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 represented using in vitro hiPSC-CM cardiotoxicity screening assays^4,5. This creates the opportunity to perform "Clinical Trials in a Dish", with significant representation from diverse populations⁶.

One critical advantage over existing animal and animal cell cardiotoxicity screening approaches is that hiPSC-CMs utilize the full human genome and offer an in vitro system with genetic similarities to the human heart. This is especially attractive for pharmacogenomics and personalized medicine - the use of hiPSC-CMs for medication and other therapy development is predicted to provide more accurate, precise, and safe medication prescriptions. Indeed, two-dimensional (2D) hiPSC-CM monolayer assays have proven to be predictive of medication cardiotoxicity, using a panel of clinically used medications with a known risk of causing arrhythmias^1,7,8,9. Despite the vast potential of hiPSC-CMs and the promise to streamline and make drug development cheaper, there has been a reluctance to use these novel assays^10,11,12.

Until now, one major limitation of widespread adoption and acceptance of hiPSC-CM screening assays is their immature, fetal-like appearance, as well as their function. The critical issue of hiPSC-CM maturation has been reviewed and debated in the scientific literature ad nauseum^13,14,15,16. Likewise, many approaches have been employed to promote hiPSC-CM maturation, including extracellular matrix (ECM) manipulations in 2D monolayers and the development of 3D engineered heart tissues (EHTs)^17,18. At the moment, there is a widely held belief that the use of 3D EHTs will provide superior maturation relative to 2D monolayer-based approaches. However, 2D monolayers provide a higher efficiency of cell utilization and increased success in plating compared to 3D EHTs; 3D EHTs utilize greater numbers of cells, and often require the inclusion of other cell types that can confound results. Therefore, in this article, the focus is on using a simple method to mature hiPSC-CMs cultured as 2D monolayers of electrically and mechanically coupled cells.

Advanced hiPSC-CM maturation can be achieved in 2D monolayers using an ECM. The 2D monolayers of hiPSC-CMs can be matured using a soft, flexible polydimethylsiloxane coverslip, coated with basement membrane matrix secreted by an Engelbreth-Holm-Swarm mouse sarcoma cell (mouse ECM). In 2016, reports showed that hiPSC-CMs cultured on this soft ECM condition matured functionally, displaying action potential conduction velocities near adult heart values (~50 cm/s)¹⁸. Further, these mature hiPSC-CMs displayed many other electrophysiological characteristics reminiscent of the adult heart, including hyperpolarized resting membrane potential and expression of K_ir2.1. More recently, reports identified a human perinatal stem cell-derived ECM coating that promotes the structural maturation of 2D hiPSC-CMs¹⁹. Here, easy-to-use methods are presented to structurally mature 2D hiPSC-CM monolayers for use in high-throughput electrophysiological screens. Further, we provide validation of an optical mapping instrument for the automated acquisition and analysis of 2D hiPSC-CM monolayer electrophysiological function, using voltage-sensitive dyes (VSDs) and calcium-sensitive probes and proteins.

