Overview of the development of cardiac differentiation methods.
Abstract
Research of genetic cardiovascular diseases has lacked of good disease models because rodents, which are primarily used, differ greatly from humans. The ability to derive human induced pluripotent stem cells (hiPSCs) from patients carrying inherited cardiac diseases has revolutionized research in the cardiovascular field. The aim for this chapter is to review the current hiPSC reprogramming methods and methods for differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes. The chapter focuses on the published hiPSC models for hypertrophic cardiomyopathy (HCM) and discusses the challenges related to modeling this interesting disease using hiPSC technology.
Keywords
- cardiac differentiation
- cardiomyocyte
- disease modeling
- human induced pluripotent stem cell
- hypertrophic cardiomyopathy
1. Introduction
The derivation of human embryonic stem cells (hESCs) [1] and, more recently, the invention of human induced pluripotent stem cells (hiPSCs) [2] have opened new opportunities for research and cellular therapies in regenerative medicine. These cells, collectively called human pluripotent stem cells (hPSCs), have the ability to self-renew indefinitely and to differentiate into derivatives of all three germ layers [1, 2]. Thus, hPSCs provide a potential source of cells for regenerative medicine applications as well as in vitro modeling of genetic diseases and drug screening.
Traditionally, hiPSCs have been reprogrammed from skin fibroblasts by virally transferring four pluripotency factors, specifically octamer-binding transcription factor 3/4 (
hPSCs can be differentiated into cardiomyocytes under laboratory conditions. During the recent years, the cardiac differentiation methods have developed from embryoid body (EB) formation [6] and co-culturing hPSCs with mouse endodermal-like cells (END-2) [7] to more direct differentiation on a monolayer using different growth factors and small molecules [8–10].
Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases, with a worldwide prevalence of 1:500. In HCM, the cardiac muscle tissue mainly in the interventricular septum is thickened. The most severe symptoms of HCM are progressive heart failure and sudden cardiac death [11]. HCM is caused by more than 1400 mutations, which reside primarily in genes coding for sarcomeric proteins [12]. The clinical phenotype of the disease is variable, and most of the patients carrying the mutations live their lives without any symptoms [11]. Currently, there have been five reports published using HCM patient-specific hiPSCs for modeling the disease [13–17]. Results from these reports will be reviewed thoroughly in this chapter.
2. Human pluripotent stem cells
hPSCs are defined as undifferentiated cells which have the ability to self-renew indefinitely and to differentiate into derivatives of all three germ layers: endoderm, mesoderm, and ectoderm [1, 2]. The first hESC line was derived by Thomson and co-workers in 1998 [1]. Traditionally, hESCs are derived from the inner cell mass (ICM) of the blastocyst but early blastomeres or morula stage embryos have also been used [18, 19].
For a long time, the scientific community believed that cell differentiation was a one-way route and that there was no turning back when a cell had passed specific differentiation stages or reached the fully differentiated state. However, in 2006, Yamanaka and co-workers were able to reprogram already fully differentiated mouse cells back into the pluripotent stage via retroviral induction with specific pluripotency factors:
Because of their unique properties, hPSCs can be maintained in the laboratory for extended periods of time in their undifferentiated state and differentiated into various cell types, including cardiomyocytes [6], retinal pigmented epithelial (RPE) cells [25], neural cells [26], and hepatocytes [27] (Figure 1). Thus, hPSCs represent a limitless cell source for regenerative medicine applications as well as for studying developmental processes and genetic diseases.
The groundbreaking discovery of hiPSCs has opened completely new opportunities for disease modeling and drug screening also in the cardiac field. Conventionally, cardiac diseases have been modeled using animal models or genetically engineered cell lines. These models often correlate poorly with the results from human studies. In addition, obtaining cardiac tissue directly from patients for research purposes is difficult, and adult human cardiomyocytes dedifferentiate rapidly under cell culture conditions and lose their characteristic properties. With the hiPSC technique, we are able to derive cells directly from a patient and transfer the same genetic information and mutations into hiPSC-derived cardiomyocytes. Therefore, hiPSCs have great potential to revolutionize the research of cardiovascular diseases.
