Arrhythmogenic Risks of Stem Cell Replacement Therapy for Cardiovascular Diseases

Abstract

Ischemic heart disease and congestive heart failure are major contributors to high morbidity and mortality. Approximately 1.5 million cases of myocardial infarction (MI) occur annually in the United States; the yearly incidence rate is approximately 600 cases per 100,000 people. Although significant progress to improve survival rate has been made by medications and implantable medical devices, damaged cardiomyocytes are unable to be recovered by current treatment strategies. After almost two decades of research, stem cell therapy has become a very promising approach to generate new cardiomyocytes and enhance function of heart. Along with clinical trials with stem cells conducted in cardiac regeneration, concerns regarding safety and potential risks have emerged. One of the contentious issues is electrical dysfunctions of cardiomyocytes and cardiac arrhythmia after stem cell therapy. In this review, we focus on the cell sources currently used for stem cell therapy and discuss related arrhythmogenic risk.

Introduction

Most functional cardiomyocytes in the adult heart are terminally differentiated and are unable to re-enter the cell cycle for regeneration. Hence, despite the regenerative capacity of cardiac progenitor cells in neonatal heart, damage to mature cardiomyocytes from ischemic injury, such as myocardial infarction (MI), is irreversible in adult heart and eventually leads to progressive heart failure. Traditional medications and therapies with implantable devices only slow down the progress of diseases, such as ischemic heart disease and chronic heart failure, but are unable to repair the damaged heart. Heart transplantation faces the challenge of insufficient donors and severe side effects developed from immunosuppressive drugs, which restrict the wide adoption for heart transplantation. Stem cell therapy is emerging as an exciting therapeutic approach to strengthen cardiac function, either by introducing new cardiomyocytes or providing niche signals for recovery of damaged heart muscle through paracrine, angiogenic, and anti-apoptotic effects (Dimmeler et al., 2005; Galdos et al., 2017; Laflamme and Murry, 2005; Murry et al., 2005; Narmoneva et al., 2004). Results from basic research and initial clinical trials indicate that stem cell therapies improve contractility and left ventricular ejection fraction (LVEF) by harmonizing cellular niche of the local infarcted area, cardiomyocyte generation, and vascular regeneration, which enhance heart function and prognosis to certain extent.

Based on the rationale that the myocardial function would be improved by repopulating the damaged areas with new functional cells, a variety of cell types used for cardiac regenerative therapy have been characterized in two major categories (Figure. 1): pluripotent stem cells (PSCs), and adult stem and progenitor cells. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced-pluripotent stem cells (iPSCs), are capable of self-renewal and differentiation into all cell types of the three germ layers. On the other hand, adult stem cells, which have the capability of self-renewal and differentiation into limited cell types, are characterized as the cells existing in tissues or organs. Various autologous adult stem and progenitor cells have been utilized in heart regenerative studies, such as mesenchymal stem cells (MSCs), cardiac progenitor cells (CPCs), and skeletal myoblasts (SkMs). In addition, induced cardiomyocytes (iCMs) by direct reprogramming provide an alternative approach to generate cardiomyocytes. Achieving effective integration with host cells and establishing effective myocardial electrical conduction are the key requirements of stem cell therapy. To better understand the status quo of stem cell therapy in the treatment of cardiovascular disease, we would like to review the cell types for cardiac transplantation and discuss related challenges of the cell therapy that may cause arrhythmia and suggest some solutions to them.

Figure 1. Types of cells being used for cardiac cell therapy.

iPSC, induced-pluripotent stem cells; ESC, embryonic stem cells; MSC, mesenchymal stem cell; SkM, skeletal myoblasts; CPC, cardiac progenitor cell; iCM, induced cardiomyocyte.

