Extracellular Matrix Mediated Maturation of Human Pluripotent Stem Cell Derived Cardiac Monolayer Structure and Electrophysiological Function

Abstract

Background

Human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electrical impulse propagation velocity and immature action potential profiles.

Methods and Results

Here we have identified an optimal extracellular matrix (ECM) for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal ECM combination have impulse propagation velocities ~2X faster than previously reported (43.6±7.0 cm·s⁻¹ n=9) and have mature cardiomyocyte action potential profiles including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s, N=5 monolayers). In addition the optimal ECM promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1 and Connexin43) and myofilament markers (cTroponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase (FAK) activation prevented structural maturation.

Conclusions

Maturation of human stem cell derived cardiomyocyte monolayers is achieved in a one week period by plating cardiomyocytes on PDMS coverslips rather than on conventional 2D cell culture formats such as glass coverslips or plastic dishes. Activation of integrin signaling and FAK are essential for significant maturation of human cardiac monolayers.

Introduction

The generation of induced human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offers a revolutionary platform to study physiological/pathophysiological processes and cardiac arrhythmia mechanisms, and for the development of new therapies in vitro.^{1, 2} Experimentally, they offer a human system that can be used to study drug cardiotoxicity and the molecular mechanisms of mono/poly genetic diseases.^{3, 4} Recently, we reported on the generation of large (1cm diameter) electrically coupled hiPSC-CM monolayers.⁵ Despite forming a functional syncytium, impulse propagation remained very slow with conduction velocities (CV) near 25cm s⁻¹, which is 2–3 fold slower than normal propagation in adult hearts.^{6, 7} The slow CV of hiPSC-CM monolayers may be attributed to the immature electrophysiological phenotype of the hiPSC-CMs generated to date.^8–11 At present this poses a major limitation to the utility of these cells for research and therapeutic purposes.^12–14

The extracellular matrix (ECM) plays an important role in stem cell fate decisions, normal development and cardiogenesis.^15–17 A recent report demonstrated the critical role of the ECM for highly efficient cardiac directed differentiation of human pluripotent stem cells.¹⁷ In addition to providing structural support for the developing myocardium, the matrix also contains important signaling molecules. Bioengineering techniques are currently being developed to create artificial structural matrices using biocompatible synthetic materials that have similar stiffness to the native matrix.^18–20 For instance, it was recently shown that ECM stiffness could modulate gene expression in neonatal cardiac myocytes.²⁰ Interestingly, the softer ECM produced greater and more highly organized gap junctions (connexin43, Cx43) than stiff surfaces. Here we tested the hypothesis that hPSC-CM monolayers can be structurally and functionally matured rapidly by plating them on a soft silicone surface, and investigated the role of integrin signaling in the maturation process.

Methods

iCell™ Human iPSC-CM Monolayers

Cryopreserved vials (liquid nitrogen) of iCell™ human cardiac myocytes were obtained from Cellular Dynamics International, Inc (Madison, WI). iCell™ cardiac myocytes are highly purified (>98%) hiPSC derived cells that are cryopreserved after 30–31 days of cardiac directed differentiation. Cells were thawed and subsequently plated on bovine fibronectin-coated (20 μg/mL; Life Technologies) glass coverslips, on bovine fibronectin-coated (20 μg/mL; Life Technologies) transparent PDMS membranes, matrigel (500 μg/mL; BD biosciences) coated glass coverslips, or matrigel (500 μg/mL; BD biosciences) coated transparent PDMS membranes at a density of 125,000 cells per monolayer (1,500 cardiomyocytes/mm²) in differentiation medium.^{5, 10} In all cases a 100μL droplet of cells was plated on each ECM. Differentiation medium (EB20), consisting of 80% DMEM/F12, 0.1 mmol/L nonessential amino acids, 1 mmol/L l-glutamine, 0.1 mmol/L β-mercaptoethanol, 20% FBS and 10μmol/L blebbistatin, was used to culture the cells to promote attachment. Each ECM was added as a 100μL drop of solution in the center of each coverslip, this routinely produced a 1cm diameter circle of ECM on which the cardiomyocytes were plated following a 30min incubation period and aspiration of liquid. PDMS silicone sheeting was obtained from SMI (Specialty Manufacturing, Inc. Saginaw, MI) with 40D (D, Durometer or ~1000kPa) hardness and cut to 18×18 mm coverslips. After 24 hours, the media was switched to RPMI (Life Technologies) supplemented with B27 (Life Technologies). The cells were subsequently cultured for 4–7 days at 37°C, in 5% CO₂ before phenotype analysis. Use of human pluripotent stem cells and derived cardiomyocytes was approved by the HPSCRO (Human Pluripotent Stem Cell Research Oversight) Committee of the University of Michigan.