2.1. Generation of human induced pluripotent stem cells
In the first conversion of mouse and human fibroblasts into iPSCs, Yamanaka’s group used the viral transduction of four transcription factors (
Viral transduction can be achieved using either integrating or non-integrating viral vectors. In 2007, two distinct research groups published the first generation of hiPSCs. In the first paper, the delivery was accomplished using retroviral pMXs vectors [2], while in the second paper, the transduction was performed using lentiviruses [31]. The proteins, which are needed for the additional rounds of virus replication and packaging, are deleted from the pMXs vectors. Retroviral vectors are able to target cells according to their envelope pseudotype and they only transduce actively dividing cells. Lentiviruses, in contrast, can also transduce non-dividing cells [28]. Both retro- and lentivirally transferred genes are expected to be silenced during the reprogramming process through methylation and epigenetic modification [32]. Sometimes, however, the process is incomplete, which results in partially reprogrammed hiPSC lines [20, 33, 34].
To overcome the problems related to transgene integration into the host genome, excisable vectors have been engineered based on, for example, Cre-recombinase-mediated excision [4]. In this method, the sequence of the gene to be integrated into the genome is inserted between two loxP sites in the LTR (long terminal repeat) region of the vector. The integrated transgenes can be afterwards excised from the genome by transfecting the hiPSCs with Cre-recombinase [4]. Reprogramming factors in distinct vectors are integrated at independent sites in the genome, which can lead to genomic instability and genome reorganization when Cre-recombinase is introduced into the cells. Therefore, polycistronic vectors, which express all reprogramming factors in one vector separated by 2A sequences, are favored when using this method [35, 36].
Non-integrative viral methods include the use of Sendai viral and adenoviral vectors. With these two non-integrative viral methods, the Sendai virus has turned out to be more efficient for the generation of hiPSCs. Sendai virus vectors replicate their single-stranded RNA in the cytoplasm without entering the nucleus of the infected cell [3]. In addition, they are able to infect a wide variety of cell species and tissues by attaching to sialic acid receptors, which are present on the surface of various cell types [3]. Adenoviral vectors, in contrast, contain DNA, which is transported to the nucleus of the target cell. However, this adenoviral DNA is not integrated into the genome, and the expression of the adenoviral genes is thus transient [37, 38].
Non-viral methods are based on delivering genes (DNA), RNA copies of the genes or proteins to target cells. Different delivery carriers, such as transposons, and methods, including electroporation and transfection reagents, have been reported. The previously mentioned polycistronic vectors can also be transferred into target cells without viral delivery through electroporation [4, 36]. The PiggyBac transposon and transposase system is another integrative non-viral method used in hiPSC generation [39]. In this method, the transposase enzyme cleaves the delivered genes from specific cleavage sites in the PiggyBac vector and transfers them into the target genome. The same enzyme can be used to excise exogenous genes from the genome after reprogramming [39].
Methods based on episomal vectors, messenger RNA (mRNA) molecules, or purified protein have been developed for non-integrative non-viral hiPSC production. Episomal vectors derived from Epstein–Barr virus (oriP/EBNA1) can be used to introduce reprogramming factors into cells without a need for viral packaging [5, 40]. The expression of the exogenes is transient and they disappear from the transfected cells during culture [5]. Lastly, nanoparticles have been used to improve the efficiency of reprogramming, particularly with mRNA molecules and proteins [41, 42].
3. Differentiation of human pluripotent stem cells into cardiomyocytes
The heart is the first functional organ that develops during embryogenesis. It develops from the mesoderm, although signals from adjacent cell populations, especially from the endoderm, have a significant role in cardiogenesis. The entirety of the development of the human heart is not yet fully understood; however, many molecular events and factors taking part in the early stages of cardiomyogenesis have been identified. The three main growth factor families thought to participate in early mesodermal induction and cardiomyogenesis are the wingless/INT proteins (WNTs), the fibroblast growth factors (FGFs), and members of transforming growth factor-beta (TGFβ) superfamily, which include bone morphogenetic protein 4 (BMP4), Nodal and Activin A [43]. The expression of these factors or their inhibitors in the adjacent endoderm occurs at different times, and their combination eventually leads to the induction of the cardiac mesoderm. After receiving these initial signals, development is directed to more specific and highly conserved cardiogenesis [43–46].
In vitro differentiation methods mimic the phases of heart development, from the mesoderm to the cardiac mesoderm, cardiac progenitors, and finally, cardiomyocytes [44]. This process is regulated by alternating activation and inhibition of the signaling pathways participating in the cardiomyogenesis (Figure 3). Current cardiac differentiation methods are based on EB differentiation in suspension, co-culturing the cells with END-2 cells or inducing cardiac differentiation with different growth factors on a monolayer. The details of the most important differentiation methods are collected in Table 1.
