Electrophysiological and calcium-handling development during long-term culture of human-induced pluripotent stem cell-derived cardiomyocytes

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for personalised medicine and preclinical cardiotoxicity testing. Reports on hiPSC-CM commonly describe heterogenous functional readouts and underdeveloped or immature phenotypical properties. Cost-effective, fully defined monolayer culture is approaching mainstream adoption; however, the optimal age at which to utilise hiPSC-CM is unknown. In this study, we identify, track and model the dynamic developmental behaviour of key ionic currents and Ca2+-handling properties in hiPSC-CM over long-term culture (30–80 days). hiPSC-CMs > 50 days post differentiation show significantly larger ICa,L density along with an increased ICa,L-triggered Ca2+-transient. INa and IK1 densities significantly increase in late-stage cells, contributing to increased upstroke velocity and reduced action potential duration, respectively. Importantly, our in silico model of hiPSC-CM electrophysiological age dependence confirmed IK1 as the key ionic determinant of action potential shortening in older cells. We have made this model available through an open source software interface that easily allows users to simulate hiPSC-CM electrophysiology and Ca2+-handling and select the appropriate age range for their parameter of interest. This tool, together with the insights from our comprehensive experimental characterisation, could be useful in future optimisation of the culture-to-characterisation pipeline in the field of hiPSC-CM research. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-022-00973-0.


Experimental Methods hIPSC reprogramming and culture
The human induced pluripotent stem cell (hiPSC) line UMGi014-C clone 14 (isWT1.14) was derived from dermal fibroblasts of a healthy male donor. They were cultured in feeder-free conditions using the integration-free CytoTune iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) with reprogramming factors OCT4, KLF4, SOX2, c-MYC.
All protocols for biopsy procurement, and somatic cell reprogramming and differentiation were approved by the ethics committee of the University Medical Center Göttingen (10/9/15). hiPSC were maintained in feeder-free and growth-factor free conditions with Stem MACS IPS-Brew XF medium (Miltenyi Biotec) refreshed daily at 37°C. Cells were cultured in 1:120 Matrigel TM (BD Biosciences) coated 6-well plates and passaged every 3-4 days upon reaching confluence. During passaging, cells were briefly washed with versene (Thermo Fischer Scientific), then incubated with fresh versene for 2-3 minutes. 1 ml of fresh Stem MACS medium was then used to flush each well to ensure full monolayer detachment. Cells were counted using a CASY counter. For continuing expansion cultures, fresh Matrigel TM coated 6-well plates were filled with new Stem MACs solution supplemented with 1:2000 ROCK inhibitor Y27632 (Stemolecule). Roughly 1-2 million cells were added to each well prior to incubation and continued daily maintenance with Stem MACS at 37°C.
Subsequent application of 2.5 µM IWP2 (Sigma-Aldrich) for a further 48 hours stimulates WNT signalling cessation. Contraction was observed around d7. At d8 medium was changed to a 'Culture Medium' containing: RPMI 1640 with Glutamax, and 2% B27 (Thermo Fisher Scientific). Cardiomyocyte purification by lactate selection was performed between d15 and d20 with a 'Selection Medium' containing RPMI 1640 without glucose (Thermo Fisher Scientific), 0.5 mg/ml human recombinant albumin, 0.2 mg/ml L-ascorbic acid 2-phosphate, and 4 mM lactate (all Sigma-Aldrich). Subsequently, hiPSC-CM were maintained with culture medium every 2-3 days. Between d27 and d30, purified iPSC-CMs were digested with TrypLE (Thermo Fisher Scientific) and seeded on round borosilicate glass 10 mm diameter #0 coverslips at a density of 15,000 cells/cm -2 . Coverslips were incubated in culture medium with 1:2000 ROCK inhibitor Y27632 (Stemolecule) 24 hours. Subsequently, medium was changed to pure culture medium and refreshed every 2-3 days.

