A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes

1The generation of induced pluripotent stem cells (iPSCs) forever changed the field of regenerative medicine, basic and translational biomedical researches. Human iPSCs became an appealing alternative to embryonic stem cells. Since Yamanaka’s breakthrough, many efficient protocols have been developed for generating cardiomyocytes derived from human iPSCs (hiPSC-CMs).

2One of the unresolved problems is that hiPSC-CMs remain largely immature when compared to human adult cardiomyocytes. Such immaturity hinders their usage on many aspects, e.g., pharmacological and toxicological screening and cardiovascular disease modeling. Transplanting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) or non-human primate iPSC-CMs into non-human primates model of myocardial ischemia-reperfusion resulted in substantial remuscularization, but non-fatal ventricular arrhythmias were observed, which could be a result of transplanting immature PSC-CMs that have automaticity. To address the issue of immaturity, many different approaches have been examined on enhancing the maturation of hiPSC-CMs.

3In this review, we briefly list the main differences between immature, fetal-like hiPSC-CMs and adult cardiomyocytes (Figure 1). Then, we discuss the current methods used to promote hiPSC-CMs maturation (Figure 2). In the second part, we describe the details of the evolving 3D culture model – it’s structure, the effect on hiPSC-CMs maturation, incorporation with different maturation methods, limitations, and future perspectives.

4Researchers in the stem cell field agree that hiPSC-CMs are immature, but there is no consensus about how to evaluate their degree of maturation. Therefore, in addition to developing ways to increase iPSC-CMs maturity, methods on how to assess maturation are required. Here, we summarize characteristics of adult cardiomyocytes and differences to hiPSC-CMs (Figure 1).

5It takes up to 10 years for cardiomyocytes to acquire adult phenotypes in structure and ploidy in a human heart. Adult cardiomyocytes are well-aligned, rod-like, multinucleated/tetraploid cells, with highly organized sarcomeres, well developed sarcoplasmic reticulum (SR) and transverse tubules (T-tubules), and have intercalated disks with mature mechanical and electrical junctions. Such phenotypical maturation is still lacking in hiPSC-CMs, which tend to be small, mononucleated, rounded cells with disorganized sarcomere. Moreover, they have shorter sarcomeres, poorly developed SR, and no T-tubules. Structural features can be used to evaluate the degree of hiPSC-CMs maturity because some of these features are characteristic to mature cardiomyocytes.

6Adult cardiomyocytes only beat when stimulated with a force around 40–80 mN/mm2, conduction velocity around 60 cm/s and upstroke velocity about 150–350 V/s. In hiPSC-CMs, these parameters are around 0.08–4 mN/mm2, 10–20 cm/s, and 10–50 V/s, consecutively. Moreover, hiPSC-CMs display mixed action potential (AP) morphologies that can be categorized as atrial, nodal, or ventricular-like AP. Although hiPSC-CMs generate important cardiac currents such as INa, ICa,L Ito, IKr, and IKs, they lack IK1 that is essential for stabilization of the resting potential. This deficiency might be particularly important when hiPSC-CMs are used to study long QT syndrome. Human ether-a-go-go related gene (hERG) encode a subunit of IKr channel, and mutation in hERG or blockade of IKr cause long QT syndrome. Without IK1, hiPSC-CMs rely on IKr for the maximum diastolic potential (MDP) that is markedly depolarized with IKr blockers. The other characteristic is the spontaneous beatings of hiPSC-CMs. If current generated by HCN4, which is restricted to pacemaker cells in vivo, depolarize MDP and make hiPSC-CMs beat. Measuring the electrophysiological parameters and assessing the generation of IK1 current is a promising tool that can be utilized to assess the maturation of hiPSC-CMs, though it would be technically challenging.

7In adult cardiomyocytes, T-tubules and SR are well developed to regulate Ca2+ induced Ca release (CICR) and fast excitation-contraction coupling (ECC). The inflow of Ca2+ via L-type channels triggers the release of Ca2+ from the SR through the ryanodine receptor (RyR) channels. T-tubules, invagination of the cell membrane, near L-type Ca2+ channel and RyR in adult cardiomyocytes. In the relaxation phase, Ca2+ is returned to the SR through sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and is extruded from the cell through the Na+–Ca2+ exchanger (NCX). The sharp and uniform increase of intracellular Ca2+ concentration in adult cardiomyocytes is important for the synchronized contraction in multiple sarcomeres. In hiPSC-CMs, T-tubules are absent and SR is underdeveloped with low expression of SERCA and other key proteins. As a result, hiPSC-CMs rely on L-type channels for the increase of Ca2+ and ECC is slow.