Optical Mapping

Optical action potentials in figure 1 were recorded using FluoVolt™ membrane potential probe (F10488, Life Technologies). For calcium transient and propagation experiments, monolayers were loaded with the intracellular Ca²⁺ indicator, rhod-2AM (10 μmol L⁻¹, Life Technologies) or fluo-4AM (5 μmol L⁻¹, Life Technologies) as indicated. After a 30 minute incubation time, the cells were washed in Hanks balanced salt solution (HBSS, Life Technologies) for an additional 30 minutes before optical mapping recordings. All human cardiac monolayers displayed pacemaker activity and the spontaneous action potentials or calcium waves were recorded using a CCD camera (Red-Shirt Little Joe, Scimeasure, Decatur, GA, 200 fps, 80×80 pixels) with the appropriate emission filters and LED illumination.²¹ Movies were filtered in both the time and space domain and conduction velocity (CV) was measured as described previously.^{22, 23}

Figure 1.

Electrical wave propagation in mature hiPSC-CM monolayers. A. Left, optical activation map of spontaneously initiated electrical wave propagation in an iCell™ cardiomyocyte monolayer cultured on PDMS+matrigel. Right, single pixel signals of optical action potentials recorded from the pacemaker site and a more distal site in the monolayer. We used a CCD camera and the voltage sensitive dye FluoVolt™. B. Action potential impulse propagation velocity slowed as pacing frequency increased. The conduction velocities were: 0.7Hz= 42.4±2.3cm s⁻¹, 1Hz=36.4±1.5cm s⁻¹, 1.2Hz=33.8±0.7cm s⁻¹, 1.5Hz=29.2±0.8cm s⁻¹, 1.8Hz=27.0±0.71cm s⁻¹, 2 Hz=25.9±0.9cm s⁻¹, and 2.5Hz=23.3±0.87cms⁻¹. C. Action potential duration calculated at 80% repolarization (APD₈₀) shortened as pacing cycle length shortened. APD₈₀ values were: 0.7Hz=629.9±16.6ms, 1Hz=569.2±14.9ms, 1.2Hz=511.2±14.4ms, 1.5Hz=452.7±9.8ms, 1.8Hz=398.1±6.9ms, 2.0Hz=363.2±3.3ms, and 2.5Hz=293.3±7.3ms. n=6 monolayers for 0.7–1.8Hz and n=4 for 2.0 and 2.5Hz. Inset shows representative action potential recordings at different frequencies. All data are presented as mean ± SEM.

Biochemical analysis of hPSC-CM

Details of Western blotting, flow cytometry, immunofluorescence and image analysis can be found in the expanded supplemental methods.

Results

ECM Effects on iCell™ hiPSC-CM Monolayer Impulse Propagation

In figure 1 (left) we provide the first demonstration that purified iCell™ cardiomyocytes cultured as monolayers on PDMS+matrigel achieve a high degree of electrical maturity, with average action potential propagation velocities as high as 55 cm s⁻¹. It is important to note however that while iCell™ cardiomyocytes are highly purified; one always encounters mixtures of different cardiomyocyte phenotypes, including atrial-like, ventricular-like and pacemaker-like myocytes.^{9, 10, 24} This is reflected in figure 1A by the different optical action potential (AP) configurations (right) in the monolayer. Pacemaker-like cells at the site of impulse initiation undergo slow diastolic depolarization at a steady rate until the threshold potential is reached and an action potential is generated. More distally, ventricular-like APs have stable resting membrane potentials and respond to the propagating impulse with very rapid upstrokes. In figures 1B and C, we characterized action potential propagation velocity and duration over electrical pacing frequencies ranging from 0.7 to 2.5Hz. Figure 1B shows conduction velocity restitution as one would expect with faster conduction at lower frequency (greater cycle length) of stimulation. Figure 1C demonstrates the APD restitution of mature hiPSC-CM monolayers where APD gets shorter as pacing frequency increases. Thus, the biomatrix combination of matrigel+PDMS promotes functional electrophysiological maturation of hiPSC-CMs in as little as a week. To establish whether the softer ECM provided by PDMS+matrigel promoted maturation significantly more than stiffer matrices, we tested the combinations presented in figure 2A by quantifying calcium transient (CaT) propagation across the monolayer. First, the rate of spontaneous pacemaker activity was recorded for each of the 4 conditions. There was no difference in the spontaneous activation rate between the groups with the averages being the following: i. fibronectin+glass=0.25 ± 0.05 Hz n=10, ii. fibronectin+PDMS=0.22 ± 0.03 Hz n=4, iii. matrigel+glass=0.24 ± 0.09 Hz n=14, and iv. matrigel+PDMS=0.2 ± 0.07 Hz n=9 (mean±SEM, One way ANOVA, P>0.9999). Shown in panel B of figure 2 are representative color CaT activation maps of wave propagation for each of the experimental conditions. The fastest CV was observed in human cardiac monolayers cultured on matrigel+PDMS (figure 2B, iv). The quantification of CV in each condition is shown in panel C of figure 2. The average CVs were: fibronectin+glass=21.6±6.8 cm·s⁻¹ n=10, fibronectin+PDMS=24.6±6 cm·s⁻¹ n=4, matrigel+glass=22.0±4.0 cm·s⁻¹ n=14, and matrigel+PDMS=43.6±7.0 cm·s⁻¹ n=9 (mean±SEM, One way ANOVA, see figure and legend for details). Upper 95% confidence interval for the matrigel+PDMS group is 47.8 cm·s⁻¹. Point stimulation of monolayers (15–20V, 5ms duration, 1Hz) in each condition was performed in a separate group of experiments to determine differences in CV. The average CVs during 1Hz pacing were: i. fibronectin+glass=24.2±1.8 cm·s⁻¹ n=6, ii. fibronectin+PDMS=28.1±1.5 cm·s⁻¹ n=4, iii. matrigel+glass=28.4±3.2 cm·s⁻¹ n=5, and iv. matrigel+PDMS=37.1±1.7 cm·s⁻¹ n=6 (figure 2D, mean±SEM). Thus CV was faster during spontaneous pacemaker activations as well as during 1Hz electrical pacing in human cardiac monolayers cultured on matrigel coated PDMS coverslips. In parallel experiments iCell cardiomyocytes were plated on laminin or collagen purified from matrigel to test whether these individual components of matrigel would support confluent 2D monolayer maturation on PDMS. The results in supplemental figure 1 show that monolayer confluence was not maintained over seven days in culture on these purified components of matrigel. Optical mapping of action potential propagation was continuous in each of the conditions tested (i.e., laminin, collagen), suggesting that cellular reorganization was occurring, rather than cell death. Conduction velocity was fastest in monolayers plated on matrigel compared to those on collagen or laminin (supplemental figure 1C). Furthermore cTnI, the mature myofilament marker was only induced when monolayers were plated on matrigel (supplemental figure 1D). Surface chemistry of cell culture surfaces can also impact on cellular phenotype²⁵, and the surface chemistry of glass is distinct from PDMS. Therefore, additional experiments were performed where glass coverslips were silanized to make their surface chemistry comparable to PDMS. Results of optical mapping 2D monolayers cultured on silanized glass indicate that the differences in surface chemistry cannot account for the rapid conduction velocity that we observed on PDMS (supplemental figure 2).