Measurement of basal inward-rectifier K + current (IK1)
Measurement of IK1 was performed using the whole-cell ruptured-patch configuration.
The coverslip was superfused with a bath solution at 37°C containing (mmol/L): NaCl 120, KCl 20, MgCl2 1, CaCl2 2, glucose 10, HEPES 10; pH = 7.4. A high (20 mmol/L) extracellular K + concentration depolarises the IK1 reversal potential and allows for indepth dissection of inward currents at -100mV. Basal current was measured in voltage-clamp configuration at 0.5 Hz using a protocol with a holding potential at -80mV followed by a depolarising ramp pulse from −100 to +40 mV. IK1 was identified as the current responsive to Ba 2+ application (1-mmol/L). Signals were amplified with an Axopatch 200B microelectrode amplifier and acquired and analysed using pClamp-Software V 10.7 (both from Axon Instruments Inc., Foster City, USA).
Membrane currents were corrected for membrane capacitance (a measure of cell size) and expressed in pA/pF. The coverslip was superfused with a bath solution at room temperature containing (mmol/L): NaCl 5, HEPES 10, MgCl2 1, CsCl 10, glucose 10, CaCl2 0.5, and TEA-Cl 120 (pH = 7.4, adjusted with CsOH). INa was measured in voltage-clamp configuration at 0.5 Hz using a protocol with a holding potential at -80mV, followed by a 100 ms step to -110 mV to increase the availability of Na + channels. 30 ms steps from -80 to +20 mV were used to elicit channel opening to generate current-voltage (I-V) curves. Peak INa was identified as current that was blocked by tetracaine, as described by Poulet et al. [4]. INa,L was measured by restoring 120 mmol/L NaCl to the bath solution with a voltage-clamp protocol holding at -120 mV, followed by a 5 ms activating step to +50 mV (in order to maintain voltage control) before a 300 ms pulse at -30 mV as suggested by Sosalla et al. [6]. Both INa and INa,L were recorded in the presence of Nifedipine (1 µmol/L). INa,L was identified as tetrodotoxin-sensitive current and the integral of the current was measured between 50 and 250 ms after the -30 mV pulse. Signals were amplified with an Axopatch 200B microelectrode amplifier and acquired and analysed using pClamp-Software V 10.7 (both from Axon Instruments Inc., Foster City, USA). Membrane currents were corrected for membrane capacitance (a measure of cell size) and expressed in pA/pF.

Measurement of the rapid component of the delayed rectifier current (IKr)
Measurement of IKr was performed using ruptured whole cell configuration in a high performance automated patch clamp platform (SyncroPatch 384; Nanion Technologies GmbH). Cells were isolated using the same TrypLE process defined above and stored in suspension in the SyncroPatch cell hotel prior to measurements. hiPSC-CM were measured at room temperature using partial borosilicate glass, single-aperture, 384-well planar chips (NPC384T 1x S-type). Seal resistance, series resistance and cell capacitance were acquired from each well via a test pulse.
PatchControl 384 (Nanion Technologies GmbH) software digitized and acquired the data. A voltage clamp protocol consisting of a holding potential of -80 mv followed by a 2 s step to 60 mV with steps of 10 mV was employed for I-V acquisition as described previously [3]. External solution contained (in mmol/L): CsCl 144, CaCl2 2, MgCl2 2, glucose 5, HEPES 10 (pH = 7.4 adjusted with CsOH). Internal solution contained (in mmol/L) CsCl 20, EGTA 10, HEPES 10, CsF 110 (pH = 7.2 adjusted with CsOH) in accordance with previous protocols [3]. Cs + was used as a charge carrier due to its selectivity for the hERG channel. After measurement of IKr in reference external solution, 25 µmol/L E-4031 was added to block hERG channels and ascertain the molecular basis of the current. Tail current amplitude was analysed using DataControl 384 software (Nanion Technologies GmbH). Membrane currents were corrected for membrane capacitance (a measure of cell size) and expressed as pA/pF.  Quantification of SERCA activity. The iMATURE user interface, as shown in Figure 8 of the manuscript, will pop-up and iMATURE is now ready for use.

Simultaneous measurement of intracellular [Ca
 Running action potential simulations [1] Click on 'Cell 1' or 'Cell 2' in the left panel.
o Insert the preferred maturation stage (in days).
o If desired, the maximum ion channel conductance of major ionic currents can be altered by adjusting the slider or by entering the desired value in the box to the right of the slider.
o Press "Run" to start the action potential simulation. This may take a few seconds.
[2] The "Output" panel shows two plots that each depict a particular variable. By