Abstract
The discovery of human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), has opened up novel paths for a wide range of scientific studies. The capability to direct the differentiation of hPSCs into functional cardiomyocytes has provided a platform for regenerative medicine, development, tissue engineering, disease modeling, and drug toxicity testing. Despite exciting progress, achieving the optimal benefits has been hampered by the immature nature of these cardiomyocytes. Cardiac maturation has long been studied in vivo using animal models, but finding ways to mature hPSC cardiomyocytes (hPSC-CMs) is only in its initial stages. In this review, we discuss progress in promoting the maturation of the hPSC-CMs, in the context of our current knowledge of developmental cardiac maturation and in relation to in vitro model systems such as rodent ventricular myocytes. Promising approaches that have begun to be examined in hPSC-CMs include long-term culturing, three dimensional tissue engineering, mechanical loading, electrical stimulation, modulation of substrate stiffness and treatment with neurohormonal factors. Future studies will benefit from the combinatorial use of different approaches that more closely mimic nature’s diverse cues, which may result in broader changes in structure, function, and therapeutic applicability.
Keywords: Human Pluripotent Stem Cell-derived Cardiomyocytes Maturation, Disease Modeling, Pharmacologic Screening
Introduction
In the last 6 years, multiple labs have reported the successful directed cardiomyocyte differentiation from hPSCs (reviewed in1). These stem cell-derived cardiomyocytes beat spontaneously, express the expected sarcomeric proteins and ion channels, and exhibit cardiac-type action potentials and calcium transients. They show similar functional properties to the cardiomyocytes in the developing heart with comparable excitation-contraction coupling mechanism and neurohormonal signaling2. After transplantation into infarcted hearts of mice, rats, and guinea pigs, hPSC-CMs engraft, form gap junctions with the host cardiomyocytes, reduce adverse remodeling, and enhance cardiac mechanical function3, 4. In spite of these promising results, many lines of evidence indicate that under the conditions currently utilized, hPSC-CMs do not exhibit the morphological and functional characteristics of adult cardiomyocytes.
One might reasonably ask, why do we need to mature these human cardiomyocytes? Indeed, researchers have used immature rodent cardiomyocytes as model systems for decades without ever maturing them to an adult phenotype. There are several answers to this question. First, adult rodent cardiomyocytes are readily available for study, whereas adult human cardiomyocytes are difficult to obtain. Second, learning how to mature human cardiomyocytes may teach us how our own hearts mature in post-natal life. Third, mature human cardiomyocytes may better reflect the physiology of the adult heart and therefore be more useful in disease modeling and drug screening. Finally, for myocardial repair, it is desirable to have hPSC-CMs whose electrical and mechanical properties more closely resemble those of native myocardium. Such matured cells would be expected to pose less arrhythmic risk and have enhanced contractile performance.
In this review, we will discuss the state of the current knowledge regarding the maturation, disease modeling, and drug screening prospects of hPSC-CMs. Because maturation of hPSC-CMs is a relatively new field, we will discuss it in the context of what is known in other systems including in vivo animal models and isolated rodent cardiomyocytes. We will address the factors that have been shown to influence cardiomyocyte maturation and also discuss issues that need to be addressed to generate bona fide mature human heart tissue, starting with a comparative discussion of immature and adult cardiomyocytes in relation to what we currently know regarding hPSC-CMs. Please note that all three types of cardiomyocytes (ventricular-, atrial-, and nodal-type) can be obtained from human pluripotent stem cells. Since virtually all studies to date have not used purified subtypes, we refer here to mixed cardiomyocyte populations. Cardiomyocyte subtype differentiation and purification is under intensive investigation5, 6, and its advances will benefit this area.
I. Overview of Cardiomyocyte Maturation
Morphology
As heart development progresses, cardiac muscle cells undergo a complex series of structural changes that ultimately lead to their adult phenotype (Fig. 1 and Table 1). The growth of the embryonic/fetal heart is primarily achieved by cardiomyocyte proliferation7. In the post-natal heart, hypertrophic growth becomes predominant and the increase in cardiomyocyte size for both rodents and humans can be 30–40 fold8. It is worth mentioning that increase in cell size may also result from pathological in addition to physiological hypertrophic responses. In this review, we consider cardiomyocyte maturation to involve physiological hypertrophy, since it is part of normal heart development. For a review discussing physiological vs. pathological hypertrophy, we refer the readers to9. The hESC-CMs have been reported to be around 600 μm2 on average, which is significantly smaller than an adult cardiomyocyte that has spread out with prolonged culture. Membrane capacitance is directly proportional to cell surface area. The capacitance of the hESC-CMs is 17.5 ± 7.6 pF10, in comparison to the ~ 150 pF typically reported for adult human ventricular myocytes11. Geometric considerations indicate that the cells show even greater differences in volume than cell area. In any case, cell size is an important parameter since it influences impulse propagation, maximal rate of action potential depolarization and total contractile force12.
Fig. 1.
Morphological differences between an immature hPSC-CM and adult rat cardiomyocyte. A (hPSC-CM) and B (adult rat): Overview of contractile cytoskeleton with alpha-actinin staining (green) and blue nuclear counterstain. C (hPSC-CM) and D (adult rat): cellular ultrastructure by electron microscopy. Note that there are significant differences with respect to cell size, length to width ratio, mitochondria quantity, size and morphology, appearance of T-tubules (arrows), and elongated nuclei. (Scale bar in A and B: 25 μm, C and D: 0.2 μm) Fig. 1B and 1C were kindly provided by Scott Lundy and Dr. Michael A. Laflamme.
Table 1.
Summary of the differences between immature and adult cardiomyocytes. Data refer to hPSC-derivatives when possible.