8In adult cardiomyocytes, mitochondria volume increases, and the oxidative capacity is increased, which represents to switch in metabolic substrates from glucose to fatty acid. To examine glycolysis and fatty acid oxidation, the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) are often used, respectively. During early heart development, around 80% of energy is produced by glycolysis. When cardiomyocytes become mature, fatty acid β-oxidation increases and becomes a major source for energy production. In a rabbit, the metabolic switch occurs during early postnatal growth. As hiPSC-CMs remain immature, they rely on glycolysis rather than fatty acid β-oxidation.

9Identifying the genes involved in human cardiomyocytes maturation is still an ongoing process. But the overall expression pattern of maturation-related genes identified in mice and humans are mostly similar. Isoform transitions of sarcomeric genes occur from fetal to adult period. Cardiac myosin heavy chain (MHC, also known as MYH) has two isoforms. These are α-isoform (α-MHC, also known as MYH6) and β-isoform (β-MHC, also known as MYH7). In adult cardiomyocytes, the β-isoform is predominant. To note, the isoform switch occurs from β-isoform to α-isoform in mouse hearts. Troponin I (TnI) has three isoforms [slow skeletal (ssTnI), fast skeletal (fsTnI), and cardiac (cTnI)] encoded by TNNI1, TNNI2, and TNNI3, respectively. In adult cardiomyocytes, cTnI is highly expressed although ssTnI is the primary isoform in hiPSC-CMs. Titin (TTN) has three major isoforms. These are N2B, N2BA and fetal cardiac titin (FCT). In adult cardiomyocytes, N2B is mainly expressed whereas N2BA is predominant in hiPSC-CMs. Moreover, hiPSC-CMs show low expression levels of important cardiac genes such as SERCA2 (sarcoplasmic reticulum ATPase), CAV3 (caveolin 3), KCNH2 (potassium voltage-gated channel), and other adult cardiomyocytes genes.

10To assess hiPSC-CMs maturation, measuring the TNNI3 to TNNI1 ratio is one way. To achieve more precise measurement of maturation, transcriptome-based approaches were proposed, including a gene regulatory network-based and a relative expression orderings-based scoring method. Single-cell transcriptome analysis could also predict the developmental ages of cardiomyocytes. However, most of these approaches are limited to mouse PSC-CMs as limited transcriptome data is available for full-spectrum of human developing hearts including late fetal and early postnatal periods. Thus, further work must be done to generate a valid, agreed upon, maturation index for hiPSC-CMs.

11In order to be able to fully utilize hiPSC-CMs for clinical or research purposes, especially for drug discoveries and disease modeling, they must acquire an adult-like maturation state. In this part, we discuss different approaches used to enhance the maturation of hiPSC-CMs (Figure 2).

12It takes years for cardiomyocytes to fully mature in vivo, which prompted a hypothesis that prolonged culture time would promote maturation of hiPSC-CMs. To date, hiPSC-CMs were cultured up to a full year to test their maturity. With prolonged culture, hiPSC-CMs displayed more mature phenotypes in morphology (larger cell size), structure (myofibril density, alignment, microscopically visible sarcomere), and physiology (calcium handling and β-adrenergic response). The cells expressed maturation-related cardiac genes such as MYH7 with isoform switch. Interestingly, extending the cultures to 180 days resulted in more tightly packed myofibrils with the appearance of mature Z-, A-, H-, and I-bands, but not M-bands. M-bands, a key feature of sarcomere structure, are finally developed after 360 days of culture. These results consolidate the fact that prolonged culture generates more mature cardiomyocytes. However, it poses a question if it is possible to yield cells mature enough in a realistic, and financially appropriate culture time frame.

13Thyroid hormone, known to have a crucial role in cardiac development and cardiovascular physiology, displayed strong enhancement of hiPSC-CM maturation. Triiodothyronine (T3) treatment makes hiPSC-CMs bigger, more elongated morphology with longer sarcomeres. T3-treated hiPSC-CMs displayed increased mitochondrial activity and improved calcium handling along with higher contractile force.

14Glucocorticoids are essential for maturation of fetal heart structure and function. Endogeneous glucocorticoids work by stimulating glucocorticoid receptor (GR) on fetal cardiomyocytes/vascular smooth muscle to promote myofibril assembly and organization. Adding glucocorticoid analog, dexamethasone, to T3 in culture further improve hiPSC-CMs maturation.