Cell types used for cell therapy of cardiac regeneration

1. Human embryonic stem cells (hESCs)

ESCs are isolated from inner cell mass of blastocyst stage of embryos. They are capable of self-renewal with high regenerative potential and pluripotency to differentiate into all the cell types present in three germ layers, including cardiomyocytes. Because of lacking sufficient developmental cues to direct ESC differentiation in vivo, implantation of undifferentiated ESCs forms teratomas with limited lineage-specific cells. De novo cardiomyocyte generation from hESCs have three major steps: mesodermal-cardiac lineage differentiation, subtype specification, and maturation of cardiomyocytes. hESC-derived cardiomyocytes (hESC-CMs) are good cell sources for cardiac regeneration. After transplantation into infarcted hearts of animal models, they improve cardiac function (Caspi et al., 2007; Laflamme et al., 2007; Liu et al., 2018; Shiba et al., 2012; Shiba et al., 2016; van Laake et al., 2007). Because they express many genes of the ion-channels, gap-junctions and cardiac muscles similar to adult cardiomyocytes (He et al., 2003; Jonsson et al., 2010; Kehat et al., 2001a; Kehat et al., 2001b), hESC-CMs is a promising cell type for myocardial replacement. In addition to ethical issues, immune compatibility, and teratoma formation, a major risk of using hESC-CMs in the current clinical setting is immature electrophysiologic characteristics-associated arrhythmogenicity (Chong et al., 2014; He et al., 2003; Liao et al., 2010; Mummery et al., 2003; Shiba et al., 2016). For example, hESC-CMs give a prolonged action potential (AP) delay; larger maximal diastolic potential (MDP) of depolarization and resting potential; irregular gap junction alignment; and altered spontaneous action potential in pacemaker cells; and increased mortality resulted from occurrence of ventricular arrhythmia(He et al., 2003; Liao et al., 2010; Mummery et al., 2003). A recent study of the non-human primate Macaca nemestrina showed that hESC-CMs provide robust and durable improvement in cardiac function in the infarcted heart, and graft-induced arrhythmias result from pacemaker-like activity rather than abnormal conduction in macaque monkeys (Liu et al., 2018).

2. Human induced pluripotent stem cells (hiPSCs)

Similar to hESCs, hiPSCs have the capability of self-renewal and differentiation into all cells of the three germ layers. In 2006, Takahashi et al. first reported that mouse skin fibroblasts can be reprogrammed into pluripotent stem cells, by ectopic-expression of sex-determinacy region protein 2 (Sox2), octamer-binding transcription factor 4 (Oct4), Kruppel like factor 4 (Klf4) and master regulator of cell cycle entry and proliferative metabolism (cMyc) genes (Takahashi and Yamanaka, 2006). Subsequently, hiPSCs are successfully generated from human fibroblasts by reprogramming(Takahashi et al., 2007). It has been reported that hiPSC-CMs displayed similarities as hESC-CMs in molecular expression, structured morphology and contractility (Mauritz et al., 2008; Shiba et al., 2016). The generation of patient-specific iPSCs by reprogramming technology is an exciting field because hiPSCs have wider applications and less ethical concerns. In addition to cardiac regeneration, cardiomyocyte subtypes (atrial, ventricular and pacemaker) are important for cardiotoxicity testing, drug screening, drug validation and electrophysiology applications, such as a model of atrial fibrillation, a common type of arrhythmia. Human iPSC-CMs spontaneously beat in vitro and are characterized by the expression of cardiac-specific markers, including cardiac troponin T (cTnT). Atrial cardiomyocytes derive from hiPSCs express Troponin T, atrial natriuretic peptide (ANP) and atrial myosin light chain 2 (MLC2a), whereas human iPSC-derived ventricular cardiomyocytes express sarcomeric α-actinin and β-myosin heavy chain (β-MHC) (Lee et al., 2017). Whether hESC-CM and hiPSC-CMs proved a long-term improvement of cardiac function in infarcted hearts may depend on engraftment contribution of mature cardiomyocytes(Liu et al., 2018) or indirect paracrine effects (Tachibana et al., 2017; Zhu et al., 2018). Electrical mapping studies showed that instead of reentrant pathway, impulse generation in the graft region from either pacemaking or depolarizations may be the underlying cause of hESC-CM-induced arrhythmias (Liu et al., 2018). Further study to decrease susceptibility to pacing-induced arrhythmias should improve clinical outcomes.

