PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circ Arrhythm Electrophysiol. Author manuscript; available in PMC 2013 August 30.
Published in final edited form as:
PMCID: PMC3757253
NIHMSID: NIHMS504111

Mechanism-based facilitated maturation of human pluripotent stem cell-derived cardiomyocytes

Deborah K. Lieu, PhD,1,2,# Ji-Dong Fu, PhD,3,# Nipavan Chiamvimonvat, MD,2,4 Kelvin W. Chan Tung, MSc,8 Gregory P. McNerney, PhD,9 Thomas Huser, PhD,9 Gordon Keller, PhD,8 Chi-Wing Kong, PhD,1,5,6,7 and Ronald A. Li, PhDcorresponding author1,5,6,7

Abstract

Background

Human embryonic stem cells (hESCs) can be efficiently and reproducibly directed into cardiomyocytes (CMs) using stage-specific induction protocols. However, their functional properties and suitability for clinical and other applications have not been evaluated.

Methods and Results

Here we showed that CMs derived from multiple pluripotent human stem cell lines (hESC: H1, HES2) and types (induced pluripotent stem cell or iPSC) using different in vitro differentiation protocols (embryoid body formation, endodermal induction, directed differentiation) commonly displayed immature, pro-arrhythmic action potential (AP) properties such as high-degree of automaticity, depolarized resting membrane potential (RMP), Phase 4- depolarization and delayed after-depolarization (DAD). Among the panoply of sarcolemmal ionic currents investigated (INa+/ICaL2+/IKr+/INCX+/If+/Ito+/IK1-/IKs-), we pinpointed the lack of the Kir2.1-encoded inwardly rectifying K+ current (IK1) as the single mechanistic contributor to the observed immature electrophysiological properties in hESC-CMs. Forced expression of Kir2.1 in hESC-CMs led to robust expression of Ba2+-sensitive IK1 and more importantly, completely ablated all the pro-arrhythmic AP traits, rendering the electrophysiological phenotype indistinguishable from the adult counterparts. These results provided the first link of a complex developmentally arrested phenotype to a major effector gene, and importantly, further led us to develop a biomimetic culturing strategy for enhancing maturation.

Conclusions

By providing the environmental cues that are missing in conventional culturing method, this approach did not require any genetic or pharmacological interventions. Our findings can facilitate clinical applications, drug discovery and cardiotoxicity screening by improving the yield, safety and efficacy of derived CMs.

Keywords: human embryonic stem cells, cardiomyocytes, maturation, electrophysiology, arrhythmogenicity

Introduction

Loss of non-regenerative, terminally differentiated cardiomyocytes (CMs) is irreversible; myocardial repair is further hampered by a severe shortage of donor cells and organs. CMs can be differentiated from human (h) embryonic stem cells (ESCs) that can propagate indefinitely in culture while maintaining their pluripotency1-9. Therefore, hESCs may provide an unlimited ex vivo source of CMs for clinical application and drug testing. While existing efforts mostly focus on the derivation of heart cells from hESCs, it is imperative that these derived CMs are functionally mature in ways similar to their adult counterparts before the desired therapeutic outcome can be achieved. In fact, hESC-CMs exhibit embryonic- or fetal-like electrophysiological properties2, 9-10. For instance, hESC-derived ventricular CMs exhibit spontaneously firing action potentials (AP), in contrast to the normally quiescent-yet-excitable phenotype of adult. Indeed, we previously demonstrated that transplantation of a node of electrically-active hESC-CMs, consisting of a mixture of ventricular, atrial and nodal cells, could collectively serve as a surrogate pacemaker in vitro and in vivo5. Thus, immature hESC-CMs are potentially arrhythmogenic after transplantation. Recently, more efficient methods for directed cardiac differentiation have been established. Although it is well accepted that their cardiac derivatives are similarly functionally immature, their electrophysiology has not been systematically examined. Understanding their electrophysiology is critical for the safety of clinical application, since abnormal pulse formation and reentry of action potentials (APs) can be arrhythmogenic. Moreover, there is a need to develop protocols for rescuing the immature phenotypes for their eventual clinical and other applications (e.g., cardiotoxicity screening and heart disease models that accurately reflects the adult heart).

Methods

Culture and Directed Differentiation

HES2 human ESC line (ESI, Singapore) was cultured and differentiated by co-culturing with the immortalized endoderm-like END2 cells as previously reported2, 7. H1 human ESC line (WiCell, Madison, WI) was cultured and differentiated by embryoid body (EB) formation1, 8-9 as described previously. Human induced pluripotent stem cells (iPSC ES4skin-clone 3), a kind gift from Dr. James Thomson (Wisconsin, Madison), were also maintained as described11. IPSC-CMs were derived via EB formation following the H1 hESC differentiation protocol. D3 murine ESCs were cultivated and differentiated into spontaneously beating CMs as previously described12.

For directed differentiation (dd) of HES2 hESCs into the cardiac lineage, cardiogenic EBs (or mesodermal cardiospheres) were generated for 24 hours in the presence of 0.5 ng/ml BMP4 in STEMPRO 34 media as described13. From days 1-4 the EBs were induced with 10 ng/ml BMP4, 5 ng/ml bFGF and 3 ng/ml activin A and then from days 4-8, they were cultured in 150 ng/ml DKK1 and 10 ng/ml VEGF. From day 8 onwards the EBs were maintained in the presence of 10 ng/ml VEGF, and 5 ng/ml bFGF. Cultures were maintained in a 5% CO2/5% O2/90% N2 environment for the first 10–12 days and were then transferred into a 5% CO2/air environment. Directed differentiation typically results in aggregates consisting of 40-50% CMs as assessed by expression of cardiac troponin T using FACS. For driven maturation, ddhESCs-CMs of 24-28 days were plated for electrical stimulation at 2.5 V/cm with 5 msec pulse width at 1 Hz for 14 days before experiments.