15These results highlight the importance of these chemical cues and call for studying more molecules and combinations that may further enhance maturation of cardiomyocytes.

16A hallmark of postnatal cardiomyocyte maturation is switching their metabolism from glycolysis to fatty acid oxidation. Recently, the glucose-free and lactate-containing medium were identified to eliminate non-cardiomyocyte and enrich hiPSC-CMs, while hiPSC-CMs are usually cultured in glucose-containing medium. Replacing glucose with galactose and fatty acids – more specifically, palmitate, oleic acid, linoleic acid, and carnitine – enhanced maturation of hiPSC-CMs. The switch of energy source not only increased mitochondrial number and metabolisms but also enhanced morphological, structural and physiological maturation. On the other hand, culturing hiPSC-CMs in high glucose medium inhibits their structural and functional maturation by promoting nucleotide biosynthesis, which is attributed to a reduction of cardiac glucose uptake and increased nucleotide deprivation during late gestational and early postnatal stages.

17Cells interact with each other through direct cellular contact or indirect paracrine factors secreted by the neighboring cells in a heart, and cellular interaction has been implicated in cardiac maturation. To mimic these cellular interactions in vitro, hiPSC-CMs were cocultured with non-cardiomyocytes, such as human mesenchymal stem cells (MSCs) and endothelial cells. Human MSCs secrete VEGF, bFGF, SDF-1, and GM-CSF to mediate differentiation and electrical coupling of hiPSC-CMs. In contrast, endothelial cells express extracellular matrices (ECMs; collagens I and III, fibronectin, thrombospondin-4) to increase sarcomere length of hiPSC-CMs. ECMs alone had some effects on enhancing structural and functional maturation of hiPSC-CMs.

18Extracellular matrices regulate tissue stiffness, and the stiffness of a heart increases gradually in vivo as a result of collagen accumulation. This process increases the ability of the heart to pump blood due to its increased stiffness. Compared to hearts (∼10 kPa), cell culture dishes are much stiffer (∼1 MPa), which prompted a hypothesis that soft matrices might be better for the maturation of hiPSC-CMs. Polydimethylsiloxane (PDMS), hydrogels or polyacrylamide (PAA) were often used to create such culture conditions. Soft surfaces (6∼10 kPa) tuned sarcomere tension and contractility, and hiPSC-CMs on it generated more force (0.1 μN) than those on a hard surface (35 kPa, 0.01–2 μN). PDMS and PAA are also used to regulate topology of cell morphology and forcing hiPSC-CMs into rectangular shape results in better maturation. In the agreement with this, the vascular structure also forces hiPSC-CMs in a rectangular shape and made them more mature.

19Biophysical stimulation is absent under a standard culture condition. In a heart, cardiomyocytes are regularly exposed to electrical stimulation and mechanical stress. Applying a continuous electrical stimulation yielded hiPSC-CMs with rod-like morphology, enhanced cellular alignment, and more organized sarcomeres. Furthermore, subjecting combined synchronized electrical and mechanical stimulation on hiPSC-CM resulted in enhanced localization of N-cadherin toward cell membrane, sarcomere shortening, and reduced transmembrane calcium current, suggesting more mature phenotype.

20Instead of mimicking in vivo environment in vitro, an in vivo environment can be used to provide all necessary signals including unknown ones to hiPSC-CMs. There are some disagreements what developmental stages of the heart can be appropriate – neonate, adult, or adult heart after infarction, however, hiPSC-CMs in vivo are more matured than ones in vitro. Transplantation to neonatal hearts generated adult-like mature hiPSC-CMs within 2 months of transplantation, suggesting that the maturation speed is accelerated in a rat heart and it is defined by non-cell autonomous manner. Furthermore, hiPSC-CMs from a patient of arrhythmogenic right ventricular dysplasia/cardiomyopathy recapitulated disease phenotypes with the neonatal transplantation method.

21Cells are aligned three-dimensionally in vivo rather than a monolayer. In the last decade, 3D culture methods have been advanced and become an appealing alternative to the conventional 2D monolayer culture for the maturation of hiPSC-CMs. As 3D tissues also resemble native cardiac architecture, 3D tissues of dilated cardiomyopathy hiPSC-CMs could recapitulate disease phenotypes that 2D cultures failed. Here, we discuss the usefulness of 3D cultures on the maturation of hiPSC-CMs.

