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

Abstract

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 I_Ca,L density along with an increased I_Ca,L-triggered Ca²⁺-transient. I_Na and I_K1 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 I_K1 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.

Keywords: Action potential; Calcium handling; Cardiovascular; Ion channel; Maturation; Stem cell.

Fig. 1

Overview of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) differentiation. A Schematic overview of the differentiation protocol utilised in this study (upper), and the process of long-term continuous culture on glass coverslips (lower). Early (young) hiPSC-CM underwent experimentation between 30 and 46 days after differentiation whilst late (old) hiPSC-CM were measured between day 47 to 80. B Immunofluorescent staining of hiPSC-CM at d29. C Flow cytometry analysis of hiPSC-CM at d29. D Longitudinal section area of early and late hiPSC-CM (left), corresponding cell capacitance (middle) and T-tubule density (right), estimated through a ratio of capacitance to longitudinal section area of each cell. E Representative photomicrographs of early (left) and late (right) hiPSC-CM. Scale bar represents 10 µm. Data are mean ± SEM. Symbols represent separate differentiations. n/N = number of hiPSC-CM/differentiation

Fig. 2

I_Ca,L-triggered Ca²⁺ transients (CaT) in isolated early and late human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). A Representative simultaneous recordings of I_Ca,L (upper) and triggered CaT (lower) in early (left) and late hiPSC-CM (right). Inset: voltage-clamp protocol. B Peak I_Ca,L. C Current–voltage relationship curve for I_Ca,L. D Diastolic and systolic [Ca²⁺]_i (left) and resulting CaT-amplitude (right). Data are mean ± SEM. *P < 0.05 ***P < 0.001 versus early hiPSC-CM culture by Welch’s t test or Student’s t test (D left). Symbols represent separate differentiations. n/N = number of hiPSC-CM/differentiation

Fig. 3

Caffeine-induced Ca²⁺ transients (cCaT) with corresponding transient-inward currents (I_NCX) to assess sarcoplasmic reticulum (SR) Ca²⁺ content in isolated early and late human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). A Representative cCaT (upper) and corresponding I_NCX (lower) in early (left) and late hiPSC-CM (right). B SR Ca²⁺ load, quantified as cCaT amplitude (left), or integrated membrane current (Charge; right). C Rate constants of Ca²⁺ transport k_syst (far left), k_caff (centre left), k_SERCA (calculated as the difference between k_syst and k_caff; centre right) and the resulting relative proportions of NCX and SERCA-mediated cytosolic Ca²⁺ removal in early and late hiPSC-CM (far right). D Peak I_NCX. E Representative western blots showing the expression of NCX1 and SERCA2a against CSQ2. F Quantification of NCX1 and SERCA2a expression relative to early hiPSC-CM (3 independent experiments per group). Data are mean ± SEM. *P < 0.05 versus early hiPSC-CM culture by Welch’s t test or Mann–Whitney U test (B). Symbols represent separate differentiations. n/N = number of hiPSC-CM/differentiation

Fig. 4

Peak Na⁺ current (I_Na) and late Na⁺ current (I_Na,L) in isolated early and late human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). A Representative I_Na in early (left) and late hiPSC-CM (right). Inset: voltage-clamp protocol. B Current–voltage relationship for I_Na. C Representative I_Na,L in early (left) and late hiPSC-CM (right) in the absence (Baseline) or presence of 10 µmol/L tetrodotoxin (TTX). Inset: modified voltage-clamp protocol to accentuate late current (as described in Poulet et al. [56]). D I_Na,L integral. Data are mean ± SEM. *P < 0.05 versus early hiPSC-CM culture. Symbols represent separate differentiations. n/N = number of hiPSC-CM/differentiation

Fig. 5

Rapid component of the delayed-rectifier K⁺ current (I_Kr) and basal inward-rectifier K⁺ current (I_K1) in isolated early and late human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). A Representative I_Kr in early (left) and late hiPSC-CM (right). Inset: voltage-clamp protocol. B Maximum tail I_Kr defined as E4031-sensitive current. C Representative recordings of I_K1 in early (left) and late hiPSC-CM (right) during a depolarising ramp pulse protocol (inset). D Peak I_K1 defined as Ba²⁺-sensitive current. E Representative western blots showing the expression of Kir2.1 against CSQ2 (same gel as Fig. 3). F Quantification of Kir2.1 expression relative to early hiPSC-CM (3 independent experiments per group). Data are mean ± SEM. *P < 0.05 ***P < 0.001 versus early hiPSC-CM culture. Symbols represent separate differentiations. n/N = number of hiPSC-CM/differentiation

Fig. 6

Overview of maturation-dependent changes in cellular Ca²⁺ dynamics and electrophysiology which drive action potential (AP) characteristics in experimental and in silico human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). A Plots of the experimental data for all measured electrophysiological cellular parameters in aging hiPSC-CM. The red line indicates the in silico output of expected results demonstrating non-linear maturation-dependent characteristics. B Simulated steady-state AP traces at 0.5 Hz in d30 and d70 modelled hiPSC-CM. The stimulus current (I_stim) was set to − 120 μA/μF and the hyperpolarising current (I_hyper) was set to 0.2 μA/μF. C Representative experimental AP traces in early (left) and late hiPSC-CM (right). D Comparison of experimental and model AP properties during 0.5 Hz pacing: resting membrane potential (RMP; left) and AP duration at 90% repolarisation (APD₉₀; right). Experimental data are mean ± SEM. *P < 0.05. Symbols represent separate differentiations. n/N = number of hiPSC-CM/differentiation

Fig. 7

The role of inward-rectifier K⁺ current (I_K1) and inward Na⁺/Ca²⁺-exchanger (NCX) current in maturity-dependent action potential (AP) shortening in in silico human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). A Steady-state AP simulations over increasing ages with I_K1 clamped at 30 days of maturation (solid lines), as well as during acute inhibition of NCX at d60 (dashed line). Note: automaticity is observed at d60 and d65. The stimulated AP at d65 is short due to the incomplete repolarisation of the preceding spontaneous AP. B AP duration at 90% repolarisation (APD₉₀; left) and resting membrane potential (RMP; right) at increasing stages of development in the absence of I_K1 maturation, as well as at d60 with acute NCX inhibition (black/white bars). Note: RMP and APD₉₀ values for d60 and d65 in the absence of NCX inhibition are not shown due to abnormal automaticity

Fig. 8

Screenshot of the induced pluripotent stem cell-derived cardiomyocyte maturity evaluator (iMATURE) software tool. The iMATURE tool incorporates the experimentally-observed maturity-dependent changes on cardiac ion channels (i.e., I_Na, I_NaL, I_Ca,L, I_Kr and I_K1) and Ca²⁺-handling proteins in the Kernik hiPSC-CM model [36]. The tool enables the simulation and comparison of two maturity levels simultaneously under different experimental conditions. It also enables rapid evaluation of the effects of inhibition of major ionic currents