Adenovirus-Mediated Gene Transfer of Single Derived-CMs

The full-length coding sequence of human Kir2.1 was cloned into the multiple-cloning site of pAd-CMV-IRES-GFP (pAd-GFP) to generate pAd-CMV-GFP-IRES-Kir2.1 (pAd-Kir2.1), where GFP allows for identification of positively transduced cells. Adenoviruses were generated by Cre-lox recombination of purified ψ5 viral DNA and shuttle vector DNA as previously described14. The recombinant products were plaque purified, expanded and purified by CsCl gradient, yielding concentrations on the order of 1010 PFU/ml. For transduction, adenoviral particles were added to single ddhESC-CMs (20~25 days), EB-hESC-CMs (7+14~21days), END2-hESC-CMs (16~20 days), EB-iPSC-CMs (7+21~24days) and EB-mESC-CMs (7+4 days) at a concentration of ~2×109 PFU 15.

Electrophysiology

HESC- and hiPSC-CMs were dissociated into single cells with 1 mg/ml collagenase II and plated on 0.1% gelatin-coated glass coverslips. Electrophysiological experiments were performed using whole-cell patch-clamp technique with an Axopatch 200B amplifier and the pClamp9.2 software (Axon Instruments Inc., Foster City, CA). Pipette solution was consisted of (mM): 110 K+ aspartate, 20 KCl, 1 MgCl2, 0.1 Na-GTP, 5 Mg-ATP, 5 Na2-phospocreatine, 1 EGTA and 10 HEPES, with pH of 7.3. The external Tyrode's solution consisted of (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES with pH of 7.4. Voltage- and current-clamp recordings were performed at 37°C within 24 to 48 hours after adenovirus transduction. ESC-CMs were categorized into pacemaker, atrial or ventricular phenotypes based on the maximum diastolic potential (MDP), maximum rate of rise of the AP and AP duration (APD). In general, pacemaker cells always generated spontaneous APs exhibiting a more depolarized MDP, slower maximum rate of rise and the shortest APD. Atrial cells with a triangular AP profile have a faster rate of rise than pacemaker cells, more hyperpolarized MDP or resting membrane potential (RMP), and intermediate APD. Ventricular cells have the most hyperpolarized MDP or RMP, a fast rate of rise and the longest APD that exhibited a prolonged AP phase 2. Different protocols as given in insets were employed to elicit the corresponding currents. 5 mM nifedipine, 30 mM tetrodotoxin (TTX), 1 mM Ba2+, 30 mM ZD7288, 10 mM E4031 and 30 mM Chromanol 293B were used to define the ICaL, INa, IK1, If, IKr, and IKs, respectively. All blockers were purchased from Sigma.

Formulation of Cardiac Electrophysiology Mathematical Model

Ionic currents and membrane potential of ventricular CMs were formulated based on an embryonic chick ventricular cell model16 and according to the algorithms that we previously reported17. In our model, the six ionic currents initially included based on previous reports18 were slow inward Ca2+ current (ICa), slow delayed K+ current (IKs), rapid delayed rectifier K+ current (IKr), pacemaker current (If), background current (Ib), and seal-leak current (Iseal). The kinetics of the currents was derived empirically from experimental data16. IK1 was absent in our base model to simulate our experimental data and subsequently manipulated to predict the effects of Kir2.1 overexpression. The computations were performed in Matlab (Mathworks, Natick, MA) using a variable order ordinary differential equation solver plus a built-in backward-difference method, with relative tolerance of 10-8 and absolute tolerance of 10-4.

Confocal Ca2+ Imaging

Single-cell Ca2+ transients of Fluo-4-loaded ddhESC-CMs were recorded with a spinning disk confocal microscope (Yokogawa CSU10) at ~180 frames per second with a 40× microscope objective at room temperature in Tyrode's solution consisting of (mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose and 10 HEPES at pH 7.4. Ca2+ transients were elicited by a field stimulator at 0.2 Hz and 40 V with 90 ms pulse duration. The Ca2+ transient changes were quantified by the background subtracted fluorescent intensity changes normalized to the background subtracted baseline fluorescence using Image J.

Quantitative PCR

mRNA was extracted from derived CMs using RNeasy Kit (Qiagen, Valencia, CA). cDNA were reverse transcribed from mRNA using Quantitect Reverse Transcription Kit (Qiagen). Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) was used for qPCR analysis with a BioRad iCycler (BioRad, Hercules, CA). The expression level was normalized relative to GAPDH using the ΔΔCT method.

Transmission Electron Microscopy

Unpaced control and electrically conditioned beating cardiospheres from two separate differentiation batches were manually dissected out, then fixed with Karnovsky's fixative at room temperature. Specimens were further fixed with 1% oxmium tetroxide for 90min. After acetone dehydration, the specimen were infiltrated with epoxy resin and allowed to polymerize at 70°C overnight. Sections 60nm thick were stained with uranyl acetate. A total of 15 unpaced control and 13 electrically conditioned cells were imaged with a FEI/Philips CM120 TEM (Philips/FEI, Endhoven, Netherlands).

Statistics

Data are shown as mean±SEM. Unpaired Student's t test or Chi-square (χ2) test were used for statistical analysis where p<0.05 was considered statistical significant.