22Conventional 2D cultures fail to recapitulate the complexity of the in vivo cellular crosstalk, tissue architectures, and extracellular microenvironments with forced and unwanted apical-basal polarity. On the other hand, 3D cultures have more similarity to the in vivo extracellular microenvironment, support better cellular interactions, and allows for biochemical and physical stimuli to reach the cells in an evenly distributed manner. In 3D cultures, hiPSC-CMs display structural, functional and metabolic maturation compared to that in 2D cultures. They show improved myofibrillar alignment and sarcolemma remodeling which led to better Ca2+ handling. Moreover, hiPSC-CMs in 3D culture exhibited faster maturation analyzed by transcriptome, while 2D cultures hinder the maturation or cause maturation arrest.

23Different strategies are used to produce 3D cardiac tissues. One way is seeding hiPSC-CMs in to designed scaffolds or embedding them in hydrogel. To produce scaffold, photolithography is often used, however, it requires clean room and specialized equipment that are not often available for biomedical research labs. 3D printing technologies have been evolving rapidly, and digital light processing (DLP)-based printing is now used to fabricate scaffolds or molds to embed cells in hydrogel directly in a cell culture dish. Another method is layering hiPSC-CMs into multi-layered cardiac tissue constructs, which successfully recapitulate Torsade de Pointes in vitro.

24Overall, there is a growing effort to learn the appropriate technology and materials to design a reproducible, efficient and affordable 3D culture system, such advancement will be a great step toward the generation of mature, functional cardiomyocytes.

25Although 3D cultures of hiPSC-CMs have great potential to make more matured cardiomyocytes, it is insufficient to do so by itself, and a combination of the above-mentioned maturation-promoting methods worked in 2D must be utilized within a 3D setting, which includes cell-cell interaction (e.g., fibroblasts), hormones, and electrical and physiological stimulation. Below, we will provide a brief review of the electrical and physiological stimulation applied to 3D cultures to reach the ultimate goal.

26One of the epoch-making studies was done by. Embedding hiPSC-CMs with collagen gel into PDMS channel to form a wire-like structure and exposing the tissues to electrical stimuli generate functionally more matured cardiac tissue. To further augment electrical conduction, electrically conductive silicon nanowires or carbon nanotubes were incorporated into hiPSC-CMs spheroids to form an electrically conductive environment that is later enhanced by the addition of an exogenous electrical stimulation. The regimens of electrical stimulation were explored by several groups to achieve T-tubule formation and positive force-frequency relationships. More sophisticated stimulation has been explored, and a high-intensity training regimen of electrical current with gradually increasing frequency, from 2 to 6 Hz within 2 weeks, followed by another week of stimulation at 2 Hz, achieved hiPSC-CMs with adult-like gene expression, well-developed ultrastructure including T-tubule, better calcium handling and contraction force.

27In the mechanical aspect, passive stretch or application of afterload on 3D tissue of hiPSC-CMs (namely, engineered heart muscle or engineered heart tissue) promoted its structural and functional maturation. A passive stretch is enough to facilitate metabolic switches in hiPSC-CMs. Moderate afterloads are beneficial on cardiomyocyte maturation, while higher afterloads may be detrimental and cause pathological changes. The combinations of both electrical and mechanical stimuli – either cyclic stretch or static stress – were also explored. In such conditions, hiPSC-CMs displayed a more matured signature than a single stimulus.

28As we summarized, the maturity of hiPSC-CMs is getting better by numerous efforts. However, methods still need further improvement to reach the desired degree of maturity. Other aspects lacked in this field are to determine definitive maturity of hiPSC-CMs rather than relative measurements and some mechanistic insights why cells are maturing with particular stimuli. Transcriptome-based assay followed by bioinformatics would be one way to define the maturity and uncover mechanistic insights, though more transcriptome data of human hearts in late fetus and postnatal periods is required. As we noted, human cardiomyocytes require years to complete their maturation in a human heart. Therefore, a question remains to be elucidated if and how we can generate adult-like mature hiPSC-CMs at an affordable time and cost. In a specific condition in vivo, the maturation process is accelerated, thus, hopefully, the issue will be resolved in the near future.

29RA summarized the publications for this mini review and drafted the manuscript. RA, TA, and HU wrote the manuscript. RA and NC drew the figures. HU finalized the manuscript.

30The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.