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Low-attachment | DMEM + ko-SR | – | First generation of EBs from hESCs | – | Itskovitz-Eldor et al. [6] |
Low-attachment | DMEM + FBS | – | First functional cardiomyocytes from EBs | 30% (cTnI, FC) | Kehat et al. [47] |
V-96 well | RPMI + FBS/ HSA |
PVA, BMP4, bFGF, insulin, AA | Forced aggregation strategy | 60–90% (cTnI, FC) |
Burridge et al. [48] |
3D microwell | DMEM/F-12 + FBS | – | Engineered 3D microwell coated with Matrigel | 18–20% (beating EBs) |
Mohr et al. [49] |
Low-attachment | StemPro34 | AA, BMP4, bFGF, Activin A, VEGF, DKK1 | Serum-free, GF-based EB method | 40–50% (cTnT, FC) |
Yang et al. [50] |
Low-attachment | StemPro34 | BMP4, AA, IWR-1, blebbistatin, Activin A |
Serum-free, GF-based EB method | 90% (cTnT, FC) |
Karakikes et al. [51] |
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END-2 | DMEM + FBS | – | First co-culture with END-2 cells | 35% (beating wells) |
Mummery et al. [7] |
END-2 | DMEM + ko-SR | AA | Addition of AA and serum-free medium in END-2 co-culture | 5–20% (α-actinin, ICC) |
Passier et al. [52] |
Low-attachment | END-2 CM | SB203580 | Differentiation with medium conditioned on END-2 cells |
22% (α-MHC, cytospin) |
Graichen et al. [53] |
Low-attachment | END-2 CM | Prostaglandin I2, AA, SB203580 | Differentiation of EBs in END-2 CM, inhibitory effect of insulin |
6–9% (α-MHC, cytospin) |
Xu et al. [54] |
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Matrigel | RPMI + B27 | Activin A, BMP4 | First monolayer differentiation method | 30% (β-MHC, ICC) |
Laflamme et al. [9] |
Matrigel | RPMI + B27 | Activin A, BMP4, bFGF | Sandwhich method, Matrigel is applied below and on top of cells | 40–98% (cTnT, FC) |
Zhang et al. [55] |
Matrigel | RPMI + B27 | CHIR99021, IWP2/IWP4 | Protocol exploiting small molecules modulating Wnt signaling pathway | 80–95% (cTnT, FC) |
Lian et al. [10] |
Vitronectin | RPMI + albumin | AA, CHIR99021, WNT-C59 | Serum- and animal-component free, defined differentiation protocol | 80–90% (cTnT, FC) |
Burridge et al. [8] |
The first hESC-derived cardiomyocytes were isolated from spontaneously formed EBs [6, 47]. EBs are spherical, multicellular, three-dimensional aggregates, which are formed when hPSCs are detached from the feeder cell layer or the culture matrix and cultured in suspension. In these EB structures, the hPSCs spontaneously differentiate into all three germ layers. In more recent versions of EB differentiation protocols, the aim has been to generate more uniformly-sized EBs, for example, via forced-aggregation using centrifugation [48, 56] or by culturing hPSCs in microwells coated with Matrigel prior to EB formation [49]. Due to the increased knowledge of the heart development in recent years, more guided EB differentiation methods using different growth factors and serum-free basal medium have been developed. Yang and co-workers were able to generate a population of cardiac progenitor cells by inducing EB differentiation with Activin A, BMP4, bFGF, vascular endothelial growth factor (VEGF), and Dickkopf homolog 1 (Dkk-1) [50]. With the protocol of Karakikes et al. [51], almost 100% of the EBs were beating and 90% of the cells were cTnT positive. In this protocol, the EB formation is induced by BMP4 and blebbistatin, which inhibits the actin-myosin contraction and suppress the dissociation-induced apoptosis. After this initial step, the EBs are induced to cardiac lineages by ascorbic acid, BMP4, Activin A, and finally with IWR-1 [51].
As discussed above, anterior endoderm is located directly posterior to mesoderm in the early development and the signals from adjacent endoderm initiate the early cardiogenesis. Based on this fact, Mummery et al. developed a differentiation method in 2003, in which the hPSCs are plated on top of mitotically inactivated END-2 cells [7, 52]. END-2 cells are derived from mouse P19 embryonal carcinoma cells, and they provide cell-to-cell contacts and produce factors that induce cardiac differentiation. However, medium conditioned on END-2 cells alone has been used in cardiac differentiation, which suggest that cell-to-cell contacts might not be needed in the cardiac induction [53, 54]. The exact mechanism how END-2 cells induce the cardiac differentiation is still unclear. However, a valuable details of cardiac differentiation has been discovered using these cells. For example, END-2 cells have been shown to remove insulin from the medium and insulin has been shown to inhibit the differentiation of hPSCs into cardiac lineages [54, 57]. Although, the efficiency of END-2 co-culture method is quite low, the method has turned out to be reliable in generating cardiomyocytes from various hPSC lines.
