Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.

Methods and Results Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment.

METHODS

An expanded Methods section is available in the Supplemental Materials . Differentiation of hiPSC-CMs from hiPSCs Lentiviral reprogramming was used to generate three hiPSC lines from skin fibroblasts of three healthy individuals in a 7-member family cohort 25 . An additional three hiPSC lines were generated with a previously-published Sendai virus reprogramming protocol using peripheral blood mononuclear cells from three healthy individuals 26 . These 6 hiPSC lines were differentiated into hiPSC-CMs using a 2D monolayer differentiation protocol and were maintained in a 5% CO 2 /air environment as previously published 25 , 27 . Briefly, hiPSC colonies were dissociated with 0.5 mM EDTA into single-cell suspension and resuspended in E8 media (Life Technologies) containing 10 µM Rho-associated protein kinase inhibitor (Sigma). Approximately 100,000 cells were replated into 6-well dishes pre-coated with Matrigel (BD Biosciences). Next, hiPSC monolayers were cultured to 85% cell confluency. Cells were then treated for 2 days with 6 µM CHIR99021 (Selleck Chemicals) in RPMI+B27 supplement without insulin to activate Wnt signaling and induce mesodermal differentiation. On day 2, cells were placed on RPMI+B27 without insulin and CHIR99021. On days 3–4, cells were treated with 5 µM IWR-1 (Sigma) to inhibit Wnt pathway signaling and induce cardiogenesis. On days 5–6, cells were removed from IWR-1 treatment and placed on RPMI+B27 without insulin. From day 7 onwards, cells were placed on RPMI+B27 with insulin until beating was observed. At this point, cells were glucose-starved for 3 days with RPMI (no glucose)+B27 with insulin to purify hiPSC-CMs, as cardiomyocytes can selectively metabolize fatty acids as a source of cellular energy 28 . Following purification, cells were cultured in RPMI+B27 with insulin. When replating hiPSC-CMs for downstream use, cells were dissociated with 0.25% trypsin-EDTA (Life Technologies) into a single-cell suspension and seeded on Matrigel-coated plates.

CVB3-Luc infections and antiviral treatments

Stocks from a previously-published CVB3-Luc strain expressing Renilla luciferase were stored at −80°C until needed 29 . IFNβ1 (Life Technologies), ribavirin (MP Biochemicals), PDTC (Sigma), and fluoxetine (Sigma) stocks were dissolved in water. Before CVB3-Luc infection, day 30–35 post-differentiation hiPSC-CMs were pretreated with antiviral compounds for 12 hours unless noted otherwise. Bioluminescence imaging Day 30–35 post-differentiation hiPSC-CMs were plated in RPMI+B27 with insulin on Matrigel at a density of 40,000 cells per well of a 96-well plate. At the time of CVB3-Luc infection, 6 µM Enduren extended duration coelenterazine (Promega) was added. Following infection, bioluminescence imaging was conducted using a Xenogen IVIS 100 Imaging System. Living Image software (Perkin Elmer) was used for image analysis. Ca 2+ imaging Dissociated day 30–35 post-differentiation hiPSC-CMs were reseeded in Matrigel-coated 8-well Lab Tek II chambers (Nalge Nuc International) and were treated with 5 µM Fluo-4 AM and 0.02% Pluronic F-127 (Molecular Probes) in Tyrode’s solution for 15 minutes at 37°C. Cells were washed with Tyrode’s solution afterwards. Ca 2+ imaging was conducted using a Zeiss LSM 510Meta confocal microscope (Carl Zeiss AG) and analyzed using Zen imaging software. Spontaneous Ca 2+ transients were obtained at 37°C using a single-cell line scan mode.

Cell metabolism and viability assays WST-1 reagent

(Abcam) was used to determine hiPSC-CM metabolism and viability following antiviral treatment. After 48-hour treatment with antiviral compounds, 10 µL of WST-1 reagent was added to 100 µL RPMI+B27 with insulin on day 30–35 post-differentiation hiPSC-CMs. After 24 hours, a microplate reader (Promega) was used to quantify conversion of tetrazolium salt WST-1 into formazan dye at 420–480 nm absorbance. Absorbance reading correlated directly with cell viability.

Gene expression and immunocytochemistry

For qRT-PCR, RNA was extracted with the miRNeasy kit (Qiagen). cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and real-time PCR was conducted on an Applied Biosystems 7900HT Fast Real-Time PCR System. Primers are listed in Online Table I . For additional gene expression analysis, a GeneChip® Human Gene 2.0 ST DNA Microarray was used (Affymetrix). Immunostaining was performed according to previous protocols 23 . Imaging was performed using a DMIL–LED microscope (Leica Microsystems) or a Zeiss LSM 510Meta confocal microscope (Carl Zeiss AG) using Zen imaging software.

Statistical Methods

Data are presented as mean ± SEM. Comparisons were conducted via student’s t-test with significant differences (*) defined by p