Results

Pro-arrhythmic Properties of Derived CMs are Independent of Cardiogenesis Protocol and Origin of Stem Cell Lines

Single ddhESC-CMs from cardiogenic EBs differentiated for 20-25 days were electrophysiologically assessed by whole-cell patch-clamp techniques. The ddhESC-CMs were classified by their ability to generate AP into spontaneously firing and quiescent ddhESC-CMs, and also classified into CM subtypes by the observed signature ventricular, atrial and pacemaker APs (Figure 1A). Unlike healthy adult CMs, 26% of all ddhESC-CMs displayed a high degree of automaticity or spontaneous firing of APs (Figure 1C). While the remaining ddhESC-CMs were quiescent, stimulation could elicit a single AP characteristics of ventricular or atrial subtype (Figure 1A, right), indicating that their excitability remained intact; however, these quiescent-yet-excitable ddhESC-CMs displayed a prominent “phase 4-like” depolarization (Figure 1A, arrows), a known pro-arrhythmic triggering substrate that is also not seen in mature contractile adult CMs19-21. The ddhESC-CM population overall exhibited a distribution of 42% ventricular, 53% atrial and 5% pacemaker CMs (Figure 1B; n=70). Delayed-after depolarization (DAD) was observed in 23% of quiescent ventricular ddhESC-CMs (Figure 1D). Furthermore, the resting membrane potentials (RMPs) were significantly more depolarized (cf. Figure 4E) than ~-80mV typical of normal adult19-21 and comparable to ~-53mV typical of immature human fetal ventricular CMs of 18 weeks (our unpublished data).

Figure 1
Electrophysiology of pluripotent stem cell derived CMs. A) Representative tracings of spontaneously firing (left) and quiescent (right) ventricular (top), atrial (middle) and pacemaker (bottom) action potentials (APs) recorded from ddhESC-CMs. The arrows ...
Figure 4
Effects of IK1 presence on electrophysiology of pluripotent stem cell derived-CMs. A) In silico analysis of IK1 effects on the maturation of ventricular embryonic electrophysiological phenotypes. B) Action potentials (APs), If and IK1 of Ad-Kir2.1-transduced ...

Given the potential for myocardial repair, we next focused on analyzing ventricular ddhESC-CMs. To understand the electrophysiological basis of the observed AP phenotypes, we functionally profiled the individual ionic components present (Figure 1E and Figure 2). Ventricular ddhESC-CMs robustly expressed INa, ICaL and Ito13, which are comparable to adult ventricular CMs. If that is typically not seen in healthy adult ventricular CMs (except in immature or pathophysiological states such as hypertension and heart failure) was highly expressed in both atrial and ventricular ddhESC-CMs with a higher current density for the atrial relative to ventricular subtype, but their steady-state activation curves were identical (Figure 2A, B). ICaL was present at higher current density in ventricular relative to atrial ddhESC-CMs with similar steady-state inactivation properties (Figure 2C, D). However, the inwardly rectifying K+ current (IK1) and slow (IKs) components of the delayed rectifier that are present in high current density in adult CMs for repolarization were not present in ventricular ddhESC-CMs. Although not as robust as the adult counterpart, the rapid component of the delayed rectifying K+ current (IKr) was expressed in both atrial and ventricular ddhESC-CMs with a slightly higher current density in the latter (Figure 2F).

Figure 2
Current-voltage, activation and inactivation relationships of currents in ddhESC-CMs. A) Current-voltage (I-V) relationships of If in ventricular (n=6) and atrial (n=6) ddhESC-CMs. B) Steady-state activation curves of If in ventricular (n=6) and atrial ...

Inductive interactions among the three primitive germ layers figure prominently in embryogenesis22, the first step of ddhESC-CMs derivation involved directed mesodermal differentiation13. Considering the method of induction as well as the specific hESC line may influence the type of CMs that develops23, we next examined and compared electrophysiology of hESC-CMs derived by two distinct methods: 1) endodermal induction with END2 co-culturing of the HES2 (END2-hESC-CMs)2 and 2) EB formation of the H1 hESC lines (EB-hESC-CMs)1. Like the ddhESC-CMs, immature AP properties such as automaticity, phase 4-like depolarization, depolarized RMP, the presence of DAD and If, and the absence of IK1 were all commonly observed in both END2- (Figure 3A-C) and EB-hESC-CMs (Figure 3D). Even human induced pluripotent stem cell11 (iPSC)-derived CMs by EB formation behaved similarly (Figure 1F). Taken together, these data suggest that the immature, pro-arrhythmic AP properties are genuine developmental hallmarks of early stage human CMs derived in vitro. Unfortunately, derived CMs failed to mature further even after culturing for >100 days24. These data hint at the intriguing possibility that a crucial physical component that drives hESC-CM maturation in vivo is missing in the conventional method of in vitro culture.

Figure 3
Electrophysiological properties of hESC-CMs derived from the HES2 and H1 lines. A) Representative action potential (AP) waveforms of spontaneous and quiescent ventricular, atrial and pacemaker END2-hESC-CMs as indicated (left), showing percentage distribution ...

IK1 Is a Major Mechanistic Contributor to the Immature AP of hESC-CMs

In normal fetal CM development, one of the major electrophysiological changes is a progressive increase in IK1 and concomitant reduction in If25. In heart failure, the fetal gene program is re-initiated in adult CMs to cause electrical remodeling 26, such that IK1 becomes down-regulated whereas If is reciprocally upregulated, thereby predisposing the afflicted individuals to lethal arrhythmias. Along this line, our data collectively hint at three possible mechanisms for the immature AP phenotypes: the presence of If, the absence of IK1, or both. To understand the basis of electrical immaturities and thereby develop a strategy for maturing and eliminating the undesirable proarrhythmic traits of derived CMs, we next performed an in silico analysis of their AP profiles (Figure 4A)16-17. By incorporating all the ionic components identified (Figure 1E), our ventricular model sufficed to reproduce the experimentally determined AP parameters. When the maximum conductance of IK1 (GK1) was increased from 0, the experimentally observed level, to 3.6 nS (i.e. 1/8 of that of adult ventricular cells) and subsequently to 7.2 nS, the firing rate decreased accordingly. When GK1 was 10.8 nS or higher, the spontaneous firing ceased along with RMP hyperpolarized to the adult level; these IK1-silenced cells remained excitable and could generate a normal ventricular AP when triggered by a stimulus. Silencing could not be achieved by If suppression alone; thus, according to our model, the absence of IK1 appears to be a major mechanistic contributor of pro-arrhythmic automaticity.