3). Mesenchymal stem cells (MSCs)

Mesenchymal stem cells (or multipotent stromal cells) were initially found in the bone marrow, and later discovered in various tissues, such as adipose tissue, umbilical cord, and umbilical cord blood (UCB) (da Silva Meirelles et al., 2006). Since the discovery, more than 20,000 papers have been published on the subject of MSC biology and their clinical applications, including treatment of cardiovascular diseases (Ji et al., 2017). The primary feature of MSCs is their expression of a cluster markers: CD90, CD105, CD44, CD106, CD166, CD73 and CD29 while not expressing CD45, CD34, CD14, CD11b, CD19 and CD31 (Dominici et al., 2006). Due to tissue specificity and isolation-culture protocols, subpopulations of MSCs have been described with the potential heterogeneity (Blazquez-Martinez et al., 2014; D’Ippolito et al., 2004; Jiang et al., 2002a; Jiang et al., 2002b; Rossini et al., 2011; Varma et al., 2007; Yoon et al., 2005). MSCs have a broad differentiation potential and are capable of differentiation into bone (osteocytes), cartilage (chondrocytes), adipocytes, endothelial cells, cardiomyocytes, β-pancreatic islets cells and hepatocytes. MSCs are considered an attractive option for cell therapy because of their autologous cell sources (bone marrow and adipose tissue). One of the most striking features of MSCs is their low immunogenicity due to the absent expression of molecules involved in allogeneic tissue rejection, such as major histocompatibility complex class (MHC) I and class II (HLA-DR), CD40 ligand and CD80/86 (Berglund et al., 2017; Le Blanc et al., 2003; Majumdar et al., 2003). Because of their immune-privilege potential, allogeneic MSCs can be used as “off the shelf” products from umbilical cord or and umbilical cord blood (UCB).

Preclinical studies of MSCs in animal models indicated that transplantation of MSCs helps cardiac function recovery after myocardial infarction (Quevedo et al., 2009; Wang et al., 2011). Although MSCs are capable of differentiation into cardiomyocytes in vitro and in vivo, the level of direct MSC contribution to cardiomyocyte regeneration is low (Hare et al., 2012; Makino et al., 1999; Pei et al., 2017; Quevedo et al., 2009; Toma et al., 2002). Many clinical trials have been conducted for MSCs to treat cardiac disease (based on studies found in clinicaltrials.gov website). Future application of MSC therapy for ischemic heart diseases is waiting for convincing results of phase-III clinical trial. Study of electrophysiological behavior of BM-MSCs indicated that MSCs have a −40mV to −30mV resting membrane potential(Heubach et al., 2004). They also have delayed rectifier channels, and express particular L-type Ca²⁺ channel, but do not express Na⁺ and inward rectifier K⁺ channel(Heubach et al., 2004). When MSC co-culturing with ventricular myocytes in vitro, gap junction mediated through Cx43 are formed between MSCs and ventricular myocytes, resulting in electrical coupling of MSCs with myocytes (Chang et al., 2006; Valiunas et al., 2004). Compared to SkMs, MSCs preserved electrical viability and impulse propagation in the border zone and reduced the risk of arrhythmia after transplantation in following MI(Mills et al., 2007), suggesting that MSC therapy is less proarrhythmic and electrically more stable transplantation than of SkMs. It is interesting to note that slowed conduction has been observed at the edge of infarction. Despite gap junction formation between MSCs and myocytes, most MSCs maintained their state of undifferentiation and low excitability (Mills et al., 2007). The cause of low level of ventricular arrhythmic rate after MSC engraftment in myocardium following MI presumably is because of increased tissue heterogeneity by unexcitable MSCs ^{(Askar et al., 2013; Wang et al., 2011)}. Mechanistically, MSC therapy to improve cardiac function is not a direct contribution of new cardiomyocyte generation. The beneficial effects of MSCs on cardiac function probably is due to their immunomodulatory properties to provide paracrine factors to protect cardiovascular cell survivial and/or improve different pathological conditions (Beigi et al., 2013; Cao et al., 2005; Chung et al., 2015; Mirotsou et al., 2007; Premer et al., 2015; Suzuki et al., 2011). Compared to other hMSCs, Type C hMSCs do not have hEAG1 activity and hence have lower vulnerability to re-entry as compared to those hMSCs that show hEAG1 activity. So injecting Type C hMSCs might be a better option to improve chances of successful cardiac cell therapy (Mayourian et al., 2016).