Parameters | Immature Cardiomyocytes | Adult Cardiomyocytes | |
---|---|---|---|
Morphology | cell shape | Circular | Rod-shaped |
membrane capacitance | 17.5 ± 7.6 pF10 | ~ 150 pF11 | |
Sarcomere | Structure | Disarrayed | Highly organized |
Length | ~1.6 μm80 | ~2.2 μm | |
Myofibrillar Isoform | Titin | N2BA | N2B |
Troponin I | ssTnI | cTnI | |
MHC | Beta > alpha | Beta ≫ alpha | |
T-tubules | No | Yes | |
Mitochondria | Irregular reticular network in cytoplasm; Occupies a small fraction of cell volume | Regularly distributed; Occupies ~20–40% of cell volume | |
Metabolic Substrate | Glucose | Fatty acid | |
Multinucleation | Mononucleated | ~25% multinucleated | |
Electrophysiological Properties | Upstroke velocity | ~ 50 V/sec80 | ~250 V/sec11 |
Resting membrane potential | ~−60 mV80 | ~−90 mV11 | |
E-C coupling | Partially developed (Please refer to the “calcium handling and E-C coupling” part of the main text) | Mature | |
Contractile Force | ~ nN range/cell | ~ μN range/cell | |
Gap Junction Distribution | Circumferential | Polarized to intercalated disks | |
Conduction Velocity | ~0.1 m/sec78 | 0.3 – 1.0 m/sec | |
Responses to β- Adrenergic Stimulation | Chronotropic response Lack of inotropic reaction72 | Chronotropic response Inotropic reaction |
In addition to size, the shape of the cardiomyocytes also has important functional implications including excitation-contraction coupling. It is well known that adult cardiomyocytes have elongated anisotropic shapes (Fig. 1B) with a length to width ratio of 7 – 9.513 and are aligned in the context of cardiac tissue. In vivo, immature cardiomyocytes are rod-shaped, similar to the adult ones, but when cultured in vitro, the immature cardiomyocytes flatten and spread in all directions while the adult ones maintain their cylindrical morphology in short term culture14. Thus far, under commonly used conditions, hPSC-CMs have irregular shapes (Fig. 1A and15–17) and they do not typically show alignment in two-dimensional cultures.
The Contractile Apparatus
The sarcomere is the fundamental unit for cardiomyocyte contraction. Monitoring the expression levels of sarcomeric proteins such as cardiac troponin T, cardiac troponin I, alpha actinin and beta-myosin heavy chain provides a basic assessment of specialization and maturation of hPSC-CMs. RT-PCR and immunocytochemical analyses from various groups have confirmed the expression of sarcomeric genes in hPSC-CMs16–18. Since relatively immature cardiomyocytes express these proteins, the detection of sarcomeric genes should not be utilized as the only assessment of maturation. Electron microscopy has shown that hPSC-CMs have immature ultrastructural characteristics with various degrees of sarcomeric organization (Fig. 1C and19, 20). During cardiomyocyte development, sarcomeric structure becomes more organized and sarcomeric length increases to facilitate force-generation. In a relaxed adult human cardiac muscle cell, sarcomere length is about 2.2 μm21, whereas immature hPSC-CMs have a sarcomere length of approximately 1.65 μm22.
Myofibrillar Isoform Switch
During development, several myofibrillar protein isoforms undergo switching, which modulates the contractile function of cardiomyocytes. Titin, which is involved in the maintenance of sarcomere integrity and elasticity, has been shown to shift from relatively compliant isoforms (with a size range 3200–3700 kDa, designated N2BA) to a shorter and stiffer form (3000 kDa, designated N2B). This alteration regulates the passive tension of maturing cardiomyocytes23–25. In human post-natal left ventricles, N2B is the dominant titin form26. Troponin I also undergoes a developmentally regulated isoform switching that affects contractility. Developing hearts of many species, including humans, contain an isoform designated slow skeletal troponin I (ssTnI), whereas adult hearts contain cardiac troponin I (cTnI)27–30. Functional analyses indicate that the troponin complex containing cTnI has decreased Ca2+ sensitivity for tension production, compared with complexes containing ssTnI31–33. For myosin heavy chain (MHC), it is well-known that in rodents there is a switch from beta-MHC before birth to alpha-MHC after birth34, which correlates with a postnatal heart rate increase. In human hearts, there are more beta-MHC than alpha-MHC at all stages, but there is more alpha-MHC in fetal than adult hearts35, correlating with the postnatal heart rate decrease. To our knowledge, the issue of contractile protein isoform switching remains open in the context of the maturation of hPSC-CMs. Because of the clear impact of myofibrillar protein isoform switching in cardiac function, future studies will benefit from taking this parameter into consideration.
Transverse tubules (T-tubules)
T-tubules are the membrane invaginations along the Z-line regions, with regular spacing (~2 μm) along the longitudinal axis of adult mammalian ventricular myocytes (Fig. 1D). Because they represent a key component of excitation contraction coupling (ECC) in adult-sized myocytes, T-tubule appearance is a hallmark of cardiomyocyte development36. Fetal rat cardiomyocytes lack T-tubules, but by postnatal days 6 to 9 there are some T-tubule-like subcellular structures in the subsarcolemmal region. The myocytes of 12- to 14-day neonatal rats contain T-tubules that extend to the cell interior though still irregular. At around one month after birth, the T-tubule system is fully developed and displays the pattern of an adult cardiomyocyte. T-tubules make it possible for an adult cardiomyocyte to have rapid electric excitation, initiation, and synchronous triggering of sarcoplasmic reticulum (SR) calcium release, and therefore, coordinated contraction throughout the entire cytoplasm. Studies from various groups have shown that hESC-CMs have few to no T-tubules in 2D culture20, 37, 38, and further experiments showed that these cardiomyocytes had unsynchronized Ca2+ transients, as reflected by non-uniform calcium dynamics across the cell and greater calcium peak amplitude in the cell periphery than at the central region.