The increase in understanding the regulatory signaling pathways related to cardiogenesis in conjunction with advances in hPSC culture methods has enabled the development of more defined and guided monolayer-based differentiation methods. Monolayer methods begin with the feeder cell-free culture of hPSCs, in which the feeders are substituted with Matrigel or Geltrex, or matrix composed of different extracellular matrix (ECM) proteins. The advantage of monolayer differentiation methods is that the cells are in uniform monolayers and there are no diffusional barriers, which would prevent the function of growth factors. Thus, differentiation should be easier to control and reproduce, than in EB or in co-culture methods [44]. In the first monolayer method published in 2007 by Laflamme and co-workers [9], cells were directed toward cardiac differentiation by a combination of Activin A and BMP4. One of the more recent methods, the so-called sandwich method, is based on the combination of ECM with growth factor signaling [55]. Cells were seeded on Matrigel matrix, and after reaching 90% confluence, Matrigel was added on top of the cells. The sequential application of Activin A, BMP4, and bFGF on the matrix sandwich resulted in 40–90% pure cardiomyocyte populations [55].
As in the case of hPSC culture development, cardiac differentiation is also moving toward easily scalable, chemically defined, and xeno-free conditions. A group of small molecules has been identified and applied to replace the recombinant cytokines and unknown factors in the serum. These molecules either activate or inhibit the WNT and TGFβ-signaling pathways. Two recent publications are based on the sequential activation and inhibition of the WNT signaling pathway. At first, the formation of the mesoderm is induced using small molecules, such as CHIR99021 and BIO for WNT signaling activation [10, 58]. After that, more specific cardiac differentiation is induced by inhibiting the WNT signaling pathway using small molecules, such as KY02111 and IWP2 [10, 58].
In 2014, Burridge and co-workers [8] published a chemically defined cardiac differentiation method. The medium consisted of Roswell Park Memorial Institute (RPMI) basal medium supplemented with HSA and L-ascorbic acid. Cardiac differentiation was further induced by the sequential activation and inhibition of WNT signaling by CHIR99021 and WNT-C59, respectively. They also tested various defined matrices (E-cadherin, vitronectin, vitronectin peptide, laminin-521, laminin-511, fibronectin, and fibronectin peptide) in combination with differentiation medium. Laminins were the most promising, but because they are extremely expensive for large-scale applications, vitronectin was selected for further studies. The final protocol resulted in 80–90% pure cardiomyocyte populations for the multiple hPSC lines tested [8].
Although the differentiation methods for hPSC-derived cardiomyocytes have developed greatly, the efficiency of differentiation varies to a large degree when using different methods and cell lines and none of the methods result in a homogenous population of cardiomyocytes [8, 44, 59]. Homogenous cell populations would be needed, for example, to obtain reliable results from drug-screening assays [44]. Thus, there is a need for efficient purification methods.
Manual dissection [7] and Percoll gradient separation [60] were the first published methods for cardiomyocyte purification. However, the yield of pure cardiomyocytes with these methods was quite low. There are a few cell surfer markers that can be used for fluorescence activated cell sorting (FACS)-based purification of cardiomyocytes. These markers include signal regulatory protein α (SIRPA), which is expressed both in cardiac progenitor cells and in hPSC-derived cardiomyocytes [61], and vascular cell adhesion molecule 1 (VCAM1), which functions in leukocyte-endothelial cell adhesion but is also expressed in hPSC-derived cardiomyocytes [62]. One of the most recent method takes advantage of cardiomyocytes’ ability to use lactate as an energy source. When culturing cells in glucose-depleted and lactate-abundant conditions other cells than cardiomyocytes will not survive [63]. Another interesting option is the microRNA (miRNA) switch technology, in which the cells are purified based on their miRNA activity. Heterogenous cell population is transfected with synthetic mRNA, which comprise cell type specific miRNA target site and fluorescent protein. Cells in which the target miRNA is absent will translate the fluorescent protein and can be sorted out from the cardiomyocyte population. Alternatively, miR-Bim-switch can be used, which selectively induces the apoptosis of cells in which the miRNA is not present [64].