4). Cardiac progenitor cells (CPCs)

The adult heart is traditionally considered as a terminally differentiated organ, which is deprived of cardiomyocyte regeneration capability after MI and increases cardiac load by myocardial thickening as a measure of compensation. Studies of animal models indicated that c-kit⁺ cells isolated from bone marrow or adult heart give rise to new muscle tissue after transplantation into damaged myocardium, suggesting that endogenous cardiac progenitor cells (CPCs) may exist and have regenerative potential (Beltrami et al., 2003; Bergmann et al., 2009; Strauer et al., 2002). CPCs can be assessed for their multipotency based on side population and presence of surface antigens like c-kit, stem cells surface antigen-1 (sca-1), Islet1 and Flk1. The idea of resident CPCs in adult heart is controversial and debatable. One of the first human trials, a phase I randomized trial of autologous c-kit⁺ CPCs in ischemic heart failure indicated that CPC-treated patients showed improvements in ejection fraction and a reduction in infarct size at four months post-infusion (Chugh et al., 2012). However, several studies demonstrated that c-kit⁺ cells in the heart are unable to contribute to new cardiomyocytes ^{(Molkentin, 2014; van Berlo et al., 2014; van Berlo and Molkentin, 2016)}. Recently, two groups of researches reported that sca-1⁺ cells rarely became cardiomyocytes and instead become endothelial cells in the heart ^{(Neidig et al., 2018; Zhang et al., 2018)}. It is possible that these c-kit⁺ cells or sca-1⁺ cells secrete paracrine factors that help repair injured heart cells, a similar mechanism underlying MSC effect on improvement of cardiac function.

5). Skeletal myoblasts (SkMs)

Skeletal myoblasts (SkMs) are progenitor cells existing in skeletal muscles, and they have key functions in promoting growth, repair and maintenance of adult skeletal muscles. Because these cells are of autograft origin, easy to extract and multiply, lack carcinogenic risks, and show wide tolerance to ischemia and excellent intrinsic contractility characteristics, autologous SkMs were the earliest candidates in clinical studies for myocardial repair (Menasche et al., 2003; Smits et al., 2003). It has been demonstrated that SkMs partially survive after being transplanted into infracted myocardial tissue and improve LV function (inhibit LV dilation, increase contractility and EF) and reverse LV remodeling (Saito et al., 2012; Shirasaka et al., 2016). SkMs ectopically expressed either serine/threonine kinase 1 (Akt-1) or placental growth factor significantly enhance cardiac function and ventricular remodeling in infarcted heart (Gmeiner et al., 2011; Siepe et al., 2011). Despite their effect on cardiac repair after transplantation, however, SkMs have limited potential to differentiate into cardiomyocytes (Reinecke et al., 2002), suggesting their indirect paracrine effects. Results from an animal study (Siepe et al., 2005) and a human study of 70 patients (Menasche, 2005) showed that SkMs have the longer survival potential after integration into host cardiomyocytes, although they cannot differentiate into cardiomyocytes, and they do not form Cx43⁺ gap junctions nor electrical coupling with host cardiomyocytes. Their skeletal muscle-like phenotype electrophysiologically is different from host cardiomyocytes, resulting in variations in dispersion and increased arrhythmogenic risks. Mills et al.(Mills et al., 2007) systematically evaluated electrophysiological characteristics and arrhythmogenic risks in post-MI mice after SkM therapy. Compared to untreated control, the SkM transplantation results in lower electrical stability at the infarcted area and marginal tissues and decreased amplitude of action potential in marginal areas, leading to ventricular arrhythmia (Mills et al., 2007). Due to lack of gap junctions between host cardiomyocytes and transplanted myoblasts, the improvement of cardiac function is not caused by electromechanical coupling between grafted and host cells (Leobon et al., 2003).