Mitochondria and Metabolic Substrate
Mitochondrial structural and functional changes are critical components of maturation during heart development. In immature cardiomyocytes ex vivo, mitochondria distribute throughout the cytoplasm in a reticular network, and they only account for a small fraction of the cell volume. Ultrastructurally, the inner membrane does not exhibit well-formed cristae39. Interestingly, the mitochondria permeability transition pore, whose opening leads to pathologic condition that initiates cell death in adult hearts40, is open in embryonic mouse hearts and its closure results in mitochondrial structural and functional maturation41, evidenced by increased mitochondria length and mitochondrial membrane potential, and decreased reactive oxygen species production. As development proceeds, mitochondria develop more mature lamellar cristae, and in the adult cardiomyocytes, they are regularly distributed. Mitochondria occupy ~20–40% of the adult myocyte volume, and are distributed throughout the cell in a crystal-like lattice pattern42. Since they are restricted by tightly packed distribution along myofibrils or beneath the sarcolemma, mitochondrial motility is highly limited. Corresponding with the structural changes from an immature to a mature state, the oxidative capacity of the mitochondria increases, as reflected in a metabolic substrate switch43. During early cardiac development, glycolysis is a major source (80%) of energy. As cardiomyocytes mature and become terminally differentiated, mitochondrial oxidative capacity increases, with fatty acid β-oxidation (80%) becoming a major source of energy. In hPSC-CMs19, long and slender mitochondria are clustered next to nucleus or in the cell periphery. Accordingly, hiPSC-CMs rely on glycolysis rather than fatty acid β-oxidation44.
DNA Synthesis, Multinucleation and Nuclear ploidy
During early stages of development, cardiomyocytes exhibit a robust proliferative capacity. Studies in diverse model systems have shown that as the heart matures, this proliferative capacity diminishes and, before cell cycle withdrawal, many cardiomyocytes exhibit binucleation and, in some instances, extensive polyploidy8. Work by the groups of Field, Bishop, Gerdes and others revealed that during murine development, DNA synthesis occurs in two distinct phases: the early fetal phase is associated with cell division, while the second phase leads to binucleation7, 45, 46. At birth, few cardiomyocytes are binucleated, but within 2 weeks after birth, binucleated cardiomyocytes reach the adult level (~75% in rodents)46. The timing of binucleation varies among different species. In sheep, there is a sharp increase in binucleation before birth47. In humans, cardiomyocytes proliferate for the first few months after birth and the estimate of binucleation percentage of 25%8. In addition, mature human cardiomyocyte nuclei commonly exhibit polyploidy, which is the result of DNA replication without nuclear division or cytokinesis. Ploidy in human cardiac nuclei can reach 8N in healthy hearts (diploid = 2N), with levels as high as 64N in overloaded hearts48, 49.
Electrophysiological properties
To generate the adult cardiomyocyte action potential, it takes the orchestrated activity among several ionic channels. During cardiac development, expression and function of distinct channel types occur over time. Indeed, studies in animal models show that channels undergo fetal and postnatal developmental changes that lead to the acquisition and maintenance of a mature cardiac electrophysiological phenotype. In freshly isolated cells, transient outward current (Ito) density has been shown to double at day 15 compared with day 5 rat neonatal cardiomyocytes50. Only IKr is functionally expressed in the young canine ventricles whereas both IKr and IKs are present in adult canine myocardium, suggesting a greater dependence on IKr for repolarization in young dogs51. Due to the low level of IK1 channel, the resting membrane potential is higher (~−60 mV) in immature cardiomyocytes than their mature counterparts (~−90 mV)11. In addition, lower levels of the sodium channel Nav1.5 and the L-type calcium channel in immature cardiomyocytes lead to slower upstroke velocity and to a lack or a shorter plateau phase, respectively52.
Calcium Handling and Excitation-Contraction Coupling
One of the most widely characterized functional parameters in hPSC-CMs is calcium handling. Multiple reports have shown that hPSC-CMs express critical Ca2+ handling proteins and exhibit [Ca2+]i transients2, 17, 18, 38, 53. However, the maturation status and the basic mechanism that regulates the ECC of these cells remains controversial. Early studies indicated that in hPSC-CMs, [Ca2+]i transients and contraction were insensitive to drugs that interfere with SR Ca2+ release or reuptake54. Further analyses of the expression of proteins known to be involved in Ca2+ handling could not detect key SR regulatory proteins such as calsequestrin or phospholambam. This study suggested that in hPSC-CMs, the rise in [Ca2+]i transients is the result from trans-sarcolemmal entry via calcium channels and that internal Ca2+ stores do not contribute significantly to this process. A subsequent report identified two different subpopulations of hESC-CMs, one that was caffeine-sensitive and another insensitive53. Follow up studies by this group showed that compared to control cells, hESC-CMs transduced with the regulatory protein calsequestrin had increased transient amplitude, upstroke velocity and transient decay, suggesting that calsequestrin has a significant impact in the functional maturation of hESC-CMs55. Extensive evidence from two other groups support the notion that hPSC-CMs have functional SR Ca2+ stores and that, unlike cardiomyocytes in model species such as rat56, 57 rabbits58, 59 and mice60, 61, the establishment of a tight and localized control of ECC is an early event in the maturation process of hPSC-CMs2, 38. Two more recent reports characterizing hPSC-CMs have shown the expression of key regulatory proteins such as calsequestrin and have also provided evidence supporting the presence of functional SR-dependent Ca2+ handling17, 18. It is possible that differential Ca2+ handling properties may arise from variability in the maturation status of different hPSC-CMs analyzed. As will be discussed later, the derivation of cardiomyocytes from hPSCs is far from standardized, and different protocols may yield cells at different stages. Another possibility is that the differences result from inherent differences in the maturation rates of cardiomyocytes derived from diverse cell lines.
Contractile Force
For both immature and mature cardiomyocytes, contractile force is one of the least studied parameters. At the tissue level, Hasenfuss et al62 reported a peak twitch tension for strips of human myocardium of 44 ± 11.7 mN/mm2, and similarly 56.4 ± 4.4 mN/mm2 for rat myocardium. In studies from our own group63, hPSC-CMs in collagen constructs generated about 0.08 mN/mm2 (at a passive tension of 0.4 mN/mm2), which is about 550-fold less than the adult human myocardium. The collagen constructs made of NRVM generated 0.4 to 0.8 mN/mm2 at a resting tension of 0.1 to 0.3 mN/mm64. These studies may underestimate force generation, because the collagen gels in which the cells are embedded are compliant and may stretch rather than transmit all force, and the cell density of these constructs is lower than native tissue. Nevertheless, the force generation of engineered human or rat myocardium is clearly much less than adult myocardium.
