Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for drug testing and modelling genetic disorders. Abnormally low upstroke velocity is a current limitation. Here we investigated the use of 3D engineered heart tissue (EHT) as a culture method with greater resemblance to human heart tissue in comparison to standard technique of 2D monolayer (ML) format. INa was measured in ML or EHT using the standard patch-clamp technique. INa density was ~1.8 fold larger in EHT (−18.5 ± 1.9 pA/pF; n = 17) than in ML (−10.3 ± 1.2 pA/pF; n = 23; p < 0.001), approaching densities reported for human CM. Inactivation kinetics, voltage dependency of steady-state inactivation and activation of INa did not differ between EHT and ML and were similar to previously reported values for human CM. Action potential recordings with sharp microelectrodes showed similar upstroke velocities in EHT (219 ± 15 V/s, n = 13) and human left ventricle tissue (LV, 253 ± 7 V/s, n = 25). EHT showed a greater resemblance to LV in CM morphology and subcellular NaV1.5 distribution. INa in hiPSC-CM showed similar biophysical properties as in human CM. The EHT format promotes INa density and action potential upstroke velocity of hiPSC-CM towards adult values, indicating its usefulness as a model for excitability of human cardiac tissue.


Generation and culture of human induced pluripotent stem cellderived cardiomyocytes in engineered heart tissue and monolayer format
The undifferentiated hiPSC line C25 (kind gift from Alessandra Moretti, Munich, Germany) was expanded in FTDA medium 1 and differentiated in a three step protocol based on growth factors and a small molecule Wnt inhibitor DS07 (kind gift from Dennis Schade, Dortmund, Germany) as previously published 2  For culture in conventional ML format, 4x10 5 hiPSC-CM were plated on gelatin-coated 24well plates. For optimal comparability, hiPSC-CM in EHT and ML were cultured under the same conditions in a 37 °C, 7% CO 2 , 40% O 2 humidified cell culture incubator with the same medium consisting of DMEM (Biochrom; F0415), 10% heat-inactivated horse serum (Gibco 26050), 1% penicillin/streptomycin (Gibco 15140), insulin (10 µg/ml; Sigma I9278) and aprotinin (33 µg/ml; Sigma A1153). All experiments were performed in parallel from the same batch of cells. EHTs started coherent and stable beating at day 10-14 after casting.

Patch-clamp experiments
After a 24-29 day culturing period, hiPSC-CM in EHT and ML were isolated with collagenase II (200 U/ml, Worthington, LS004176) for 5 and 3 hours, respectively, and re-plated on gelatin-coated coverslips for 24-48 h in order to maintain adherence under perfusion 3 . 25-31 days after differentiation the hiPSC-CM were used in the patch-clamp experiments. I Na recordings were performed as described previously 4 . In brief, borosilicate glass microelectrode pipettes (tip resistances 1.5-3.0 MOhm) were used to record I Na in whole-cell After placement of the cover slip into the recording chamber we washed out the residual culture medium for at least 5 minutes before starting the experiment. We did not correct for offset potential. After rupture of the cell membrane a standard protocol (holding potential at -80 mV; conditioning pre-pulse to -110 mV for 1000 ms, depolarisation to -30 mV for 30 ms at a frequency of 0.5 Hz; see inset Figure 1C) was applied until I Na became stable to within ~30 s. After equilibration further voltage-protocols for the I-V relationship and steady-state inactivation (at 0.5 Hz, see inset Figure 2C) and recovery of inactivation (0.2 Hz, see inset Figure 2D) were performed within 180 s post cell rupture.
Current amplitude was measured as the difference between peak inward current and current at the end of the depolarising step. Steady-state inactivation curves for I Na were obtained by plotting the normalised current amplitude at the test potential as a function of the conditioning potential (V m ). A Boltzmann function was fitted to the data for each experiment and characterised using the half-maximum voltage of inactivation (V 0.5 inact ) and the corresponding slope factor (k inact ): I/I max =1/(1+exp (Vm−V0.5inact)/kinact) ). Activation curves were calculated from I-V curves for each experiment using the equation G=I/(V m − E rev ) (G=peak Na conductance; I=current at the test potential V m ). The apparent reversal potential E rev was obtained by linear regression of two data points close to E rev for each experiment. The relation between normalised peak conductance G/G max and membrane potential V m could be described by the Boltzmann equation: G/G max =1/(1+exp ((V0.5act−Vm)/kact) ).

Immunofluorescence and immunohistochemistry
Immunofluorescence and immunohistochemistry were performed as described previously 2 .

Data analysis
The ISO2 software (MFK, Niedernhausen, Germany) was used for data acquisition and GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used for data analyses.
Curves were fitted to data points from individual experiments and all data were compared using unpaired t-tests and for groups greater than 2 One-way ANOVA followed by Tukey corrections. Two-way ANOVA was used to assess repeated measurements of currentvoltage relationship (Figure 2A). All analyses were two-tailed and a p<0.05 was considered to be statistically significant. Group data are presented as mean±SEM. Figures 2C and D show sigmoidal functions fitted to the mean data, which show minimal differences to V 0.5values averaged from individual fitted experiments (Table 1).

Supplementary Figure 1.
Example of time course of the peak I Na upon the exposure to 1, 10 and 30 µmol/L TTX in a non-cumulative manner. Steady state was reached after ~5 s of TTX wash-in and after ~10 s of wash-out.

Supplementary Figure 2. Expression of Na channel isoforms during differentiation from stem cells to hiPSC-CM.
Transcript levels of various Na channel isoforms were quantified by qPCR in 3 technical replicates of stem cells during day 0 to 20 of differentiation to cardiomyocytes. Expression levels are normalized to GUSB. Sequences of primers are provided in Supplementary Table  1. CT stands for cycle threshold of PCR amplification. CT >32 cycles were defined as no expression.