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 rehydrate the MECM plates with Hank's balanced salt solution (HBSS) or phosphate-buffered saline (PBS) containing calcium and magnesium, for 1 h prior to cardiomyocyte plating (200 µL of buffer per well of a 96-well plate).
2. Wash the MECM plates 2x with HBSS or PBS containing calcium and magnesium for 1 h prior to cardiomyocyte plating (200 µL of buffer per well of a 96-well plate) and keep the wells hydrated.
3. Prepare a 37 °C water bath.
4. Remove the cardiomyocyte tubes from the liquid nitrogen tank, transfer the tubes to dry ice, and slightly open the tube caps to release pressure.
   NOTE: Releasing pressure in the tubes is extremely important! If too much pressure builds inside the tubes, they could explode.
5. Reseal the tube caps and place them in the water bath to thaw for 4 min.
   NOTE: Allow them to thaw completely, to avoid cell damage due to partial thawing.
6. After the cells thaw, spray the tubes with 70% ethanol before opening. Transfer the cells into 15 mL conical tubes with a 1 mL pipette. Slowly drip 8 mL of plating medium, agitating the tube each time 1 mL is added, to allow the cells to adjust to changes in osmolarity.
   1. Wash the cryovial with 1 mL of plating medium using a 1 mL glass pipette. Then, slowly drip the wash into the 15 mL conical tube.
7. Centrifuge the tubes at ~300 × g for 5 min. Aspirate the supernatant and resuspend the pellet in 1 mL of plating medium. Remove an aliquot and perform live cell counting with a hemocytometer. Add additional plating medium to obtain 7.5 × 10⁵ cells/mL.
   NOTE: Approximately 10 mL of cell suspension is required to prepare 96 wells.
8. Dispense 100 µL of cell suspension per well of an MECM-coated 96-well plate using a multichannel pipette.
   NOTE: Make sure to avoid cell precipitation and obtain uniform cell density in all wells while plating.
9. Incubate the cells at 37 °C, 5% CO₂ for 2 days prior to changing the medium to maintenance medium (200 µL/well). Change the maintenance medium on day 5 after thawing. Perform EP assays on day 7 or later, as described previously^8,9. Change the medium every other day when opting for extending the cell culture.

2. hiPSC cardiac-directed differentiation and hiPSC-CM purification

1. Warm 1x commercially available ethylenediaminetetraacetic acid (EDTA) solution, HBSS without calcium and magnesium (HBSS--), and 6-well plates coated with solubilized basement membrane matrix secreted by an Engelbreth-Holm-Swarm mouse sarcoma cell (mouse ECM) to room temperature.
2. Mark the differentiated colonies by phase-contrast microscopy and aspirate/ablate. Wash each well with 1 mL of HBSS--. Perform two washes in wells containing >10 differentiation spots.
3. Aspirate the HBSS-- and add 1 mL of EDTA solution to each well. Incubate the plates for up to 5 min at 37 °C. Check the plates after 3 min and look for translucent white, visible colonies.
4. Aspirate the EDTA solution and add 1 mL to a single well. Dislodge the cells with 2 mL of hiPSC medium by pipetting the suspension up and down repeatedly, using a 10 mL glass pipette to detach all stem cells from the well, and transfer the cell suspension to a collection tube. Repeat the aspiration and dislodging with subsequent wells.
   NOTE: Dislodge hard to lift colonies with the tip of the glass pipette.
5. Count the stem cells and adjust the volume to plate 8.0 × 10⁵ cells/well. Culture the cells on hiPSC medium (2 mL/well) until the stem cells reach 90% confluence (this time is referred from now on as D₀).
6. Prepare 2 mL of basal differentiation medium supplemented with 4 µM of CHIR99021.
7. On D₀, wash each well of a 6-well plate of stem cells with 1 mL of HBSS per well. Replace the HBSS with basal differentiation medium supplemented with 4 µM of CHIR99021.
8. On D₁, do nothing.
9. On D₂, prepare basal differentiation medium supplemented with 4 µM of IWP4.
10. Replace the medium with 2 mL of IWP4-supplemented basal differentiation medium per well.
11. On D₃, do nothing for a ventricular-specific differentiation. For an atrial-specific differentiation, aspirate the medium and add 2 mL of basal medium supplemented with 4 µM of IWP4 and 1 µM retinoic acid (RA) solution per well.
12. On D₄, aspirate the medium and add 2 mL of basal medium per well for ventricular differentiation. For an atrial differentiation, aspirate the medium and add 2 mL of basal medium supplemented with 1 µM RA solution per well.
13. On D₅, do nothing.
14. On D₆, aspirate the medium and add 2 mL of basal medium per well (for both atrial and ventricular differentiation).
15. On D₇, do nothing.
16. On D₈, aspirate the medium and add 2 mL of cardiomyocyte maintenance medium. Change the medium every other day until cell separation, or follow a chronic drug exposure plan.