At a single cell level, Korte el al.65 reported a force of ~ μN range by skinned adult rat cardiomyocytes. For hESC-CMs, Kita-Matsuo et al66 used dynamic traction force microscopy (which measures lateral force generation) to analyze individual cells that were plated onto polyacrylamide gels with an elastic modulus of 4 kPa and surfaces functionalized with chemically cross-linked gelatin. Selected cardiomyocytes contracted with an average total forces of 144 ± 33 nN. Surprisingly, the contractile force was not changed with culture time up to 90 days. Under the same conditions, NRVM contracted with a total force of 222 ± 54 nN. Liu et al 67 measured the contractile force of hESC- and hiPSC-CMs by using atomic force microscopy (which measures vertical force generation) and showed that hiPSC- and hESC-CMs beat similarly, with no differences in contractile force generation. However, the total force output is probably much higher, because the method employed could not measure the lateral modes of contraction. The fact that the force data produced were not correlated with the cell area makes it hard to compare cardiomyocyte force-generation between studies.
Gap Junction Distribution and Conduction Velocity
The adult human left ventricle myocardium has a conduction velocity of 0.3–1.0 meters/sec (m/s), while the conduction velocity in immature human heart has not to our knowledge been determined. In neonatal canine ventricular muscle, the average propagation velocity is 0.33 m/s while in the adult canine the number increases to 0.50 m/s12. In addition to the contribution of cell geometry, the distribution of gap junctions is an important factor that regulates conduction velocity. The gap junction protein connexin 43 and the adherens junction protein N-cadherin are circumferentially distributed during fetal life, and as the post-natal rodent heart matures, these proteins become progressively concentrated into intercalated disks at the ends of the cells. This sub cellular redistribution results in a much accelerated conduction velocity68. Thus far, connexin 43 and N-cadherin show circumferential distribution in hPSC-derived cardiomyocytes in 2D culture69, 70, supporting the notion of an immature phenotype.
Cardiac Gene Expression
When considering gene expression during maturation, it is worth mentioning that, when cells grow 30-fold, there is more of everything on a per-cell basis. Since most RNA and protein studies normalize expression internally, e.g. to housekeeping genes, total RNA or protein, global increases in transcript and protein content per cardiomyocyte are missed. Despite this limitation, multiple studies have shown that when compared to adult heart tissue, hPSC-CMs have lower expression of sarcomeric genes as well as genes encoding proteins for ion transport, and calcium handling. Due to space limitations, Table 2 briefly summarizes these data. For additional detailed information the readers are referred to the original papers35, 71.
Table 2.
Major cardiac genes up-regulated (by at least two-fold) in adult hearts compared to the immature hPSC-CMs.
Genes Upregulated in Adult Heart Compared to hPSC-CMs | ||
---|---|---|
Sarcomere35 | MYL2, TNNI3, ACTN2, MYH7, MYL3, TNNC1, TNNT2, MYH11, SORBS1 | |
Ion Transporters and some of their regulatory proteins 35, 71 | Sarcolemmal | KCNA4, KCNA5, KCNAB1, KCNAB2, KCND2, KCND3, KCNE4, KCNG1, KCNH2, KCNH7, KCNIP2, KCNJ2, KCNJ3, KCNJ5, KCNJ8, KCNK1, KCNQ1, KCNV1, SCN1A, SCN1B, SCN2B, SCN3A, SCN4B, SCN5A, HCN1, HCN4, CACNA1C, CACNA1D, CACNA1H, CACNA1G, CACNA2D1, CACNB2, SLC8A1, TRPC3, TRPC4, TRPC6, CFTR |
Sarcoplasmic Reticulum | ATP2A2, PLN, CASQ2, RYR2, RYR3, TRDN, ITPR1, ITPR3, ASPH, S100A1, HRC |
Responses to Calcium and Beta-Adrenergic Stimulation
With the above-mentioned differences between an immature and mature cardiomyocytes, it is not surprising that the hPSC-CMs could respond differently to pharmacologic stimulations. One study investigated the response of hESC-CMs to extracellular calcium and beta-adrenergic stimulation using beating clusters that were co-cultured with non-contractile, avital slices of neonatal mice ventricles72. Removing calcium or administration of a Ca2+ channel blocker stopped the spontaneous beating. Increasing Ca2+ concentration results in accelerated beating rate and increased developed isometric force up to a [Ca2+]ec of 2.5 mM. If [Ca2+]ec was increased further, spontaneous beating rate decreased, whereas the developed force continued to increase.
These same investigators found that the beta-adrenergic agonist, isoproterenol, induced a dose-dependent increase of the frequency of spontaneous beating. Interestingly, isoproterenol did not significantly change the developed isometric force during spontaneous contractions or during electrical stimulation at a constant rate. The lack of an inotropic reaction despite a pronounced chronotropic response after beta-adrenergic stimulation most likely indicates immaturity of the sarcoplasmic reticulum. In contrast with this study, Liu et al67 detected the inotropic effect of norepinephrine (mixed alpha- and beta-agonist) in both single hiPSC- and hESC-CMs using atomic force microscopy. Yokoo et al73 found similar increases in shortening with a video-edge detecting system. It is not clear what leads to the discrepancy, but further investigations are warranted to clarify this important issue.
II. Modulation of Cardiomyocyte Maturation
In order to maximize the therapeutic benefits of hPSC-CMs, a critical challenge is to enhance their maturation status. It takes human cardiomyocytes years to reach adult form in terms of size, shape, molecular composition, metabolism and physiological function in vivo74. In this section, we will discuss the variety of approaches employed to improve the maturation status of immature cardiomyocytes, including hPSC-CMs and rodent cardiomyocytes. The overall goal will be to mimic nature’s work, though at this stage, studies have only employed one or two biophysical cues, leading to an intermediate state. Because of space limitations the signaling pathways involved in maturation will not be discussed here; for this purpose, readers are referred to the excellent review by Heineke and Molkentin75.