3.1. Characterization of hPSC-derived cardiomyocytes
hPSC-derived cardiomyocytes can be characterized by their structural and biochemical, as well as by their functional features. The most apparent characteristic of hPSC-derived cardiomyocytes is their ability to contract spontaneously in culture [7, 47]. The organization of the internal structures can be studied and analyzed after immunolabeling the sarcomeric proteins [65]. The ultrastructural features of hPSC-derived cardiomyocytes can be studied in more detail using electron microscopy (EM). Laurila et al. has recently published a thorough review on different methods used in functional analysis of hiPSC-derived cardiomyocytes [66]. Shortly, the electrical properties of hPSC-derived cardiomyocytes can be studied on a cell cluster level using the microelectrode array (MEA) platform approach [47, 67] or on the single-cell level using the patch clamp-method [68–70]. Calcium plays a major role in excitation-contraction coupling process by which an electrical signal is transformed into a mechanical contraction [71]. The intracellular Ca2+ signaling in hPSC-derived cardiomyocytes on a single-cell level is studied in vitro using specific fluorescent probes for Ca2+ ions, such as fura-2 [72]. The mechanical beating behavior of hPSC-derived cardiomyocytes can be analyzed using methods based on video imaging [73].
4. Hypertrophic cardiomyopathy
An adult cardiomyocyte is composed of evenly distributed and organized myofibrils, which are divided into approximately 2.2-μm-long contractile units called sarcomeres. Sarcomeres are composed of thin actin and thick myosin filaments and Z-discs. The thin filaments are attached to Z-discs, which separate the sarcomeres from each other. The thin filament is composed of repeating actin molecules, troponin (Tn) complexes, and α-tropomyosin (TPM1) molecules. Tn complexes consist of TnT, TnI, and TnC. The Tn complex works together with TPM1 during cardiac contraction. The thick filament consists of myosin molecules, which are built upon two units of α- and β-myosin heavy chain (α-MHC, β-MHC) and four myosin light chain (MLC) molecules. Among the other proteins of the thick filament, myosin-binding protein C (MYBPC) plays the most important role in the contraction. It contributes to actin-myosin interactions and cross-bridge formation [74].
When the Ca2+ concentration in the cytosol increases, the Ca2+ binds to TnC leading to a conformational change in the Tn complex. This leads to TPM1 moving from its inhibitory position, allowing the head region of the MHC to bind to actin, forming a cross-bridge. Then, myosin hydrolyzes adenosine triphosphate (ATP), causing the sliding of actin and myosin filaments and muscle contraction. In addition to the structural proteins mentioned above, the sarcomere consists of many other important proteins, which together form a stabilized and organized structure [71].
Cardiomyopathies are diseases that affect the heart muscle and can lead to progressive heart failure and cardiac death. Cardiomyopathies can either be genetic or acquired, and they can be divided into groups based on their morphological and functional characteristics. Cardiomyopathies include, among others, HCM, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC) [75]. HCM is one of the most common genetic cardiac diseases, with a worldwide prevalence of 1:500, and is the most common cause of sudden cardiac death among young competing athletes. HCM is inherited in an autosomal dominant pattern, and the mutations are mainly located in the sarcomeric proteins, which are responsible for the contraction and relaxation of the cardiomyocyte. The clinical manifestation of the disease is extremely variable: it has age-related penetrance, and the clinical symptoms can vary within the same family having the same gene mutation. Together, these facts indicate that there might be other factors in addition to the actual gene mutation, for example, epigenetic and environmental factors, that determine the clinical outcomes of the disease. Although a large number of mutations have now been identified and related to HCM, the pathophysiological mechanisms of the disease are still largely unknown [11].
In HCM, the cardiac muscle is thickened (≥15 mm) most commonly in the interventricular septum separating the right and left ventricles. This thickening, i.e., hypertrophy, can lead to outflow tract obstruction, which indicates that the passage to the aorta from the left ventricle becomes narrow and obstructive, disturbing the blood flow. This narrowed outflow induces a massive overload to the heart and can lead to progressive heart failure. Other severe complications related to HCM include arrhythmias and sudden cardiac death. However, most individuals remain asymptomatic for their whole lives, and it has been estimated that the actual prevalence of the disease might even be 1:200 in the general population [11]. Penetrance indicates the percentage of mutation carriers who experience the phenotype of the disease. In HCM, the penetrance is highly variable: it can be age-related or even incomplete and related to gender [76].