Figure 2.

Effects of ECM on hiPSC-CM Monolayer Impulse Propagation. A. Four different ECM combinations were tested to determine the effects on hiPSC-CM monolayer structure and function. B. Activation maps of calcium impulse propagation in the different plating conditions. Each color represents a different activation time with time zero appearing in yellow. C. Quantification of impulse propagation. * denotes statistical difference where P<0.0001, † denotes statistical difference where P=0.003 analyzed by one way ANOVA, Bonferroni’s multiple comparisons test, n=4–14 monolayers per group. D. Quantification of impulse propagation during electrical stimulation at 1Hz. ‡denotes difference where P=0.0003, §denotes difference where P=0.01 analyzed by one way ANOVA, All data are presented as mean ± SEM.

ECM Effects on iCell™ hiPSC-CM Monolayer Action Potential and Single Cell Electrophysiology

APs are required for propagation of the electrical signal that triggers the Ca²⁺ mediated excitation-contraction coupling.^{10, 13} Therefore, hiPSC-CM monolayer APs were recorded and quantified by patch-clamp analysis in current-clamp mode. Properties of the AP such as the dV/dt_max (V/s, figures 3A&C), the maximum diastolic potential (MDP; figure 3D), and the threshold potential (take-off potential, figure 3E) provide quantitative metrics of the degree of myocyte maturity.^{9, 11, 13} We measured these functional parameters in hiPSC-CMs cultured on the extremely rigid ECM condition of fibronectin+glass (black bars, figure 3) and compared the results to hiPSC-CMs cultured on the softest ECM condition of matrigel+PDMS (red bars, figure 3). APs recorded from hiPSC-CMs grown on matrigel+PDMS displayed significantly faster upstroke velocities (65.3±8.9 V/s, N=6 monolayers, n=37 vs. 146.5±17.7 V/s, N=5 monolayers, n=24; figure 3C), more hyperpolarized MDPs (−69.9±1.7mV, N=6 monolayers, n=37 vs. −77.5±0.6mV, N=5 monolayers, n=24; figure 3D) and more hyperpolarized take-off potentials (−59.3±1.7mV, N=6 monolayers, n=37 vs.−70.5 ±1.2mV, N=5 monolayers, n=24; figure 3E), all indicative of cardiomyocyte maturation.²⁶

Figure 3.