Long Term Culture
Human neonatal cardiomyocytes require 6 to 10 years in vivo to reach their adult phenotype74. Due to obvious practical constraints, the current length of differentiation for hPSC-CMs is much shorter. Beating cardiomyocytes can be generated from hPSCs within 15 days of differentiation8. Thus, a logical approach to obtain more mature cardiomyocytes is by long-term culture. Studies have shown that similar to mouse neonatal cardiomyocytes, hPSC-CMs display autonomous hypertrophy in serum-free medium76. The cells undergo a process of cell-cycle withdrawal and ultrastructural maturation within 35 days of culture period20. Electrophysiological studies52 showed that the Ito1 and IK1, the transient outward and inward rectifier potassium currents, as well as the calcium current ICa,L and the pacemaking current If, underwent developmental maturation during 3-month culture period (Fig. 2G). Surprisingly, in these studies the generation of contraction stress did not increase by long culture time up to 3 months in66 or up to 2 months in77. Spontaneous beating rate after 2 months culture was reported to decrease in some culture conditions77 while other studies reported increased beating rate76. Conduction velocity was reported to be significantly upregulated after 2 months in culture78. M-band appearance, a hallmark of sarcomeric structural maturation, was reported in hiPSC-CMs after 1 year in culture79.
Fig. 2.
Effects of long-term culture on hPSC-CM maturation. In Panel 1A and 1B, representative hPSC-CMs immunostained for alpha-actinin (red) and filamentous actin (phalloidin, green). Nuclei (blue). C–F. The multinucleation cell percentage, sarcomere length, and cell area were significantly increased with a decrease in cell circularity. G. Electrophysiologically, older cardiomyocytes show significantly enhanced action potential upstroke velocity, a hyperpolarized maximum diastolic potential, and an increase in transient outward rectifier potassium currents. Panels A through F were modified from Lundy et al25 with permission from Dr. Michael A. Laflamme. Panel G was modified from Sartiani et al54 with permission from Dr. Marisa E. Jaconi.
Most recently, Lundy et al80 performed long-term hPSC-CM culture studies up to 120 days and found dramatic changes in cell size, anisotropy, sarcomere length, and percentage of multinucleated cardiomyocytes (Fig. 2). Functionally, the long-term cultured cells showed a doubling in shortening magnitude with slowed contraction kinetics. An increase in calcium release and reuptake rates was also reported. Electrophysiologically, the long-term cultured cells have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. These data demonstrate that hPSC-CMs mature over time both morphologically and functionally, implying either a timekeeping mechanism or a time-dependent signaling pathway. Though long-term culture enhances many aspects of hPSC-CM maturation, this approach offers limited throughput. For this reason, many labs are currently attempting to accelerate and improve this process using diverse manipulations (discussed below).
Substrate Stiffness
The environment of the cardiomyocyte changes dynamically during development. Collagen accumulation, for example, begins during embryonic development and continues until several weeks after birth. This, and other related changes, result in a threefold increase of elastic modulus (passive stiffness) from embryonic to neonatal stages in mice81 and twofold increase in rats from neonatal to adult stage82. This process of myocardium stiffening coincides with postnatal elevations in blood pressure and the heart’s increased capability of pumping blood.
Several studies investigated the effect of substrate stiffness on maturation. In NRVM on collagen-coated polyacrylamide gels with varying elastic moduli (1–50 kPa)83, extracellular stiffness close to that of native myocardium (10 kPa) significantly enhances their maturation as reflected by aligned sarcomeres, greater mechanical force (examined using traction force microscopy) and the largest calcium transients and SERCA2a expression. A similar effect of substrate stiffness on cell morphology was observed in hESC-CMs in the same culture context84. Rodriguez et al85 developed a novel approach that combines high-speed line scanning with microfabricated arrays of flexible posts. This provides temporal resolution to measure the power of NRVM cultured on post arrays of different stiffness. The study found that NRVM had a 6-fold greater twitch forces but slower twitch velocity when cultured on substrates with higher stiffness. Cardiomyocytes on stiffer arrays had more mature myofibril structure as reflected by a greater sarcomere length and Z-band width. Intracellular calcium levels also increased with stiffness during a twitch. Similar results were found by Palecek’s group77, which tracked the motion of fluorescent beads embedded in polyacrylamide hydrogels (4.4–99.7 kPa), and showed that both NRVM and hPSC-CMs generated greater mechanical force on gels with higher stiffness. Cell area was greatest on a substrate stiffness of 49.4 kPa. In contrast to the other studies, this group observed well-defined sarcomeres on all substrate stiffness; the reason for this difference is not clear. Taken together, these observations indicate that changes in substrate stiffness affect cardiomyocyte contractile force.
Cell Patterning
In vivo cardiomyocytes are exposed to physical stimuli including topographical cues to keep them elongated and rod-shaped. It has been elegantly demonstrated that cardiomyocyte shape regulates sarcomere alignment86. With a native cell length to width ratio of 7:1, cardiomyocytes exhibit directional anisotropy. Thus, one potential way to mature the cardiomyocytes is to develop advanced cell culture systems that mimic the in vivo micro-environmental topographical cues. For instance, cultivation of the cells on patterned substrates provides topographical cues that significantly enhance cell alignment87, 88. McDevitt et al89 seeded NRVM onto microcontact-printed laminin lanes 5–50 μm wide. The NRVMs assumed rod-shaped geometries with myofibrils aligned parallel to the laminin lanes, and strikingly, a bipolar localization of N-cadherin and connexin 43 resembling intercalated disks. Kim et al90 constructed an anisotropically nanofabricated substratum with polyethylene glycolpatterned with ridges and grooves ranging from 150 to 800 nm, designed to closely reproduce a nano-scale structure of the myocardial ECM composed of aligned fibrils approximate 100 nm in diameter. Although a single cardiomyocyte spans more than ten nanoridges, the cells still aligned along the direction of the topographical cue. Considerable differences were noticed between the non-aligned and aligned cell cultures in terms of cell geometry, conduction velocity, and Cx43 expression, with aligned cultures exhibiting properties more similar to the native heart. Combining surface topography and substrate stiffness cues together, Wang et al87 found that morphology and orientation of NRVM were mainly influenced by nanogrooved structures, while the contractile function of the cells was regulated by both surface topography and substrate stiffness with better contraction on the soft substrate with deep grooves.