To experimentally test our mathematical modeling results, we generated the recombinant adenovirus Ad-CMV-GFP-IRES-Kir2.1 (or Ad-Kir2.1) to mediate the expression of Kir2.1 channels that underlie IK127. In contrast to untransduced or Ad-GFP-transduced IK1-negative control, Ad-Kir2.1-transduced ventricular ddhESC- (n=7) and END2-hESC- (n=13) CMs robustly expressed Ba2+-sensitive IK1 (Figure 4B, C and G). More importantly, the percentages of quiescent ventricular ddhESC- and END2-hESC-CMs dramatically increased to 100% (Figure 4D; p<0.05) with their RMP significantly hyperpolarized (p<0.05, Figure 4E) to the adult level (p>0.05). Ad-Kir2.1-silenced cells remained excitable and could generate a single AP upon stimulation, but most importantly, the phase 4-like depolarization was completely eliminated (Figure 4B and C, arrow). Interestingly, Ad-Kir2.1-silenced ventricular ddhESC-CMs reverted back to the spontaneously firing phenotype upon the addition of an IK1 blocker, Ba2+ (Figure 4F). Kir2.1 expression also sufficed to mature the AP profiles of atrial ddhESC- and END2-hESC-CMs (Figure 5).

Figure 5
Kir2.1 overexpression confers upon atrial ddhESC- and END2-hESC-CMs the electrophysiological properties of mature CMs. Representative APs, If and IK1 of Ad-Kir2.1- transduced atrial ddhESC-CMs (A) and atrial END2-hESC-CMs (B). In both cell types, Ad-Kir2.1 ...

Similar to their human counterparts, murine (m) ventricular and atrial ESC-CMs exhibited comparably immature electrophysiological properties (Supplemental Figure 1) that could likewise be rendered adult-like by Kir2.1 expression (Supplemental Figs. 2 and 3). These data collectively indicate that our observations of electrophysiological immaturities in the derived CMs were a general phenomenon and not cell line-, species- or protocol-dependent.

Physiological Pacing Facilitates Maturation of hESC-CMs In Vitro

Despite our achievement of electrical maturation, Ad-Kir2.1-matured CMs continued to exhibit immature Ca2+-handling properties with smaller peak Ca2+ transient amplitudes and slow kinetics that are comparable to the control group (Supplemental Figure 4)28. Indeed, the expression levels of sarcomeric genes, such as MHCα, MHCβ, MLC2a and MLC2v, of the Kir2.1-silenced ddhESC-CMs even became significantly suppressed relative to control ddhESC-CMs (p<0.05; Figure 4G). Given that sarcomeric proteins of the developing heart are known to respond to changes in contractions29, our observation could be attributed to the lack of spontaneous beating activities of ddhESC-CMs in culture after Kir2.1-induced cessation of active contractions, which in turn led to the down-regulation of the contractile apparatus. Indeed in developing neurons, Kir2.1 expression can alter their excitability by escalating in response to extrinsic excitation via an activity-dependent mechanism to mediate synaptic plasticity29-30. To test if electrical activity likewise affects Kir2.1 expression in developing ddhESC-CMs, we mimicked endogenous pacing of adult heart by systematically field-stimulating cells in culture with electrical pulse of 2.5 V/cm at 5 msec pulse duration and 1 Hz frequency for 14 days. We hypothesized that 1) electrical conditioning of ddhESCCMs suffices to promote in vitro electrophysiological maturation, and that 2) the pacing-induced regular contractions can facilitate maturation of Ca2+-handling and contractile properties in a manner similar to the fetal heart development. Our experiments showed that this was indeed the case. Both electrically conditioned ventricular and atrial ddhESC-CMs were 100% quiescent with absence of phase 4-depolarization (Figure 6A, arrows; n=11). Moreover, the RMPs of the atrial and ventricular ddhESC-CMs were significantly hyperpolarized (p<0.05). Such a mature AP phenotype was never observed in >150 time-matched un-stimulated control hESC-CMs that we had recorded. Consistently, Kir2.1 expression became elevated (Figure 6A). Compared to unpaced controls, electrical conditioning similarly augmented both the electrically induced Ca2+ transient amplitude (Figure 6B; p<0.05) and sarcoplasmic reticulum Ca2+ load as shown by caffeine induced Ca2+ transients (Figure 6C; p<0.05) of ddhESC-CMs. Consistently, the expression levels of Ca2+-handling proteins typically present in adult CMs but absent or barely expressed in hESC-CMs such as calsequestrin (CSQ), junctin (Jct), and triadin (Trdn)28 as well as the t-tubule biogenesis proteins caveolin-3 (Cav3) and amphiphysin-2 (Amp2) all increased (Figure 6D-E). Consistent with an increase of ddhESC-CMs with maturing ventricular phenotype, electrical conditioning also resulted in a decrease of the atrial natriuretic factor (ANF) (Figure 6E). More importantly, the contractile proteins MHCα, MHCβ, MLC2a and MLC2v of electrically conditioned ddhESCCMs were significantly up-regulated (Figure 6F) relative to the control cells. In addition, the myofilaments became consistently more structured and organized as shown by transmission electron microscopy (TEM) (Figure 6G, see Supplemental Figure 5 for high resolution images), signifying contractile maturation. The increase in electrical conditioning-induced MLC2v expression could be attributed to a ~43% increase in the number of ventricular CMs as assessed by the number of MLC2v-positive cells relative to the tropomyosin-positive cells using a laser scanning cytometer. Similar to human cells, electrical conditioning likewise increased the expression of MLC2v and the ventricular derivatives in mESC-CMs as demonstrated by flow cytometric analysis of lentivirus generated EF1a-GFP-MLC2v-DsRed mESC line where ventricular CMs were identified by their expression of DsRed under the MLC2v promoter (Supplemental Figure 6).