There is no specific cure for this disease and all the complications related to HCM should be treated individually. Patients are typically asymptomatic for a long time. Often, the first sign is diastolic heart failure, while systolic heart failure can develop later. Treatments include beta-blockers or Ca2+-channel blockers for relieving symptoms such as chest pain and shortness of breath and implantable cardioverter defibrillators (ICDs) for those patients who have survived cardiac arrest. The actual hypertrophy can be treated by surgical myectomy, which indicates removing a small portion of the thickened cardiac tissue; via ethanol ablation, in which a myocardial infarction is induced in the septal area; or at the end-stage via heart transplantation [11]. Histologically, HCM is characterized by myocyte hypertrophy (diameter >40 μm); disorganization of myocyte bundles, individual myocytes, and sarcomeres; and fibrosis of heart tissue. Nuclei are hyperchromatic (contain an abundance of chromatin), pleomorphic (vary in size and shape), and often enlarged [77].
HCM is inherited in an autosomal dominant pattern and is caused by over 1400 mutations found in eleven or more genes coding primarily for sarcomeric proteins. Approximately 70% of patients have a gene mutation either in the
In Finland, two founder mutations in genes coding for MYBPC and TPM1 proteins account for approximately 18% of Finnish HCM cases, and these
Different animal models have been used to study HCM in vitro. These animals include cats, which have certain naturally occurring HCM mutations, and genetically engineered mice, rats, rabbits, and Drosophila [86]. Additionally, HCM patient tissues obtained from myectomy samples have been studied [87]. One of the major difficulties in studying the pathophysiological mechanisms of HCM has been the lack of tissue samples at early stages of disease development. While animal models have provided valuable insight into disease mechanisms, they contain only the mutated gene and not the rest of the genome, which might have effects on disease phenotype and progression. Thus, given that they contain the whole genomes of HCM patients, in addition to the fact that they are cell of human origin, hiPSC-derived cardiomyocytes represent a valuable new tool for modeling HCM in vitro.
4.1. Human induced pluripotent stem cell models for studying hypertrophic cardiomyopathy
To date, morphological and functional characteristics of cardiomyocytes, derived from HCM patient-specific hiPSCs, have been studied in five different reports [13–17]. The results presented in these publications are collected in Table 2. Cardiomyocytes have been obtained with different cardiac differentiation methods in each publication. In our report, we have differentiated hiPSCs into cardiomyocytes using END-2 co-culture method [15]. In addition, HCM hiPSC-derived cardiomyocytes carrying
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Family with HCM –5 pers. with MYH7-Arg663His –5 pers. without mutation (used as control) Timepoints: 20, 30, 40 days |
Cellular enlargement, multinucleation, increased myofibril content, disorganized sarcomeres Upregulation of Elevation of Nuclear translocation of NFAT Ca2+ transient irregularities Elevation of intracellular [Ca2+] Smaller SR Ca2+ release Arrhythmic waveforms including frequent DADs Irregular beating observed in video recordings |
Blockade by cyclosporin A and FK506 reduced hypertrophy Isoproterenol increased cell size and Amount of irregular Ca2+ transients and arrhythmia Isoproterenol together with propanolol abolished Ca2+ abnormalities, arrhytmia and hypertrophy Treatment with verapamil for 5 days ameliorated HCM phenotype Diltiazem abolished Ca2+ abnormalities and arrhythmia |
One patient: Two control hiPSC lines from unrelated donors |
Cellular enlargement, disorganized sarcomeres Disorganized Z lines in TEM Changes in (whole transcriptome sequencing): wnt/β-catenin pathway Notch signaling pathway FGF pathway Nuclear translocation of NFAT Decreased level of Ca2+ transient irregularities elevation of intracellular [Ca2+] Smaller SR Ca2+ release Delayed Ca2+ transient decay time Prolonged and dispersed interspike intervals and increase of arrhythmogenic events in MEA Irregular contractility in real- time cell analyzer APD prolongation Changes in the shape of AP Increased Ca2+, Na+ and outward K+ currents |
Isoproterenol elevated premature beats and irregular beating rates Isopoterenol together with metoprolol decreased beating irregularity and arrhythmia Treatment with verapamil for 4 days reduced arrhythmia and Ca2+ handling abnormalities Antihypertensive drug pinacidil induced irregular interspike intervals Treatment with trichostatin A for 3 days decreased cell size, nuclear translocation of NFAT, suppressed Ca2+ abnormalities and decreased resting [Ca2+] |