Electrical Stimulation
In vivo cardiomyocytes are constantly subjected to electrical signals that promote synchronous contractions. It has been hypothesized that electrical stimulation of hPSC-CMs would enhance their maturation, but there has been surprisingly little work done in this area. Most of what we know comes from the rat. Eight days after ex vivo electrical field stimulation91, NRVM in collagen sponges showed better cell alignment and coupling, and 7-fold increase in contraction amplitude. The stimulated cells also showed increased ultrastructural organization including increased mitochondria volume and mitochondria positioned between myofibrils, long and well aligned sarcomeres containing compact and clearly visible M and Z lines and H, I, and A bands that closely resembled those in native myocardium. Electrical stimulation of NRVM monolayers92 resulted in an increase in sodium calcium exchanger, increased action potential duration, and enhanced conduction velocity. It was also reported that electrical stimulation of NRVM for 72 hr93 leads to increased mitochondrial content and activity. Interestingly, electrical stimulation can also influence NRVM transcriptome independently of contraction94.
Combining in vivo topographical and electrical cues, NRVM88 were seeded onto substrates consisting of 0.5 μm-wide grooves and 0.5 μm-wide ridges and subjected to electrical stimulation (biphasic, 1 ms, 1.15 V/cm, 1 Hz). Cells were elongated and aligned along the microgrooves. Electrical cues enhanced cardiomyocyte elongation parallel to the electric field. Simultaneous application of biphasic electrical pulses and topographical cues led to gap junctions confined to the cell-cell end junctions rather than the punctate distribution in control cells.
Biochemical cues
Adrenergic receptor (AR) agonists
Adrenergic agonists have been studied extensively in rodent cardiomyocytes and are well known to induce hypertrophy. Traditionally, these are considered to be models of pathological hypertrophy, but it is interesting to consider their potential roles in physiological maturation. Norepinephrine is an α- and β-AR agonist that can significantly increase protein synthesis following treatment of fetal and neonatal mouse myocytes and NRVM95. In hESC-CM, administration of the α-AR/Gq agonist phenylephrine76 resulted in a significant 1.8-fold increase in cell area, 3.8-fold increase in cell number with organized sarcomere structure, and a 2-fold increase in cell volume. Total cellular protein to DNA content was also higher in PE-treated cells.
Triiodothyronine (T3)
Thyroid hormone is essential for normal cardiac development96. In the perinatal period, it regulates the isoform switching of several myocardial proteins, including myosin heavy chain and titin97. Chattergoon et al reported98 that T3 is a major inducer of cardiomyocyte maturation in the sheep fetus by increasing cell width, increasing binucleated cell percentage, reducing cell proliferation, and increasing the SERCA2a expression. Studies in murine ESC-CMs also showed that T3 increases Nkx2.5, myosin light chain-2v, alpha-MHC, SERCA2a, and ryanodine receptor-2 expression99. Electrophysiological studies showed that T3-treated cardiomyocytes exhibited decreased resting membrane potential and more adult–like calcium homeostasis properties, including a significantly larger maximal upstroke velocity, a higher maximal decay velocity, and a larger peak amplitude of caffeine-induced calcium transients.
Insulin-like Growth Factor I (IGF-I)
IGF-1 is essential for the regulation of cardiomyocyte proliferation, differentiation, postnatal growth and maturation of the heart. IGF-1 treatment of NRVM leads to upregulation of MLC-2 and troponin-I, cell size doubling, with an increase in protein synthesis100. IGF-1 has also been reported101 to influence the maturation of cardiomyocyte metabolism by enhancing the effect of the nuclear receptor/transcription factor peroxisome proliferator-activated receptor α and increasing the expression of the fatty acid oxidation enzymes medium chain acyl-CoA dehydrogenase and the muscle-type carnitine palmitoyltransferase I in NRVM. Although IGF-1 has been shown to be a mitogen for hESC-CMs102, to our knowledge it has not been studied in regulating their maturity.
MicroRNA
Analysis of miR profiles of human ESCs, hESC-CMs, fetal human, and adult human ventricular cardiomyocytes103 identified miR-1 as a potential modulator of cardiomyocyte maturation. Further studies showed that miR-1 over-expression decreased action potential duration and hyperpolarized both the resting membrane potential and the maximum diastolic potential in hESC-CMs due to increased Ito, IKs, and IKr, and decreased If. In addition, miR-1 augmented the immature Ca2+ transient amplitude and kinetics. Because of its involvement in thyroid hormone responsiveness and the myosin isoform switch, miR208 is a potential candidate that also may promote maturation104, 105.
Tissue Engineering and/or Mechanical Loading
The myocardium consists of a three-dimensional arrangement of rod-shaped cardiomyocytes, which form myofibers that are adjacent to interstitial fibroblasts, blood vessels, and extracellular matrix. Cardiomyocytes are also continuously subjected to cyclic mechanical strain induced by rhythmic heart beating. It is thus not surprising that even adult cardiomyocytes begin to dedifferentiate when they are placed in 2-dimensional culture without mechanical loading. An attractive hypothesis is that seeding immature cardiomyocytes in appropriate three-dimensional matrix and subjecting them to mechanical stress will enhance their maturation. This hypothesis was first elegantly proven by the work of Zimmerman, Eschenhagen and colleagues64. NRVMs were cast in hydrogels of collagen I plus basement membrane proteins and subjected to phasic mechanical stretch to generate engineered heart tissue (EHT). After 14 days (7 days in casting molds followed by 7 days of stretch), the cardiac cells in EHT exhibited interconnected, longitudinally oriented cardiac muscle bundles with morphological features resembling adult native tissue. It is worth noting that cardiomyocytes in the EHT exhibited a well-developed T-tubular system and dyad formation with the sarcoplasmic reticulum. The EHT displayed contractile characteristics of native myocardium with a high ratio of twitch (0.4 to 0.8 mN) to resting tension (0.1 to 0.3 mN) and a strong beta-adrenergic inotropic response. Action potential recordings demonstrated lower stable resting membrane potential, faster upstroke kinetics, and a prominent plateau phase. To our knowledge, this study generated cardiomyocytes with the closest morphological and electrophysiological properties to adult cardiomyocytes from NRVM.
