioproducts	BSS-355w	Adjust pH at 7.2. Add 1.2mM Calcium Chloride