Mature hiPSC-CM action potential and sodium channel characteristics. A. Representative action potential recordings from monolayers plated on fibronectin on glass (left) and matrigel on PDMS (right). Middle panel of A shows a faster time scale of the AP upstroke. Bottom panel of A shows the first derivative of the AP upstroke (dV/dt). B–E. AP parameters demonstrate significant electrophysiological maturation of monolayers plated on matrigel on PDMS (red). * denotes significant difference by Student’s t-test, P<0.05, N=5 monolayers −6 monolayers with n=24 or n=37 individual cellular recordings. F. Representative I_Na recordings of hiPSC-CMs cultured on fibronectin on glass (black, n=12) and cardiomyocytes cultured on matrigel on PDMS (red, n=19). G. Current-Voltage (I–V) relationship of sodium current in each condition shows elevated I_Na in cardiomyocytes cultured on matrigel coated PDMS. Comparisons made by Student’s t-test, P values as indicated. H. RT-PCR analysis of SCN5a expression. *P<0.05, Student’s t-test. All data are presented as mean ± SEM.

The faster dV/dt_max and faster impulse propagation may be attributed partially to the effect of matrigel+PDMS ECM to increase sodium current density (I_Na). Indeed, single cell patch clamp analysis of I_Na revealed significantly elevated current density in cardiomyocytes cultured on PDMS+matrigel compared to cardiomyocytes from the same batch cultured on fibronectin on glass coverslips (figure 3F&G; glass N=4, n=12 and PDMS N=4, n=19). Elevated I_Na density observed here is consistent with previous data showing that I_Na density increases in the maturing heart.²⁷ Supplemental Figure 3 shows the I_Na activation/inactivation profiles. The inactivation profile is left shifted for cardiomyocytes cultured on PDMS+matrigel (V1/2=−79.0±2.45mV, N=4, n=13 vs. −88.0±1.55mV, N=4, n=12; P=0.009), which suggests increased expression of cardiac sodium channel isoforms (i.e., SCN5A, Na_V1.5) that is known to be larger in adult than immature embryonic cardiac myocytes.^{27, 28} RT-PCR analysis confirmed elevated SCN5A gene (figure 3H) expression in iCell™ iPSC-CMs cultured on matrigel coated PDMS compared to iPSC-CMs cultured on fibronectin coated glass coverslips.

It has been reported by others that the MDP and spontaneous activity of hiPSC-CMs is critically dependent on the density of the rapid component of the delayed rectifier potassium current (I_Kr) and that I_K1 density is extremely low if not absent in hiPSC-CMs.²⁹ On the contrary, the hyperpolarized MDP of iCell™ cardiomyocytes cultured on PDMS+matrigel that we found here suggests that the I_K1 density is relatively high. Indeed, patch-clamp analysis revealed elevated I_K1 density in cardiomyocytes maintained on PDMS+matrigel biomatrix (figure 4A&B). Western blotting shows expression of Kir2.1 exclusively in cardiomyocytes cultured on PDMS+matrigel compared to the same cells cultured on fibronectin coated glass coverslips (figure 4C). To further investigate whether PDMS+matrigel promotes the expression of other potassium currents, we tested the effects of E4031 (100 nmol L⁻¹), a selective blocker of I_Kr on the spontaneous beating rate and calcium transient duration (CaTD₈₀) of hiPSC-CM monolayers cultured on matrigel coated PDMS compared to those cultured on standard rigid, plastic bottom 96 well dishes (also matrigel coated, supplemental figure 4 and supplemental movie 1 illustrate how this multi-well optical mapping platform was used). Small glass coverslips to fit into a 96 well format dish are not readily available so here we used plastic as the rigid comparator for PDMS. Figure 4D and supplemental figure 4 each show that the electrophysiology of hiPSC-CM monolayers cultured on PDMS, measured by the CaT duration, is less affected by I_Kr blockade, further supporting the evidence in figure 4 of elevated inward rectifier potassium current density in hiPSC-CMs cultured on matrigel coated PDMS coverslips. For example, 100 nmol L⁻¹ E4031 reduced spontaneous beating frequency by ~50% (0.83±0.02Hz pre E4031 vs. 0.45±0.05Hz post E4031, n=8) in monolayers plated on rigid plastic, whereas the same dose of E4031 modestly reduced beating frequency by ~20% (1.02±0.04Hz pre E4031 vs. 0.78±0.07Hz post E4031, n=5). This effect on beating frequency is likely due to the effect of E4031 to prolong the action potential and calcium transient durations. Similarly, CaTD₈₀ increased ~3X following E4031 application in hiPSC-CMs plated on plastic bottom dishes (605.9±24.3ms pre E4031 to 1873.8±278.9ms post E4031, n=8), but CaTD₈₀ only increased by ~1.34X in hiPSC-CMs plated on PDMS bottom dishes (579.8±13.9ms pre E4031 vs. 780.1±74.1ms post E4031, n=5; supplemental figure 4).

Figure 4.