Bursac’s group106 introduced 3-dimensional cell alignment cues consisting of elliptical posts within a fibrin-based hydrogel matrix. Mouse ESC-CMs were seeded, and they assembled into a dense, structurally and functionally aligned 3D syncytium with supporting non-myocytes within a total culture time similar to period of mouse embryonic development (21 days). Those cells had a conduction velocity between 22 and 25 cm/sec. It is worth mentioning these values are close to the conduction velocity of neonatal mouse hearts (about 29 cm/sec)107, while adult mouse hearts has a conduction velocity of about 62 cm/sec108. The cardiomyocytes produced significant contractile forces up to 2 mN. Since the authors reported cross sectional areas of 49 mm2, this corresponds to force production of 0.04 mN/mm2, achieving levels of functional differentiation similar to those of neonatal mouse hearts. So far, this tissue engineering approach has produced the fastest propagating electrical signals. It would be interesting to see to what extent this system could mature the hPSC-CMs.
Our group was the first to adapt hydrogel-based tissue engineering techniques to generate human engineered cardiac tissue from hPSC-CMs63. The cells were cast in 3D collagen and basement membrane protein matrix followed by uniaxial mechanical stress conditioning. Cyclic stress markedly increased cardiomyocyte alignment and hypertrophy, and proliferation rates were moderately upregulated. Addition of endothelial cells further enhanced cardiomyocyte proliferation, and addition of stromal supporting cells augmented vessel-like structure formation. Importantly, the optimized human cardiac tissue constructs generated Starling curves (Fig. 3), increasing their active force in response to increased resting length.
Fig. 3.
Tissue engineered hPSC-CMs collagen constructs were subjected to a series of stretches to increase resting tension. Note the increased twitch heights at higher preload (insets). (B) Starling curves showing a linear increase in active force in response to increased diastolic length. This figure was reproduced from Tulloch et al102 with permission.
Cell-cell interaction and cardiomyocyte maturation in vivo
During development in vivo, cardiomyocytes interact closely with several other cell types such as fibroblasts, endothelial, and smooth muscle cells. Therefore, either direct cell-cell contact or paracrine factors affect cardiomyocyte maturation. Indeed, using genetically purified early hESC-CMs, Kim et al109 showed that non-cardiomyocytes are required for the development of some intracellular calcium handling proteins. Ion channel development (HCN4) and electrophysiological maturation were also enhanced by the presence of the non-cardiomyocytes. It will be of considerable interest to identify the molecular signals that mediate this cross-talk.
Using ventricular slices of recipient hearts, Halbach et al110 performed time-course recordings on the electrophysiological maturation of fetal mouse cardiomyocytes after transplanting them into cryoinjured mouse hearts. Fetal cardiomyocytes that electrically integrated with the host hearts matured much faster than cells that were embedded in the cryoinjury, which showed no electrical integration. Twelve days after transplantation, the APD50 of the integrated cells matched the APD50 of the host cardiomyocytes, while the cells embedded in cryoinjured tissue still displayed immature electrophysiological properties. Structurally, transplanted cells displayed an increased cell area and length/width ratio. Though fetal mouse cardiomyocytes were used in this study, it provides some possible clues that may be applied to promote hPSC-CM maturation.
Recent work in direct reprogramming further shows the importance of the in vivo environment. The combination of three transcription factors (Gata4, Mef2c, and Tbx5) in vitro reprograms fibroblasts into cardiomyocyte-like cells, some of which express cardiac-specific genes, exhibit spontaneous calcium flux and beat111,112–115. These induced cardiomyocytes morphologically and functionally resemble immature hPSC-CMs rather than adult cardiomyocytes. When reprogramming was perfomed in vivo113, 116, however, the induced cardiomyocytes displayed morphological and functional properties of adult cardiomyocytes with elongated cell shape, intercalated disks with polarized connexin 43 expression, and electrophysiological parameters similar to the adult cardiomyocytes113, 116. Again, this study highlights the importance of in vivo cues to the maturation of cardiomyocytes.
hiPSCs and Cardiac Disease Modeling
The contribution of animal models to our overall understanding of diseases has been enormous. Nonetheless, significant differences exist between humans and common experimental animals, e.g. size, heart rate, ion channel contributions, distinct developmental processes, and many diseases cannot be modeled well in animals. With the advent of induced pluripotent stem cells, functional cardiomyocytes can be obtained by differentiation of human iPSCs derived from individuals with germline mutations. HiPSC-CMs from patients with cardiac defects such as Leopard syndrome117, long QT syndrome118, Timothy syndrome119, Pompe disease120, familial hypertrophic121, and dilated cardiomyopathy122 partially recapitulate these human cardiovascular diseases. These human “disease-in-a-dish” models hold great promise for elucidating pathogenesis and pursuing novel therapeutic strategies.
A recent study by Kim et al44 highlights the need for hiPSC-CMs with more mature properties to model adult-onset diseases. This group developed an arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) model from patients containing mutations in the plakophilin-2) gene. The median age for the onset of ARVD/C is 26 years. Mutant PKP2 iPSC-CMs showed abnormal plakoglobin nuclear translocation and decreased beta-catenin activity. However, only after the cells were induced to adult-like metabolic energetics from an embryonic/glycolytic state to fatty acid oxidation, they reproduced the pathological phenotypes such as exaggerated lipogenesis and apoptosis of ARVD/C. This study is the first to show that induction of adult-like metabolism has a critical role in establishing an adult-onset disease model using patient-specific iPSCs.
These disease modeling studies are only the first steps in a research field with great potential. Thus far, the majority of studies have been performed with relatively immature cells. To mature the hPSC-CM may lead to better recapitulation of disease phenotypes. In the case of establishing human ischemia-reperfusion models, for example, one would imagine that immature and adult cardiomyocytes might tolerate hypoxia differently due to their fundamental differences in metabolic substrates, and more mature cells would better predict adult human responses.
Pharmacological Studies
One of the principal reasons that drugs are withdrawn from the market after initial approval is cardiovascular toxicity that went undetected despite extensive testing in animals123. This points to the significant difference between human and animal physiology. Due to the lack of sufficient and healthy human cardiac tissue for experimental research, hPSC-CMs have been suggested as a new, attractive model. The effects of a variety of pharmacological agents have been assessed in hPSC-CMs73. These drugs have effects on beating frequency and contractility that were compatible with empirical results in the clinic, suggesting that hPSC-CMs could become an attractive tool for investigating drug effectiveness and safety. Another group used hiPSC-CMs as a platform to characterize the proarrythmic potential of 28 different compounds with established cardiac effects124. The hiPSC-CMs responded to drug treatment in a manner similar to what has been observed clinically.