Figure 6
Effects of electrical conditioning on ddhESC-CMs. A) Electrophysiology of electrically conditioned ddhESC-CMs. Action potential (AP) profiles (left) of electrically conditioned atrial and ventricular ddhESC-CMs showed the absence of phase 4-deloparization ...

Time-, age- and frequency-dependence of the pro-maturation effect of electrical conditioning

To obtain a better understanding, we next investigated the roles of the duration of cell exposure to field stimulation, age of hESC-VCMs when subjected to electrical conditioning and stimulation frequency (0.5Hz-2Hz) in the pro-maturation effect of electrical conditioning on hESC-VCMs. When compared to day 0 control, Kir2.1 expression of early-stage (20-25 days old) hESC-CMs was not different after electrical stimulation (at 1 Hz) for 7 days but became time-dependently increased at day 14 and further elevated at day 21 by ~1.7-fold (Figure 7A). When stimulated at 1 Hz, later-stage (40-50 days old) hESC-CMs displayed ~3-fold higher expression levels but such increases were not statistically different between days 7 and 21, unlike the early-stage counterpart (Figure 7B). Figure 7C shows that when pacing of early-stage hESC-CMs was ceased after stimulating for 14 days, Kir2.1 expression declined after 7 days, indicating that continuous stimulation was needed to maintain or augment the gained expression. Furthermore, electrical conditioning at 1 Hz of early-stage hESC-CMs for 14 days led to 2.3- and 1.9-fold higher Kir2.1 expression than 0.5 and 3 Hz, respectively. Collectively, these results indicate that our mechanism-based approach sufficed to induce maturation in ddhESC-derived CMs.

Figure 7
A) Effect of the duration (7, 14 and 21 days) of electrical stimulation at 1 Hz on Kir2.1 expression. Transcript expression normalized to GAPDH, and normalized to time-matched non-stimulated control (broken line)(n=9, from 3 independent batches). B) Same ...

Discussion

Although conceptually promising as an unlimited source, a number of fundamental hurdles need to be overcome before the use of derived CMs from hESCs or patient-specific iPSCs for clinical and other applications can be realized. It is well accepted that hESC-CMs are electrophysiologically immature, but the underlying mechanisms remain largely unknown. Indeed, multiple ionic currents in hESC-CMs behave differently from those of adult. In this study, we investigated various pluripotent stem cell line-derived CMs as well as different methods of cardiogenic differentiation methods. Our data collectively demonstrate that the observed immature electrophysiological properties are independent of the specific pluripotent stem cell types and lines, differentiation protocols as well as species, suggesting that a crucial environmental cue might be missing in the in vitro culturing system thereby leading to the artifactual developmental arrest.

Critical Role of IK1 in the Maturation of hESC-CMs

For maintaining an electrophysiologically stable phenotype with a hyperpolarized resting membrane potential close to the reversal potential of K+, an IK1 magnitude of at least 50% that of adult ventricular CMs is required. Although the IK1 magnitude of hESC-CMs has been shown to increase with prolonged culture of 100 days post-differentiation24, the current density in these aged cells is still below the threshold for attaining a mature ventricular electrical phenotype31. Therefore, it is impractical to maintain the cells for more than 100 days to achieve only a slight, if not entirely insignificant, improvement for clinical and other applications. Our goal here is to design strategies to effectively drive their maturation. By first exploring the basis that underlies the observed electrophysiological immaturities, we identified the lack of IK1 as one of the major mechanistic contributors of cellular automaticity. Forced expression of Kir2.1 channels in hESC-CMs alone sufficed to completely eliminate all the immature and proarrhythmic traits and thereby reproducing the adult AP phenotype. Although Ad-Kir2.1-matured hESC-CMs “corrected” automaticity immaturity, these cells continued to exhibit immature Ca2+-handling properties. Moreover, these quiescent cells have reduced expression of contractile proteins, MHCa, MHCb, MLC2a and MLC2v, as quantified by qPCR. Similarly, our recent publication demonstrated overexpression of CSQ, a high-capacity but low-affinity Ca2+-binding protein in the SR that is abundant in adult but completely absent in hESC-CMs, does indeed mature Ca2+ transients of hESC-CMs; however it failed to mature other functional aspects such as their AP properties32. Therefore, correcting a single gene at a time in immature hESC-CMs through viral transduction is insufficient and not effective in achieving E-C maturation for eventual clinical applications.