Potassium current density (I_K1) in hiPSC-CM single cells. A. Representative I_K1 recordings in single hiPSC-CM cultured on fibronectin on glass (n=7, black, top) and hiPSC-CM cultured on matrigel on PDMS (n=5, red, bottom). B. I–V relationship of I_K1 in each condition shows significantly elevated current density in hiPSC-CM cultured on matrigel on PDMS (red). Comparison by student’s t-test, p value as indicated. C. Western blot probing for Kir2.1 demonstrates expression only in hiPSC-CMs cultured on matrigel on PDMS (lane 1 in each blot, 2 individual monolayers for each condition). D. intracellular calcium flux measurements in hiPSC CMs cultured on rigid plastic bottom dishes (matrigel coated) show very significant impact of E4031 blockade on spontaneous beating frequency and calcium transient duration 80 (CaTD₈₀). Quantification shows greater effect of E4031 on the beat frequency and CaTD₈₀ in immature iPSC CMs cultured on rigid plastic bottom dishes compared to PDMS bottom dishes. Unpaired t-test, for effect on beat frequency: *P=0.000000001, †P=0.01 and for effect on CaTD₈₀ ‡P=0.0000001, §P=0.0003 data expressed as mean±SEM.

ECM Effects on Intercellular Junction Formation

Gap junctions: Cx43 expression

ECM stiffness has been shown to impact rodent neonatal myocyte Cx43 expression at the intercellular gap junctions. Previously, Forte et al. found that softer substrates impact Cx43 expression and myocyte morphology.²⁰ Consistent with previous reports using rodent myocytes, here we find that Cx43 expression is elevated in hiPSC-CM monolayers cultured on PDMS coated with matrigel compared to monolayers cultured on glass coverslips (figure 5) or fibronectin coated PDMS. We determined the effect of the different plating combinations (figure 2A) on Cx43 expression and subcellular localization. Panel A of figure 5 shows the Cx43 expression and localization (red) in hiPSC-CM monolayers plated in the various ECM conditions. The greatest Cx43 expression at the intercellular junctions is found in monolayers plated on matrigel+PDMS. This provides yet another molecular mechanism to explain the faster CV found for this biomatrix combination. In figure 5B, Western blot analysis shows that the amount of Cx43 expression was ~3X greater when fibronectin+PDMS is compared to matrigel+PDMS (0.07 ± 0.05 A.U. vs. 0.19 ± 0.10 A.U., n=5 monolayers, p < 0.05). Next we determined the effect of PDMS to promote Cx43 expression in purified UM22-2 human embryonic stem cell-derived cardiomyocyte (hESC-CM) monolayers. The UM22-2 control hESC-CMs were generated by the matrix sandwich differentiation protocol.²⁴ Immunostaining for α-actinin and Cx43 in hESC-CM monolayers also indicated robust induction of Cx43 expression and localization at the cell-cell borders by PDMS substrate (supplemental figure 5A). Similar to the iCell™ Cx43 expression, hESC-CM Cx43 expression outlines the entire cardiomyocytes when cells are cultured on PDMS. Collectively, these results demonstrate that the ECM combination of matrigel+PDMS promotes the development of functional gap junctions available for more efficient intercellular communication and faster impulse propagation in hPSC-CM monolayers.

Figure 5.

ECM Effect on Cx43 Expression and cardiomyocyte size. A. Immunostaining of α-actinin (green) and Cx43 (red). DAPI (blue) marks nuclei. Monolayers on matrigel coated PDMS exhibit the greatest amount of Cx43 at the cell-cell borders. B. Western blotting for Cx43 and total myosin confirms the immunofluorescence results of panel A. Quantification of total Cx43 protein expression shows elevated expression in hiPSC-CM cultured on matrigel on PDMS (n=5 monolayers in each condition). *Student’s t-test, P<0.05. All data are presented as mean ± SEM. C&D. hiPSC-CM (iCell™) cell size was determined 5 days post thaw by immunoflurescent staining for N-cadherin. There was no difference in hiPSC-CM size between the fibronectin+glass group and the matrigel+glass group (938.7±61.2μm², n=79 vs. 917.8±64.2μm², n=66). Myocytes plated on fibronectin+PDMS were larger (1403.4±66.9μm², n=132) and myocytes plated on matrigel+PDMS were even larger (2130.3±99.9μm², n=130) than those plated on rigid glass coverslips. One way ANOVA, Bonferroni’s multiple comparison test; †P<0.0001 and ‡P<0.001