Three patients: – –Mutation unknown Two control hiPSC lines from Unrelated donors Timepoints: 30, 60, 90 days culture as EBs |
Mildly but significantly larger cell size No time-dependent changes in cell size Myofibrillar disarray in EM and cTnT staining (>60d, >90d) cTnT and ANP protein levels higher MYBPC level lower in hiPSC-CMs with Mildly disorganized contractile form in video analysis |
Angiotensin II, IGF-1, phenylephrine No difference to non-stimulated cells Endothelin 1 (ET-1) Increased cell size and disarray Nuclear translocation of NFAT Disorganized contraction Similar response in mouse MYBPC+/- ETA-b was able to block ET-1 induced hypertrophic phenotype ETB-b had no effect |
(hiPSC lines published in Dambrot et al. [88] Three patients: –One hiPSC line from each One control hiPSC line from unrelated donor |
Decreased level of MYBPC relative to α-actinin |
Decreased traction stress Decreased traction force No difference in the cell size or contraction frequencies when compared to controls Similar findings in MYBPC knockdown hESC-derived cardiomyocytes |
Four patients: –2 pers. with –2 pers. with Two control hiPSC lines from unrelated donors Timepoints: 1w, 3w, 6w (as single cells) |
Cellular enlargement, multinucleation Upregulation of MYBPC level slightly reduced in Ca2+ transient irregularities Arrhythmic waveforms including frequent EADs and DADs |
Differences in expressions of various cardiac genes More abnormal Ca2+ signals in More prolonged APD in |
Lan et al. published the first report of HCM hiPSC-derived cardiomyocytes in 2013. In their publication, hiPSCs were established via the lentiviral infection of fibroblasts derived from five patients carrying the
In addition to cell size, myofibrillar disarray has been studied in three of the publications. However, in all publications, different methods and criteria have been used to quantify the disarray, and the results can be subjective. Han et al. showed that HCM hiPSC-derived cardiomyocytes have more disrupted sarcomeres than cardiomyocytes derived from control hiPSCs. However, the authors did not present any criteria to determine how the disruption was qualified [14]. In Tanaka et al. [17], cardiomyocytes were qualified with myofibrillar disarray if over 50% of the myofibrils intersected with each other. In Lan et al. [16], cardiomyocytes that had more than 25% of their cell area exhibiting punctate TnT distribution were considered disorganized. The overall morphology of hiPSC-derived cardiomyocytes is not mature, and their structure is often unorganized; this is also the case in cardiomyocytes derived from control hiPSCs. We did not report myofibrillar disarray due to the lack of proper quantitation criteria for this phenomenon.
Electrophysiological properties of hiPSC-derived cardiomyocytes have been studied in three of the publications. In our study, the APD90 was significantly increased in hiPSC-derived cardiomyocytes carrying the
Changes in Ca2+ handling properties are considered to be one of the earliest pathophysiological mechanisms in HCM. In our study, the proportion of cardiomyocytes with abnormal Ca2+ transients was approximately 20% for hiPSC-derived control cardiomyocytes, 42% for cardiomyocytes carrying the
The publication from Birket et al. is to date the only one focused on studying the contraction forces of HCM hiPSC-derived cardiomyocytes. They studied cardiomyocytes derived from three individual patient hiPSC lines carrying
4.2. Variable phenotype of hypertrophic cardiomyopathy
The primary cause of HCM is a mutation in a sarcomeric gene, while changes in Ca2+ handling properties, energy deficiency, ion channel remodeling, and microvascular dysfunction are thought to be the earliest pathophysiological mechanisms that play a role in disease progression [89]. However, the reasons why progressive changes initiated by these primary mutations occur in one individual and not in others are still largely unknown. Many mechanisms related to the variable phenotype of HCM have been proposed, including additional mutations, genetic modifiers, epigenetic factors, environment and function of protein quality systems. These additional disease modifiers are thought to impact on the development of mild HCM phenotype to end-stage, in which the functionality of the heart is disrupted [90]. Conventionally, HCM has been studied with animal models or patient samples obtained from surgical myectomy. However, animal models carry only the mutated gene, and although they contain the entire genome of an HCM patient, myectomy samples are obtained from patients in the late stage of HCM development. While HCM leads to progressive heart failure, the major difficulty in studying the pathophysiological mechanisms leading to HCM has been the lack of tissue at the early stages of disease development. Thus, hiPSC models represent a valuable tool to study HCM in vitro.