Mechanisms Underlying the Increased Maturation by Electrical Stimulation

Considering our collective data and the tight E-C coupling between electrophysiological and Ca2+-handling properties, we hypothesized that rhythmic electrical conditioning of hESC-CMs in vitro to mimic endogenous pacing may achieve E-C maturation through: 1) chronic pacing with field-stimulation may induce ion channel expression changes, 2) pacing-induced active contractions of electrically silenced hESC-CMs with mature APs can in turn facilitate Ca2+-handling and contractile maturation in a manner similar to the normal fetal heart development. Of note, although immature hESC-CMs do spontaneously fire APs and contract, we conjecture that these activities may be too weak, unsustained and sporadic in comparison to the physiological levels for effective facilitation of maturation in vitro. Consistent with our hypotheses, electrical conditioning robustly led to many aspects of cellular maturation of hESC-CMs, including electrophysiological maturation without phase 4-depolarization similar to Kir2.1 gene transfer, Ca2+-handing maturation with increased peak Ca2+ transient amplitude and SR Ca2+ load, structured organization of myofilaments as well as an up-regulation of contractile and t-tubule biogenesis proteins. Although the short-term electrical conditioning did mature numerous aspects of CM functions relative to control, the resting membrane potential of electrically conditioned ventricular ddhESC-CMs is still about 10mV more depolarized, with smaller peak Ca2+ transient amplitude and slower kinetics, than that of adult ventricular CMs. Future studies will be required to optimize physical parameters of electrical conditioning such as the voltage pulse, pulse duration and frequency for maximized facilitating effect.

Our observations were qualitatively similar in some ways to what have been observed in rodent CMs. For instance, neonatal rat and ESC-like P19 mouse embryonic carcinoma cell (ECC)-derived CMs stimulated to actively contract by pulsed electric field can self-align sarcomeres leading to improved contractility33-34. Mechanistically, we speculate that the rhythmic field-stimulations of hESC-CMs that otherwise intrinsically beat at low and/or irregular frequency may result in cyclic increase in intracellular Ca2+ concentration, which can increase their averaged Ca2+ residence time. Such an increase in intracellular Ca2+ may in turn contribute to the maturation effect observed via the second messenger system (e.g., by activating the calcineurin-NFAT pathway)35-38. Moreover, stretch of hESC-CMs from active contractions may also increase IK1 as previously shown in other cell type 39. Our future direction includes blocking independently the various Ca2+ influx pathways and decoupling cellular excitation from contraction to examine the involvement of mechanical contractions for dissecting out the underlying mechanisms of facilitated maturation in hESC-CMs. Furthermore, the optimal time for introducing the electrical stimulus and whether electrical conditioning-matured hESC-CMs will dedifferentiate remain to be elucidated and are also the direction of our future research.

Significance of Cell Maturation In Vitro Prior to Transplantation

More immature stem cell-derived CMs has been suggested to be more ischemic-resistant and tolerant to the hypoxic environment after transplantation40. The in vivo environment has also been demonstrated to induce electrophysiological maturation of immature CMs comparable to the host cells after transplantation but only for those that have fully integrated with the host cells41. Those transplanted CMs without electrical coupling remains electrophysiologcially immature, which can be arrhythmogenic as demonstrated in the present study. Therefore, it is also imperative to find a balance between safety and efficacy by defining what maturation status is best for in vivo transplantation since immature and mature hESC-CMs likely respond differently to ischemic and hypoxic environment. Eliminating intrinsic triggers for arrhythmia of the hESC-CMs to ensure no additional electrophysiological complications from their presence is of critical importance. The safety of these cells must be addressed before pondering their potential benefits to the recipient.

The strategy reported here, developed by first obtaining an understanding of the cellular differences and the underlying mechanisms, offers a simple non-genetic way to facilitate the E-C maturation of otherwise developmentally arrested derived CMs, thereby eliminating significant undesirable immature pro-arrhythmogenic traits. Furthermore, the successful use of derived CMs as human heart disease models and cardiotoxicity screening tools depends on their ability to recapitulate the properties of their adult counterparts. In combination with other advances in directed differentiation and cardiovascular progenitor identification13, 42-43, our approach can facilitate the clinical translation and enable more accurate human heart disease modeling, drug discovery and cardiotoxicity screening.

Supplementary Material

Supplemental

Supplemental Figure 1. Immature electrophysiological properties of mouse (m)ESC-CMs. A) Representative tracings of spontaneously firing (left) and quiescent (right) ventricular (top), atrial (middle) and pacemaker (bottom) APs of mESC-CMs. The arrows indicate a phase 4-like depolarization, a proarrhythmic substrate of automaticity. B) The percentage distribution of ventricular, atrial and pacemaker phenotypes (left) and the percentage distribution of spontaneously firing vs. quiescent characteristics (right) of total of mESC-CMs (n=60).

Supplemental Figure 2. Kir2.1 overexpression confers upon mouse (m)ESC-CMs the electrophysiological properties of mature CMs. A) A representative tracing of control ventricular mESC-CMs with DAD (left) and after Ad-Kir2.1 transduction showing absence of a phase 4-depolarization (right, arrow). Arrow heads indicate time of electrical stimulation. B) Total, Ba2+-insensitive If and Ba2+-sensitive IK1 currents recorded from the same cells in (A). C) Current-voltage (I-V) relationships of IK1 recorded from control spontaneously firing (open circles; n=3), control quiescent (solid circles; n=5), Ad-Kir2.1-transduced ventricular mESC-CMs (solid triangles; n=4) and adult mouse ventricular CMs (open triangles; n=5). D) The zoomed-in outward components of IK1 from (C). E) Kir2.1-overexpression (n=15) rendered nearly all ventricular mESC-CMs quiescent compared to control (n=24). F) Ad-Kir2.1-transduced ventricular mESC-CMs (n=15) displayed a hyperpolarized resting membrane potential relative to the control mESC-CMs (n=24) and comparable to the adult counterparts (n=15). * denotes p<0.05.