Terminal Differentiation, Hypertrophic Growth and cTnI Expression

After birth, myocytes in the heart switch from hyperplastic growth to hypertrophic growth and this is part of the natural maturation process.^{11, 30, 31} Therefore another marker of maturation of hPSC-CMs is the transition from cardiomyocytes remaining in the cell cycle to myocyte terminal differentiation and hypertrophy. The hiPSC-CMs cultured on pliable PDMS were significantly larger in size than cells plated on glass substrates (figure 5C&D). This indicates induction of developmental hypertrophy in hiPSC-CMs cultured on soft substrates. Binucleation, another marker of myocyte maturity,^{31, 32} was also apparent in hiPSC-CMs cultured on matrigel+PDMS (figure 6A&B and supplemental figure 6). The cell cycle marker Ki67 was also used to determine the differentiation state of cardiomyocytes.³³ In iCell cardiomyocytes the percentage of binucleated cells that were also Ki67+ was lower in iPSC-CMs cultured on PDMS+matrigel (figure 6B). Furthermore, using highly purified BJ hiPSC-CMs developed in our laboratory³⁴(figure 5B) we quantified the effect of PDMS to reduce the number of cardiomyocytes remaining in the cell cycle (figure 6C). One key myofilament marker of cardiomyocyte maturation is cTnI expression.11, 35 We took this parameter of maturation into consideration here by Western Blot analysis of BJ hiPSC-CM expression of cTnI when purified monolayers were cultured on glass or PDMS coverslips coated with matrigel. Significantly more robust cTnI expression was detected relative to GAPDH in purified BJ hiPSC-CMs plated on PDMS coverslips compared to glass coverslips (figure 6D), thus indicating a greater level of sarcomeric maturation and promotion of developmental isoform switching that is known to occur in the post-natal heart. Furthermore, cTnI sarcomeric incorporation was also apparent in hiPSC-CM monolayers cultured on PDMS, but not in immature monolayers cultured on Glass coverslips (supplemental figure 5C).

Figure 6.

Molecular markers of maturation. A. Flow cytometry analysis for quantification of the population of binucleated iCell cardiomyocytes. B. The proportion of binucleated cells was significantly greater in the PDMS group compared to matrigel coated glass (35.24±3.0% vs. 21.68±1.08%, n=5 per group; *denotes significant difference, t-test, P=0.003). Furthermore, the incidence of Ki67 positive binucleated cells was less in the PDMS group (13.4±1.1% vs. 20.5±1.1%, n=5 per group, †denotes significant difference, t-test, P=0.002. C. (top panel) immunostaining for Ki67 (red) and α-actinin (green) shows decreased proliferative activity in BJ-hiPSC-CMs cultured on PDMS compared to glass coverslips (0.87±0.29 CM/60X field compared to 6.2±0.90 CM/60Xfield; n=8 and n=10. ‡P=0.0001). C, (Bottom panel), immunostaining for sarcomeric actin (red) and N-cadherin (green) shows hypertrophy and elongation of BJ-iPSC-CMs cultured on PDMS compared to glass coverslips (cell area=3,678.59±171.6μm² compared to 2,071.44±116.7μm²;n=84 and n=83. §P=0.000003). D. Western Blotting for cTnI protein expression. On glass coverslips the cTnI/GAPDH ratio=0.39±0.11au and on PDMS coverslips the cTnI/GAPDH ratio=0.6464±0.005au, ║denotes significant difference, P<0.05, unpaired t-test. All data mean ± SEM.

Integrin Signaling in the Maturation Process

Integrins are transmembrane heterodimeric receptors essential for providing cell-extracellular matrix adhesion, cellular structural organization and transduction of mechanical signals from the extracellular matrix into biochemical signals in cardiomyocytes.^36–38 β1 integrins are abundant in the adult heart and participate in the hypertophic response in rodent ventricular myoyctes.³⁹ Therefore we hypothesized that integrin signaling underlies the maturation of hiPSC-CM monolayers induced by the cell culture condition of plating them on matrigel coated PDMS coverslips. First RT-PCR analysis showed that ITGB1 expression is significantly induced on PDMS coverslips compared to glass coverslips (figure 7A). Additionally, PTK2 gene expression is elevated in hiPSC-CM monolayers cultured on PDMS coverslips (figure 7B). The PTK2 gene encodes the Focal Adhesion Kinase (FAK) intracellular molecule that is a primary mediator of integrin signaling.⁴⁰ Figure 7C shows the localization of β1 integrin receptors in monolayers; there was elevated expression in monolayers cultured on PDMS.

Figure 7.

Integrin signalling via Focal Adhesion Kinase promotes maturation of hiPSC-CM monolayers. A. RT-PCR analysis indicates elevated expression of ITGA5 and ITGB1 integrin receptor genes in mature monolayers (red). RT-PCR performed in triplicate for 5 individual monolayers for each group. B. PTK2 gene expression is elevated in mature monolayers (red). C. β1 integrin localization (red) in hiPSC-CM monolayers. More extensive receptor expression is apparent on PDMS. D. Nab (Neutralizing antibody) again β1 integrin receptors blocks FAK activation and cTnI expression in iCell CMs. *significant difference, t-test, P<0.05; †t-test, P=0.02; n=3 monolayers per group. E. Pharmacological inhibition of FAK activity using FAK inhibitor-14 prevents cTnI protein expression. F. FAK inhibition prevents PDMS induced hypertrophic growth of hiPSC-CMs. Cell areas: PDMS control=4,410.8±217.3μm²; 10μmol L⁻¹ =2,547.7±104.7 μm²; 100μmol L⁻¹ =1,057.1±59.0 μm²). ‡denotes significant difference from GLASS (see supplemental figure 7), §denotes significant difference within PDMS group compared to control media, One way ANOVA, P<0.0001.