Kadota et al78 developed a reentrant arrhythmia model using hPSC-CM sheets. By optically imaging the calcium signal, the authors found that sodium channel blockers affected the velocity and propagation pattern of activating waves. High-frequency stimulation of the sheets generated reentrant spiral waves, which were terminated by current anti-arrhythmic drugs. This model may be useful for future screening and testing drugs with anti-arrhythmic potential.
One study125, however, showed that caution should be used before extrapolating the results from the hESC-CMs to the adult cardiomyocytes because lack of functional IK1 channels, enhanced If densitiy, and some other differences between them may limit the potential of the hPSC-CMs to respond to drugs, especially pro-arrhythmic triggers. This study suggested that in some cases, maturation of the electrical phenotype is necessary for future implementation in drug safety testing. As mentioned earlier in Section I, hPSC-CMs may respond to some stimulation (e.g. response to high concentrations of calcium and beta-adrenergic agonists) differently from their adult counterparts72, and most studies have currently screened one or at best only a handful of cell lines.
Conclusions and Future Directions
As illustrated in Fig. 4, hPSCs have diverse potential applications in cardiovascular biology and medicine, but to reach this potential we need to learn to promote their maturity. Cardiomyocyte maturation in vivo is regulated by diverse factors, including topographical, electrical, adhesive, mechanical, biochemical, and cell-cell interaction cues. It is natural to want to break these components down, decipher the signaling pathways that control the phenotype and try to harness them to accelerate the process. Maturation of cells, as in people, is a complex trait, and we suspect it is unlikely to be controlled by a single master pathway. Thus, regulating a single pathway may control only a subset of the overall network, and this could lead to a distorted or partially matured phenotype. To achieve a better maturation status of the hPSC-CMs, it might be necessary to expose the cells to multiple regulatory cues simultaneously. It is natural to think that the best outcome would be a fully adult phenotype. However, for cell transplantation, adult cardiomyocytes do not survive to form new myocardium, whereas fetal and neonatal cardiomyocytes do126. Therefore, for cell transplantation purposes, the best option might be to obtain a cardiomyocyte phenotype with a yet-to-be-identified optimal maturation status that leads to the best engrafting and functional improvements.
Fig. 4.
Potential application of hPSC-CMs.
hPSC-CMs can be used and already show promising results in regenerative medicine. These cells are also valuable for disease modeling and pharmacological studies, as discussed in the main text. Enhancing hPSC-CM maturation state may allow for improved therapeutic applications. As we develop methods to generate cardiomyocytes of different maturation states, we will be able to gain significant insights into human cardiac development.
As summarized in Fig. 5, while progress toward maturation of hPSC-CMs has been made, significant challenges remain. An immediate future application for hPSC-CMs (either in 2D or 3D culture) is to perform high-throughput experiments to find the interventions that efficiently promote their maturation. For such screens to be successful, however, the endpoint indicating “maturation” needs to be carefully selected, as you get what you select for. Another exciting possibility of the use of hiPSC-CMs is the potential for personalized drug screening, since different individuals can have distinct responses to a specific medicine. If the drug’s possible cardio-toxic effects could be identified in the individual’s hiPSC-CMs, this could greatly advance personalized medicine. In this case, the importance to enhance the cell maturation is self-evident if the drug is for adult-onset diseases.
Fig. 5.
Summary of current maturation approaches and phenotypes achieved so far. A and B: representative immature hPSC-CMs and intermediate hPSC-CMs, respectively. The cells were stained for alpha-actinin (green) and filamentous actin (phalloidin, red). Nuclei (blue). Elongated cell shape, more organized sarcomere structures, longer sarcomeres, and more abundant mitochondria were achieved with manipulations. Compared with immature hPSC-CMs, the intermediate cardiomyocytes display higher conduction velocity75, contractile force, and increased calcium transient kinetics25. Scale bars in A and B: 25 μm, C and E: 0.5 μm, D: 5 μm) Panel B, C, D and E were modified from Lundy et al25 with permission from Dr. Michael A. Laflamme.
For patients with genetic diseases, it is possible that mutations could be repaired and the hiPSC-CMs be used for autologous cell replacement therapy. With the current available techniques, investigation of the patient-derived iPS-CMs in 3D cardiac tissue constructs may lead to novel findings about the targeted diseases. One can also envision using hPSC-CMs for an in vitro human myocardial infarction model in which the cells are exposed to ischemic conditions. Since immature and mature cardiomyocytes respond to ischemia differently, optimizing the maturation status again will be necessary.
In summary, substantial progress has been accomplished in just a few years since the development of hESCs and hiPSCs. Successful differentiation of cardiomyocytes from hPSCs shows great promises in a number of applications such as human development, cell transplantation, disease modeling, and drug screening. Promoting maturation of human pluripotent stem cell-derived cardiomyocytes will be an essential step toward achieving these goals.
Supplementary Material
Acknowledgments
Sources of Funding
This work was supported by the National Institute of Health grants R01HL084642, P01HL094374, U01HL100405, and P01GM81619. Xiulan Yang is supported by the American Heart Association post-doctoral scholarship 12POST11940060.
Non-standard Abbreviations and Acronyms
- hPSC
human pluripotent stem cells
- hESC
human embryonic stem cells
- hiPSC
human induced pluripotent stem cellsl
- CMs
cardiomyocytes
- ssTnI
slow skeletal troponin I
- cTnI
cardiac troponin I
- MHC
myosin heavy chain
- ECC
excitation contraction coupling
- NRVM
neonatal rat ventricular cardiomyocytes
- mPTP
mitochondrial permeability transition pore
- ECC
excitation-contraction coupling
- AR
adrenergic receptor
- T3
triiodothyronin
- IGF-1
insulin-like growth factor 1
- miR
micro RNA (miR)
- EHT
engineered heart tissue (EHT)
- APD50
action potential duration at 50% repolarization
Footnotes
In November 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days.
Disclosure
CEM is co-founder and equity holder in BEAT Biotherapeutics.
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