Supplemental Figure 3. Overexpression of Kir2.1 similarly matures the electrophysiological phenotypes of atrial mouse (m)ESC-derived CMs. A) A representative tracing of APs of a control atrial mESC-CMs with DAD (left) and after Ad-Kir2.1 transduction (right). Phase 4-depolarization is absent in Ad-Kir2.1-transduce atrial mESC-CMs (arrow). B) Ba2+-insensitive If and Ba2+-sensitive IK1 currents recorded from the same cells from (A). C) Current-voltage (I-V) relationships of IK1 recorded from control spontaneously firing (open circles; n=3), control quiescent (solid circles; n=4) and Ad-Kir2.1-transduced atrial mESC-CMs (solid triangles; n=6). D) Kir2.1 overexpression (n=22) rendered nearly all atrial mESC-CMs quiescent compared to control (n=29). E) Ad-Kir2.1-transduced atrial mESC-CMs (n=22) displayed hyperpolarized resting membrane potential relative to control mESC-CMs (n=29), similar to their adult counterparts (n=8). * denotes p<0.05.

Supplemental Figure 4. Representative tracings of Ca2+ transients in control and Ad-Kir2.1-transduced mESC-CMs overexpressing Kir2.1. No significant differences in Ca2+ transients were observed.

Supplemental Figure 5. Transmission electron microscopy images of the additional A) unconditioned ddhESC-CMs showed myofibrils that are less dense and less organized than B) the electrically conditioned ddhESC-CMs. The z-lines (arrows) can be seen in both groups.

Supplemental Figure 6. Electrical conditioning increases ventricular phenotype in mESC-CMs. A) A representative flow cytometric dot plot of unpaced control and electrically conditioned CMs derived from Lv-EF1α-GFP-MLC2v-DsRed reporter mESC line showing increased population of MLC2v positive cells in the polygonal gate. B) Average percentage of MLC2v-positive and median of unpaced control and electrically conditioned mESC-CMs (n=5). * indicates p<0.05.

Acknowledgments

Funding Source: This work was supported by grants from the NIH (R01 HL72857, R01 HL85844, R01 HL85727), Department of Veteran Affairs Merit Review Grant, the Research Grant Council (TBRS T13-706/11 and GRF 103544), and the California Institute for Regenerative Medicine (CIRM).

Footnotes

Conflict of Interest Disclosures: None.

