Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Summary

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Abstract

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Introduction

Electrophysiological modeling of cardiomyocytes and the construction of efficient platforms for cardiac drug screening is essential for the development of therapeutic strategies for a variety of arrhythmic disorders. Rapid expansion of induced pluripotent stem cell (iPSC) technology has produced promising inroads into human disease modelling and pharmacological investigation using isolated patient derived cardiomyocytes (iPSC-CM). “Gold standard” techniques for electrophysiological characterization of these cells through patch-clamp (current-clamp) can quantify action potential (AP) morphology and duration, however, this method is incredibly complex and slow, and not ....

Protocol

1. Cellular preparations

NOTE: Human iPSCs used in this protocol were derived from healthy donors and differentiated in monolayers using fully defined small molecule modulation of WNT signaling and lactate purification techniques as previously described^12,13,14. iPSC-CMs were maintained every 2-3 days with a culture medium outlined below.

1. Prepare a culture medium of basal medium (RPMI 1640) and 2% supplement (B27). Store at 4 °C. Use at room temperature (RT).
2. Prepare a plating medium of basal medium (RPMI 1640), 2% supplement ....

Results

Figure 3: Optical action potential (AP) profiles of isolated native cardiomyocytes and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM). (A) Representative optical AP of a single murine cardiomyocyte (center) with Mean .......

Discussion

Here we describe a basic protocol to easily acquire detailed AP profiles from isolated iPSC-CMs suitable for electrophysiological modelling and cardiac drug screening. We detect regular, robust APs from our sparsely seeded iPSC-CMs which suggests both indicator functionality and methodological fidelity.

Due to the wide spectrum of commercial methodologies for iPSC reprogramming and lack of standardization for cardiac differentiation protocols, iPSC based models can show immense variability in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
0.25 Trypsin EDTA	Gibco	25200056
B27 Supplement	Gibco	17504044
CaCl2	Carl Roth	HN04.2
D(+)-Glucose anhydrous BioChemica	ITW Reagents	A1422
Fetal Bovine Serum	Gibco	10270-106
FluoVolt Membrane Potential Kit	Invitrogen	F10488
HEPES	Carl Roth	HN77.4
KCl	Sigma-Aldrich	6781.1
Lamanin	Sigma-Aldrich	114956-81-9
Matrigel	BD	354230
NaCl	Sigma-Aldrich	9265.2
Nifedipine	Sigma-Aldrich	21829-25-4
Penicillin/Streptomycin	Invitrogen	15140
ROCK Inhibitor Y27632	Stemolecule	04-0012-10
RPMI 1640 Medium	Gibco	61870010
Versene EDTA	Gibco	15040033
Equipment
495LP Dichroic Beamsplitter	Chroma Technology
Axopatch 200B Amplifier	Molecular Devices
Circle Coverslips, Thickness 0	Thermo Scientific	CB00100RA020MNT0
Digidata 1550B	Molecular Devices
Dual OptoLED Power Supply	Cairn Research
ET470/40x Excitation Filter	Chroma Technology
ET535/50m	Chroma Technology
Etched Neubauer Hemacytometer	Hausser Scientific
Filter Cubes	Cairn Research
IX73 Inverted Microscope	Olympus
MonoLED	Cairn Research
Multiport Adaptors	Cairn Research
Myopacer Cell Stimulator	IonOptix
Optomask Shutter	Cairn Research
Optoscan System Controller	Cairn Research
PH-1 Temperature Controlled Platform	Warner Instruments
Photomultiplier Detector	Cairn Research
PMT Amplifier Insert	Cairn Research
PMT Supply Insert	Cairn Research
RC-26G Open Bath Chamber	Warner Instruments
SA-OLY/2AL Stage Adaptor	Olympus
T565lpxr Dichroic Beamsplitter	Chroma Technology
T660lpxr Dichroic Beamsplitter	Chroma Technology
TC-20 Dual Channel Temperature Controller	npi Electronic
UPLFLN 40X Objective	Olympus
USB 3.0 Colour Camera	Imaging Source
Software
Clampex 11.1	Molecular Devices
Clampfit 11.1	Molecular Devices
IC Capture 2.4	Imaging Source
Prism 8	Graphpad