Next we blocked integrin signaling by two different approaches: 1. neutralization of β1 integrin receptor activation using isoform specific antibody, and 2. pharmacological blockade of FAK activation using FAK inhibitor-14. Importantly, lower dose of FAK inhibitor-14 did not reduce hiPSC-CM monolayer confluence significantly (supplemental figure 7). Western blotting in figure 7D indicates that FAK activation was prevented by Nab (neutralizing antibody) and also that cTnI protein expression was attenuated. Collectively this indicates that FAK activation via β1 integrin activation contributes to the expression of mature myofilament markers such as cTnI. Further, purified hiPSC-CMs (iCell™) cultured on PDMS in the presence of 10μmol L⁻¹ FAK inhibitor-14 failed to express cTnI and also expressed less β-MyHC (figure 7E and supplemental figure 8). This suggests that FAK activation underlies expression of mature myofilament markers. Additionally, we determined the role of FAK activation in the hypertrophic response of hiPSC-CMs to the soft cell culture environment on PDMS (figure 7F and supplemental figure 7). FAK inhibitor-14 prevented hypertrophic growth of hiPSC-CMs observed on PDMS coverslips. Interestingly, hiPSC-CMs grown on glass coverslips were only affected at very high dose of FAK inhibitor-14 (100μmol L⁻¹). In both conditions (Glass and PDMS) 100 μmol L⁻¹ FAK inhibitor-14 reduced monolayer confluence and hiPSC-CM expansion in culture (supplemental figure 7&8). Finally, the effects of integrin receptor activation were next studied using a monoclonal antibody that stabilizes the dimerization of α₅β₁ integrins and promotes FAK activation (MAB1969). Figure 8 summarizes the Western blot analysis of hiPSC-CM monolayers (iCell) treated with the activating α₅β₁ integrin monoclonal antibody or FAK inhibitor-14. FAK activation (phosphorylation) was induced by this specific antibody (figure 8B) and so was cardiac sodium channel protein expression (Nav1.5, figure 8C).

Figure 8.

Integrin activation promotes Nav1.5 sodium channel expression. A. Treatment of hiPSC-CM monolayers chronically with a mouse monoclonal antibody for the α5β1 integrin receptor heterodimer (fibronectin receptor) causes increase of total FAK expression and activation (B). C. Sodium channel (Nav1.5) expression is also induced by integrin activation via α5β1 integrin receptor antibody treatment. Data is expressed as Whiskers plots (mean, maximum and minimum values are shown), ANOVA was used to test for significance with *P=0.016 (A), †P=0.001 (B), ‡P=0.003 (panel C) to indicate difference.

Discussion

We have demonstrated the following in hiPSC-CM 2D monolayers: First, culturing the monolayers on a soft PDMS membrane (40D, durometers) coated with matrigel, but not fibronectin alone, increases the impulse CV to values up to 48 cm s⁻¹, which is 2X faster than previously reported for human iPSC-CM monolayers.⁵ Second, PDMS+matrigel ECM promotes electrophysiological maturation of the hiPSC-CM single cell characterized by increased inward rectifier potassium and sodium inward current densities, giving rise to a well polarized MDP and faster action potential upstroke velocity, respectively. Third, formation of intercellular gap junctions and mechanical junctions are promoted by the soft PDMS+matrigel ECM. Fourth, hiPSC-CM hypertrophy and mature myofilament isoform expression are induced when plated on pliable PDMS rather than on rigid glass coverslips. Fifth, integrin β1 and α5 receptor gene expression is induced in the maturation process and FAK activity is required for maturation. Remarkably, the electrophysiological and structural maturation of iCell™ cardiomyocytes plated on the optimal biomatrix combination as reported here occurs in as little as 1 week after re-plating cryopreserved cardiomyocytes. This represents a major advance over previous reports that have demonstrated modest maturation of stem cell derived cardiomyocytes over a period of up to 9 months.^{35, 41, 42} Importantly, the process described here does not require gene transfer or other genetic modifications that may artificially mimic the developmental process. Furthermore we have validated the maturation observed on PDMS in cardiomyocytes derived from multiple human pluripotent stem cell lines including other hiPSC lines and one hESC line.