References

1. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. The Journal of clinical investigation. 2001;108:407–414. [PMC free article] [PubMed]
2. Mummery C, Ward-van Oostwaard D, Doevendans P, Spijker R, van den Brink S, Hassink R, van der Heyden M, Opthof T, Pera M, de la Riviere AB, Passier R, Tertoolen L. Differentiation of human embryonic stem cells to cardiomyocytes: Role of coculture with visceral endoderm-like cells. Circulation. 2003;107:2733–2740. [PubMed]
3. Pera MF, Reubinoff B, Trounson A. Human embryonic stem cells. Journal of cell science. 2000;113(Pt 1):5–10. [PubMed]
4. Thomson JA, Marshall VS. Primate embryonic stem cells. Curr Top Dev Biol. 1998;38:133–165. [PubMed]
5. Xue T, Cho HC, Akar FG, Tsang SY, Jones SP, Marban E, Tomaselli GF, Li RA. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: Insights into the development of cell-based pacemakers. Circulation. 2005;111:11–20. [PubMed]
6. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. [PubMed]
7. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat Biotechnol. 2000;18:399–404. [PubMed]
8. Xu C, Police S, Rao N, Carpenter MK. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res. 2002;91:501–508. [PubMed]
9. He JQ, Ma Y, Lee Y, Thomson JA, Kamp TJ. Human embryonic stem cells develop into multiple types of cardiac myocytes: Action potential characterization. Circ Res. 2003;93:32–39. [PubMed]
10. Satin J, Kehat I, Caspi O, Huber I, Arbel G, Itzhaki I, Magyar J, Schroder EA, Perlman I, Gepstein L. Mechanism of spontaneous excitability in human embryonic stem cell derived cardiomyocytes. J Physiol. 2004;559:479–496. [PubMed]
11. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science New York, N Y. 2007;318:1917–1920. [PubMed]
12. Wobus AM, Guan K, Yang HT, Boheler KR. Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. Methods Mol Biol. 2002;185:127–156. [PubMed]
13. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM. Human cardiovascular progenitor cells develop from a kdr+ embryonic-stem-cell-derived population. Nature. 2008;453:524–528. [PubMed]
14. Hardy S, Kitamura M, Harris-Stansil T, Dai Y, Phipps ML. Construction of adenovirus vectors through cre-lox recombination. J Virol. 1997;71:1842–1849. [PMC free article] [PubMed]
15. Tse HF, Xue T, Lau CP, Siu CW, Wang K, Zhang QY, Tomaselli GF, Akar FG, Li RA. Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker hcn channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation. 2006;114:1000–1011. [PubMed]
16. Krogh-Madsen T, Schaffer P, Skriver AD, Taylor LK, Pelzmann B, Koidl B, Guevara MR. An ionic model for rhythmic activity in small clusters of embryonic chick ventricular cells. Am J Physiol Heart Circ Physiol. 2005;289:H398–413. [PubMed]
17. Azene EM, Xue T, Marban E, Tomaselli GF, Li RA. Non-equilibrium behavior of hcn channels: Insights into the role of hcn channels in native and engineered pacemakers. Cardiovascular research. 2005;67:263–273. [PubMed]
18. Boheler KR, Czyz J, Tweedie D, Yang HT, Anisimov SV, Wobus AM. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res. 2002;91:189–201. [PubMed]
19. ten Tusscher KH, Panfilov AV. Alternans and spiral breakup in a human ventricular tissue model. Am J Physiol Heart Circ Physiol. 2006;291:H1088–1100. [PubMed]
20. Wagner MB, Wang Y, Kumar R, Tipparaju SM, Joyner RW. Calcium transients in infant human atrial myocytes. Pediatr Res. 2005;57:28–34. [PubMed]
21. Wang Y, Xu H, Kumar R, Tipparaju SM, Wagner MB, Joyner RW. Differences in transient outward current properties between neonatal and adult human atrial myocytes. Journal of molecular and cellular cardiology. 2003;35:1083–1092. [PubMed]
22. Saxen L. Embryonic induction. Clin Obstet Gynecol. 1975;18:149–175. [PubMed]
23. Moore JC, Fu J, Chan YC, Lin D, Tran H, Tse HF, Li RA. Distinct cardiogenic preferences of two human embryonic stem cell (hesc) lines are imprinted in their proteomes in the pluripotent state. Biochem Biophys Res Commun. 2008;372:553–558. [PMC free article] [PubMed]
24. Sartiani L, Bettiol E, Stillitano F, Mugelli A, Cerbai E, Jaconi ME. Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: A molecular and electrophysiological approach. Stem Cells. 2007;25:1136–1144. [PubMed]
25. Nass RD, Aiba T, Tomaselli GF, Akar FG. Mechanisms of disease: Ion channel remodeling in the failing ventricle. Nat Clin Pract Cardiovasc Med. 2008;5:196–207. [PubMed]
26. Beuckelmann DJ, Nabauer M, Erdmann E. Alterations of k+ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circulation research. 1993;73:379–385. [PubMed]
27. Yang J, Jan YN, Jan LY. Determination of the subunit stoichiometry of an inwardly rectifying potassium channel. Neuron. 1995;15:1441–1447. [PubMed]
28. Liu J, Fu JD, Siu CW, Li RA. Functional sarcoplasmic reticulum for calcium handling of human embryonic stem cell-derived cardiomyocytes: Insights for driven maturation. Stem Cells. 2007;25:3038–3044. [PubMed]
29. Razeghi P, Young ME, Alcorn JL, Moravec CS, Frazier OH, Taegtmeyer H. Metabolic gene expression in fetal and failing human heart. Circulation. 2001;104:2923–2931. [PubMed]
30. Burrone J, O'Byrne M, Murthy VN. Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature. 2002;420:414–418. [PubMed]
31. Bailly P, Mouchoniere M, Benitah JP, Camilleri L, Vassort G, Lorente P. Extracellular k+ dependence of inward rectification kinetics in human left ventricular cardiomyocytes. Circulation. 1998;98:2753–2759. [PubMed]
32. Liu J, Lieu DK, Siu CW, Fu JD, Tse HF, Li RA. Facilitated maturation of ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression. Am J Physiol Cell Physiol. 2009;297:C152–159. [PubMed]
33. Au HT, Cheng I, Chowdhury MF, Radisic M. Interactive effects of surface topography and pulsatile electrical field stimulation on orientation and elongation of fibroblasts and cardiomyocytes. Biomaterials. 2007 [PMC free article] [PubMed]
34. Radisic M, Park H, Shing H, Consi T, Schoen FJ, Langer R, Freed LE, Vunjak-Novakovic G. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:18129–18134. [PubMed]
35. Kassiri Z, Zobel C, Nguyen TT, Molkentin JD, Backx PH. Reduction of i(to) causes hypertrophy in neonatal rat ventricular myocytes. Circulation research. 2002;90:578–585. [PubMed]
36. Lammerding J, Kamm RD, Lee RT. Mechanotransduction in cardiac myocytes. Annals of the New York Academy of Sciences. 2004;1015:53–70. [PubMed]
37. Lebeche D, Kaprielian R, del Monte F, Tomaselli G, Gwathmey JK, Schwartz A, Hajjar RJ. In vivo cardiac gene transfer of kv4.3 abrogates the hypertrophic response in rats after aortic stenosis. Circulation. 2004;110:3435–3443. [PubMed]
38. Zobel C, Kassiri Z, Nguyen TT, Meng Y, Backx PH. Prevention of hypertrophy by overexpression of kv4.2 in cultured neonatal cardiomyocytes. Circulation. 2002;106:2385–2391. [PubMed]
39. He Y, Xiao J, Yang Y, Zhou Q, Zhang Z, Pan Q, Liu Y, Chen Y. Stretch-induced alterations of human kir2.1 channel currents. Biochem Biophys Res Commun. 2006;351:462–467. [PubMed]
40. Boheler KR, Joodi RN, Qiao H, Juhasz O, Urick AL, Chuppa SL, Gundry RL, Wersto RP, Zhou R. Embryonic stem cell-derived cardiomyocyte heterogeneity and the isolation of immature and committed cells for cardiac remodeling and regeneration. Stem Cells Int. 2011;2011:214203. [PMC free article] [PubMed]
41. Halbach M, Pfannkuche K, Pillekamp F, Ziomka A, Hannes T, Reppel M, Hescheler J, Muller-Ehmsen J. Electrophysiological maturation and integration of murine fetal cardiomyocytes after transplantation. Circ Res. 2007;101:484–492. [PubMed]
42. Bu L, Jiang X, Martin-Puig S, Caron L, Zhu S, Shao Y, Roberts DJ, Huang PL, Domian IJ, Chien KR. Human isl1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature. 2009;460:113–117. [PubMed]
43. Moretti A, Caron L, Nakano A, Lam JT, Bernshausen A, Chen Y, Qyang Y, Bu L, Sasaki M, Martin-Puig S, Sun Y, Evans SM, Laugwitz KL, Chien KR. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell. 2006;127:1151–1165. [PubMed]