In our study, we could not detect the mutated MYBPC protein in hiPSC-derived cardiomyocytes carrying the
Protein phosphorylation is one of the most important post-translational modifications, which has also been suggested to affect disease development. For example, the reduced phosphorylation of TnI and MYBPC has been related to increased myofilament Ca2+ sensitivity, which is a common feature of HCM [95]. However, Ca2+ sensitivity has also been suggested to be a primary consequence of HCM. Additionally, Ca2+ sensitivity has been proposed to increase arrhythmia sensitivity either by increasing the Ca2+ binding affinity in the cytosol, which could lead to remodeling of action potentials and thus trigger arrhythmias, or by affecting energy consumption and increasing arrhythmia susceptibility via stress [90, 96, 97].
Some HCM patients have been shown to carry more than just one mutation in their genotype, and patients carrying multiple mutations have been associated with more severe symptoms or earlier onset of disease [76, 78]. Other genetic mechanisms include genetic modifiers, which can be either near or distantly located DNA variants that influence the expression of the mutated gene [76]. Additionally, epigenetic changes, which cannot be explained by the DNA sequence itself, have been suggested to contribute to the progression of HCM. These mechanisms include the methylation of GpC islands by DNA methyltransferases, histone modification, miRNAs and long non-coding RNAs (lncRNAs), which can lead to the altered regulation of genes [76].
4.3. Challenges in modeling hypertrophic cardiomyopathy with human induced pluripotent stem cells
The major limitation when using hiPSC-derived cardiomyocytes in disease modeling is the immature nature of these cells. The characteristics of hPSC-derived cardiomyocytes and the differences between these and adult human cardiomyocytes have recently been reviewed [98]. Compared to adult cardiomyocytes, the sarcomeric structures of hPSC-derived cardiomyocytes are unorganized, they lack clear T tubules, they have different ion channel profiles, and their Ca2+ handling is immature. In addition, the shapes of hPSC-derived cardiomyocytes vary from circular to star-shaped, while adult human cardiomyocytes are rod-shaped in vivo. Overall, the phenotype of hPSC-derived cardiomyocytes is thought to more closely resemble that of fetal cardiomyocytes than adult cardiomyocytes [98]. At the moment, much effort is being expended to obtain more mature cardiomyocytes. In addition to culture conditions and the long-term culture of cardiomyocytes, other techniques such as electrical and mechanical stimulation, as well as engineered heart tissue (EHT) structures, have been developed [99–102].
Another issue related to the study of HCM with hiPSC-derived cardiomyocytes is related to the assumption that cell types other than cardiomyocytes might be directly involved in the progression of HCM. For example, microvascular dysfunction is thought to be the primary reason for replacement-type fibrosis observed in HCM patients [90]. Thus, it is important to consider whether studying HCM at the single cell level is sufficient to obtain an exact picture of the disease mechanisms. EHT structures and cardiovascular constructs, consisting of various cell types could be useful in this context [102, 103].
As discussed above, in addition to primary HCM gene mutations, gene modifiers and epigenetic changes might also have an effect on disease development and progression. In all published reports, the phenotypes of HCM hiPSC-derived cardiomyocytes have been compared to control hiPSC-derived cardiomyocytes established from related or non-related healthy individuals [13–17]. Currently, useful gene-editing approaches, including clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nuclease (TALEN) techniques, are available, and they can be used either to create mutations or correct existing mutations in an hiPSC line, thus generating genotype-matched isogenic control lines [104]. When considering the variable phenotypes of HCM patients, these isogenic control hiPSCs would be useful when further studying HCM disease mechanisms.
5. Conclusions
Although, the current methods for studying cardiomyopathies in vitro with patient-specific cardiomyocytes are far from optimal, we and others have been able to create hiPSC models with HCM phenotype. hiPSC-derived cardiomyocytes can also be utilized to study the effects of genetic modifiers and epigenetic factors on disease progression between different individuals, which has been difficult when using animal models or samples from surgical myectomy.
However, particularly in the case of HCM associated with highly variable phenotypes, it would be important to optimize cardiac differentiation and cell culture conditions. The study design becomes highly valuable, and the culture conditions should be similar for both control and disease-specific cardiomyocytes. When experiments are thoroughly designed, the results obtained from these studies would be more robust and reliable. Nonetheless, hiPSC-derived HCM in vitro models represent a valuable tool to study the pathophysiological mechanisms of HCM as well as to test novel drug therapies developed to prevent disease progression and potentially optimize treatments in a mutation-specific manner.
Acknowledgments
We thank Anu Hyysalo for image of neural cells and Mostafa Kiamehr for image of hepatocytes. Parts of the text and some of the images have been published earlier in Acta Universitatis Tamperensis series as a part of a PhD thesis.
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