3. hiPSC-CM purification via MACS (magnetic-activated cell sorting)

1. Aspirate the cell culture medium and wash each well with 1 mL of HBSS--. Dissociate the cells by adding 1 mL of 0.25% trypsin/EDTA and incubating at 37 °C, 5% CO₂ for 10 min. Resuspend and singularize the cells in each well with 2 mL of plating medium to inactivate the trypsin/EDTA.
2. Collect the cells from the six wells into a 50 mL conical tube with a 70 µm strainer. Then, wash the strainer with 3 mL of plating medium. Count the cells.
3. Centrifuge the suspension at ~300 × g for 5 min. Aspirate the supernatant and wash the cells with 20 mL of ice-cold MACS separation buffer. Then, centrifuge again at ~300 × g for 5 min.
4. Resuspend the pellet in 80 µL of cold MACS separation buffer per 5 × 10⁶ cells. Add 20 µL of cold non-cardiomyocyte depletion cocktail (human) per 5 × 10⁶ cells. Gently mix the cell suspension and incubate on ice for 10 min.
5. Wash the sample by adding 4 mL of cold MACS separation buffer per 5 × 10⁶ cells. Centrifuge the sample at ~300 × g for 5 min and aspirate the supernatant.
6. Resuspend the pellet in 80 µL of cold MACS separation buffer per 5 × 10⁶ cells. Add 20 µL of cold anti-biotin microbeads per 5 × 10⁶ cells. Gently mix the cell suspension and incubate for 10 min on ice.
7. While the samples are incubating, place the positive depletion columns (fitted with the 30 µm pre-separation filters) on the MACS separator, and place the labeled 15 mL collection tubes below the columns. One column is needed for every 5 × 10⁶ cells.
8. Prime each column with 3 mL of cold MACS separation buffer. Mix the antibody-treated cell suspension with 2 mL of MACS separation buffer per 5 × 10⁶ cells, and add to the column.
   NOTE: Do not centrifuge! Centrifugation at this step has detrimental effects on cardiomyocyte yield.
9. Add 2 mL of MACS separation buffer to each column, and collect the flowthrough until 12 mL of flowthrough cardiomyocyte suspension is collected.
   NOTE: Never allow the columns to fully dry.
10. Centrifuge the cardiomyocytes at ~300 × g for 5 min, discard the supernatant, and suspend the cardiomyocytes in 1 mL of plating medium.
11. Count the cells to determine the concentration, adjust the volume to the desired seeding density, and plate the cells. Plate the purified cardiomyocytes on the MECM 96-well plates, as described above in steps 1.9-1.11 (7.5 × 10⁵ cells/well).

4. Optical mapping using voltage-sensitive dyes (VSDs) and calcium-sensitive fluorophores (CSFs)

1. Prepare the appropriate amount of VSD in HBSS with calcium and magnesium, by adding 1 µL of VSD dye per mL of HBSS and 10 µL of loading adjuvant per mL of HBSS.
   NOTE: Typically, a 96-well plate requires 10 mL of VSD solution.
2. Alternatively, prepare HBSS with calcium and magnesium supplemented with 5 µM of CSF. Aspirate the cardiomyocyte maintenance medium and replace with 100 µL of VSD or CSF per well of a 96-well plate. Incubate the cells for 30 min in the cell culture incubator.
3. Remove the dyes and replace with assay medium or HBSS. Equilibrate at 37 °C for the acquisition of baseline data optical mapping with a high-throughput optical mapping device.
4. Treat the cells with drugs for acute exposure testing, or map the cells that were chronically exposed to drugs of interest.
5. For cardiotoxicity testing in 96-well plates, use four doses of a compound with at least six wells per dose. Use doses ranging from below to above the effective therapeutic plasma concentration, including a dose of the clinical effective therapeutic plasma concentration.
6. Dilute the drugs in dimethyl sulfoxide, store them as stock solutions at -20 °C, and then dilute them in HBSS to the desired concentrations.
7. Make baseline electrophysiology measurements prior to drug application, as described in section 5. Once the drugs have been applied, make electrophysiology recordings at least 30 min later for chronic studies. See the following sections for optical mapping data acquisition and analysis procedures.