Mounting evidence has made it apparent that combining matrigel with softer synthetic biomaterials is a way to produce more mature hiPSC and/or ESC derived human cardiomyocytes that will be more useful for disease modeling, drug testing and for cardiac regeneration applications.^{43, 44} The current work is unique in that we have studied maturation of electrically and mechanically connected human stem cell derived cardiac monolayers that function as a syncytium. Importantly we show that confluent monolayer culture promotes more electrophysiological maturation than single cell culture approaches on soft matrigel substrates. In 2005 Baharvand et al⁴⁵ reported the importance of using either a native cardiac ECM called cardiogel or matrigel when culturing mouse ESC derived cardiomyocytes. In that study Baharvand et al reported modest maturation of murine cardiomyocytes cultured on both cardiogel and matrigel ECM, and also showed that cardiomyocytes grown on matrigel respond in a more mature cardiomyocyte manner to carbachol administration. Thus it seems that the complex composition of matrigel ECM consisting of collagen, laminin, fibronectin and other components may provide a better maturation ECM than the slightly more defined native cardiac ECM. Despite the higher level of maturation that we have reported here using matrigel coated PDMS coverslips, the use of matrigel does impose limitations. Matrigel is derived from a mouse engelbreth-holm-swarm sarcoma cell line.⁴⁶ It contains not only a random array of extracellular matrix proteins, but also a variety of growth factors. Thus, it is ill defined and may vary from batch to batch. Despite this concern, the maturation process reported here has been repeatable using over a dozen different batches of matrigel.

Previous studies have employed three dimensional cell culture techniques to mature human stem cell derived cardiomyocytes. For example, Nunes et al⁸ attempted to mature human stem cell derived cardiomyocytes by creating a 3D biowire platform. While they reported modest maturation and cardiomyocyte alignment, conduction velocity of those biowires was very slow with the most mature constructs having a conduction velocity of only ~15cm s^−1.8 Here we have generated mature 2D hiPSC-CM monolayers with conduction velocities of up to 55cm s⁻¹ and immature monolayers whose impulse propagation velocity of ~25cm s⁻¹- both of which are faster and more mature than in the biowire platform. Furthermore, here we have shown maturation in single hiPSC-CM ion channel densities (I_K1, I_Na), ion channel expression (Kir2.1, SCN5A), action potential profiles (rapid dV/dt, hyperpolarized diastolic potentials and threshold potentials), and structural maturation in monolayers (cTnI expression, Cx43 expression, and hypertrophy). In particular figure 5 demonstrates that immature hiPSC-CM monolayers depend almost entirely on I_Kr repolarizing current and this is congruent with previous reports.²⁹ On the other hand, mature hiPSC-CM monolayers cultured on PDMS express both Kir2.1 channels and robust I_K1 density (figure 4) and respond to the I_Kr blocker E4031 in a more modest manner that resembles responses of adult myocardium (figure 4).

Importantly our findings demonstrate the ability of hiPSC-CMs to terminally differentiate, form gap junctions and undergo myofilament isoform switches that may promote greater tension development and responsiveness to autonomic input. This may have impact on the development of autologous cardiac regeneration approaches using iPSC-CMs. Terminal differentiation of hiPSC-CMs is indicated by binucleation and significant reduction of Ki-67 positive cardiomyocyte nuclei (figure 6). This suggests that hiPSC-CMs have the capacity to exit the cell cycle in the right environment and may not be tumorigenic when used for cardiac regeneration therapies. There is also significant induction of Cx43 containing gap junctions when hiPSC-CMs are put into the optimal maturation environment (figure 5). Induction of Cx43 expression suggests that hiPSC-CMs have the capacity to electrically integrate into native myocardium if used for development of autologous cardiac regeneration therapies. We demonstrate robust induction of cTnI in mature hiPSC-CMs (figure 6) and that expression of mature myofilament proteins is dependent on β1 integrin receptor activation and FAK activity (figure 7). Thus, when in the proper environment hiPSC-CMs have the capacity for myofibrillar isoform switching including expression of cTnI, a critical sarcomeric component that modulates cardiac contractility on a beat-beat basis.^{47, 48}

Integrin signaling is a dynamic and complex process in cardiomyocytes depending on the ECM components and mechanical activity.⁴⁹ The expression and dimerization of integrin receptors can be triggered by specific extracellular protein components and specific ECM proteins (fibronectin vs. laminin, e.g.) to impact cardiomyocyte gene expression and function. Systematic study of integrin isoforms and dimers will be required to determine the optimal ECM for maturation of PSC-CMs. Our results indicate the capacity of hiPSC and hESC-CMs to mature in vitro; however, for cell transplantation the optimal state of maturation to use for cardiac regeneration that will lead to the best outcomes requires further investigation.

WHAT IS KNOWN.

• Human cardiomyocytes derived from stem cells routinely demonstrate a fetal-like, immature functional and structural phenotype.

• Maturation of stem cell derived cardiomyocytes is necessary to promote the utility of these cells for drug discovery and hypothesis testing.

WHAT THE STUDY ADDS.

• Human stem cell derived cardiac monolayers mature rapidly when formed on soft, pliable substrates rather than on rigid cell culture surfaces.

• Electrical impulse propagation of human stem cell derived cardiac monolayers can be as fast as ~50cm s⁻¹; similar to the velocity in the adult cardiac ventricle.

• Integrin receptor activation is crucial for significant maturation of human stem cell derived cardiac monolayers.