5. Optical mapping using genetically encoded calcium indicator (GECI)

1. Plate commercially available or MACS-purified hiPSC-CMs, as described above, using MECM-coated 96-well plates to make mature hiPSC-CMs. To form confluent monolayers, plate 7.5 × 10⁴ CMs per well of each 96-well plate. Use plating medium.
2. After 48 h in plating medium, switch to cardiomyocyte maintenance medium.
3. On day 4 after thawing and replating, add recombinant adenovirus for expressing GCaMP6m (AdGCaMP6m) to cells at a multiplicity of infection (MOI) = 5. Add the virus using CM assay medium.
   NOTE: Here, experiments using GCaMP6m were performed in iCell² cardiomyocytes from a commercial vendor.
4. On day 5, remove the adGCaMP6m medium and replace with fresh RPMI+B27 (cardiomyocyte maintenance medium).
5. On day 7, observe the CMs using microscopy or the optical mapping imager, to visualize spontaneous contractions and corresponding calcium transients.
6. On day 7 or later, for medication screening, directly transfer 96-well plates of mature hiPSC-CM monolayers expressing GCaMP6m to the optical mapping imager from the incubator for baseline data acquisition.
7. Following electrophysiology data acquisition, return the plates of the CMs to the tissue culture incubator, for measurements on a subsequent time point.
8. Following baseline recordings, apply the medications, using at least four doses of each medication and at least six wells per dose. Equilibrate the medications on the cells for at least 30 min prior to data collection. Warm the well temperature to ~37 °C prior to and during the acquisition of data.
9. Following baseline recordings of an entire plate, add isoproterenol (500 nM) to every well to enable robust drug response data. Quantify the effects of isoproterenol on monolayer beat rate, contraction amplitude (calcium transient amplitude), and calcium transient duration (see Figure 6), as described in section 5.

6. Acquisition of optical mapping data and analysis

1. Make sure that the optical mapping device camera, transilluminator, and plate heater are turned on.
2. Open the acquisition software and determine the file saving location.
3. Open the front drawer and position the plate on the plate heater.
4. Acquire a dark frame by clicking on the Dark Frame button.
5. Select Duration (10-30 s) and Frame Rate of Acquisition (e.g., 100 fps; 250 fps for higher temporal resolution), and click Start Acquisition.
6. Open the analysis software and, in the Import/Filter tab, select either Browse for a single file or Tile Multiple to reconstruct a plate.
7. Select Parameter Mode (APD or CaTD), enter Distance per Pixel, and use the Well Wizard to determine the wells' location in the image. Click Process Save to move to the next tab.
8. Open the ROIs (regions of interest) tab and choose to draw the ROIs Manually, Automatically, or Not Use ROIs at all, which would then consider the whole well for analysis. After the ROIs have been selected, click the Process/Save button to move to the next step. Check the Hide Wells and Only Show Filtered boxes to visualize the ROIs.
9. Open the Analysis tab and, on the top right side of the screen, select each Well or ROI to confirm the Accuracy of Automatic Beat Detection. Add or Remove Beats from the traces by pressing Add Beat/ Save Beats, or by selecting an Individual Beat and pressing Delete on the keyboard.
10. Optionally, use the Average Heat Maps feature to create heatmaps of selected parameters for the plate.
11. Click on the Time-Space Plot button in the Analysis tab to visualize data for each well or the ROI. After making sure that the beat detection is accurate, proceed to the Export tab and select File Format. Press Export and create a folder for the data to be exported to. Proceed to open data files and run the chosen statistical analysis routine.
    NOTE: .xlxs files are preferred as all the parameters are exported in a single file; other formats (.csv or .tsv) generate one file per parameter.

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...

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