6). Cardiomyocytes from direct reprogramming (iCMs)

After MI, the injury site in the heart forms a scar largely comprised of fibroblasts. Recently, the concept of reprogramming of fibroblasts into induced cardiomyocytes (iCMs) has been introduced (Qian et al., 2012; Song et al., 2012). More than 30 years ago, Davis et al. found that fibroblasts could be reprogrammed to skeletal muscle by ectopic expression of MyoD transcription factor (Davis et al., 1987). In research for cardiac master transcription factors, Takeuchi et al. demonstrated that ectopic expression of two transcription factors, Gata4 and Tbx5, and along with Baf60c, in mouse mesoderm induces cardiomyocyte generation (Takeuchi and Bruneau, 2009). Further studies showed that 3 transcription factors, Gata4, Mef2c, and Tbx5 (GMT), efficiently reprogram neonatal cardiac fibroblasts into induced-cardiomyocytes (iCMs).(Ieda et al., 2010) However, some of those iCMs have a defect of beating spontaneously because of lack of expression of cardiac troponin T (cTnT). Other transcriptional factors have been used to generate functional iCMs, which includes Hand2, MyoD, Nkx2.5, Myocd, Mesp1, Ets2 and ESRRG (Addis et al., 2013; Fu et al., 2013; Hirai et al., 2013; Islas et al., 2012; Nam et al., 2014; Protze et al., 2012; Wada et al., 2013). Several microRNAs (miRNAs), such as miRNA-133, miRNA-208, and miRNA-499, and miRNA-590, have reprogramming effect on iCM generation (Liu et al., 2008; Muraoka et al., 2014; Rao et al., 2009; Singh et al., 2016). Small-molecule compounds that regulate signaling pathways provide a non-integrative means to induce direct reprogramming. A combination of CHIR99021 (WNT signaling activator), ascorbic acid, forskolin, valproic acid, A83–01 or RepSox (TGF-β inhibitor), BIX01294 (methyltransferase inhibitor), AS8351 (histone demethylase inhibitor), SC1 (RK2 and Ras-GAP inhibitor), OAC2 (Oct4 activator), Y27632 (ROCK inhibitor), SU16F (PDGFRβ inhibitor), and JNJ10198409 (PDGF-BB inhibitor) has been reported to reprogram fibroblasts into iCM in vitro and in vivo (Cao et al., 2016; Fu et al., 2015; Huang et al., 2018). The approach of direct fibroblast-to-cardiomyocyte reprogramming has a great potential for in situ repair of heart after MI specially when the efficiency of reprogramming is increased (Qian et al., 2012; Song et al., 2012). The electrophysiological properties, gap junction formation, ion channel expression in iCMs, and their functional integration into injury hearts need to be further studied.

Mechanisms behind arrhythmias

Because of the nature to introduce exogenous cells into injury hearts, stem cell therapy for cardiac regeneration could lead to possible complications associated with challenges of preventing immune rejection, maintaining cell viability in vivo, retention as well as structural and functional integration of cells in the desired location. The cell therapy-related arrhythmic potential has arisen from cardiac electrical competence: 1) intrinsic electrophysiological properties of transplanted cells, 2) modulated graft-host electromechanical coupling, 3) alterations in ion channel function, 4) induced heterogeneity in cardiac tissue, and 5) altered myocardial tissue architecture and cardiac function (Ly and Nattel, 2009). The mechanisms behind these phenomena hold the key to resolving the arrhythmogenic risks following stem cell transplantation. Currently, there are three major known arrhythmia-causing mechanisms: 1) re-entry; 2) triggered activity; and 3) increased automaticity (Gaztañaga et al., 2012). These arrhythmogenic mechanisms are resulted from various abnormalities:

1. Gap-junction imperfection:

Electrical coupling between cardiomyocytes is critical for cardiac tissue excitation. Rapid spreading of intra-myocardial signals achieves synchronized contraction. Electrical coupling is based on the gap junction formed mainly by connexin Cx43 (Peters et al., 1995). Normal electrical conduction can only be ensured by effective formation of gap junctions among transplanted cells to improve contractility between graft and host myocardial cells. The lack of or loss of Cx43 is associated with diminished electrical coupling, functional conduction delay, re-entry induction and formation of arrhythmias (de Diego et al., 2008; de Groot et al., 2003; Procida et al., 2009). A reduced intercellular Cx43 coupling causes increased heterogeneity of repolarization and can contribute to a pro-arrhythmic substrate (Wiegerinck et al., 2008). Cx43 is expressed in hESC-CMs and hiPSC-CMs, leading to a good integration with host myocytes and improvement of marginal excitability of infarcted areas (Germanguz et al., 2011; He et al., 2003; Jonsson et al., 2010; Kehat et al., 2001a; Kehat et al., 2001b). Since Cx43⁺ gap junctions between SkMs and host cardiomyocytes are not detected in injured hearts after transplantation of SkMs, they are unable to form any electromechanical junctions with the native myocardium, resulting in slowed conduction and induced arrhythmia after SkM transplantation(Reinecke et al., 2000). Advancing electromechanical coupling by hyperthermia or mechanically preconditioned by engineered tissue constructs may improve Cx43 expression in SkMs and couple to host cardiomyocytes(Antanavičiūtė et al., 2014; Treskes et al., 2015).

2. Low cardiac excitability of transplanted cells:

Cardiac excitability generates an action potential (AP) at cardiomyocytes membrane in response to depolarization and to transmit an impulse along the membrane. Ion channels play an important role in regulating excitation-contraction coupling. Alteration of sodium (Na⁺), potassium (K⁺), and calcium (Ca2⁺) channels has been detected in heart failure (Nattel et al., 2007). Effective electrical conduction and AP spreading are crucial for ventricular synchronous contraction, which requires valid transfer of electrical charges to the excitable myocardial tissues during AP depolarization. If the ion channels responsible for charge displacement have low density in the transplanted cells, the myocardial cells will have low excitability and lead to invalid depolarization in transplanted cells, resulting in functional conduction blocks. Low excitability of stem cells will cause conduction block. In addition, transplanted cells may express paracrine factors to alter impulse conduction and ion channel expression of host cardiomyocytes (Pedrotty et al., 2009). Evaluation of ion channel distribution in cells to be transplanted can help to identify which cells are functionally capable of transplantation.

3. Electrophysiological heterogeneity:

Bioelectrical heterogeneity is considered the main cause of lethal arrhythmia. Normal AP duration (APD) of ventricular cardiomyocytes consists of five distinct phases (Figure 2). Phase 2 is typically a plateau stage in which the membrane potential remaining almost constant (the near balance of charge moving into and out of the cell). Abnormal APD, especially due to dispersion or increase of repolarization, generates arrhythmia. When the transplanted cells have a different APD from host myocardial cells, it will result in arrhythmia. Cardiac differentiation of hiPSCs predominantly generates ventricular-like hiPSC-CMs (Burridge et al., 2014; Lian et al., 2012), which have relatively shorter APD than adult ventricular cardiomyocytes. Therefore, transplanted hiPSC-CMs enter the excitable period while the host myocardial cells are still refractory, resulting in local conduction is disrupted leading to ventricular fibrillation (Blazeski et al., 2012; Ma et al., 2011). In the case of transplanted SkMs that develop into matured myotubes with shorter (20ms) and higher amplitude AP, their mismatching with the host myocardial AP also results in higher possibility of inducing ventricular fibrillation (Fernandes et al., 2006). Because different sources of hMSCs and their isolation/culture methods, the cardiac function improvements of transplanted hMSCs are inconsistent. A computational modeling of electrical interactions between hMSCs and cardiomyocytes can be used to predict possible arrhythmogenic effects on APD, conduction velocity, and increased vulnerability to re-entry(Mayourian et al., 2016), therefore, predict the quality of hMSCs.

Figure 2.

AP and ionic currents in pacemakers and venticular cardiomyocytes

4. Enhanced automaticity:

Automaticity is an intrinsic property of all myocardial cells. This occurrence of spontaneous activity is normally prevented by the natural hierarchy of pacemaker function (Figure 3). After engraftment, the normal regular spontaneous sinus rhythm can be lost because the transplanted cells have a higher spontaneous activity than the original endogenous pacemaker cells, leading to abnormal automaticity and ventricular arrhythmias (Almeida et al., 2015; Chong et al., 2014; Shiba et al., 2016; Smit and Coronel, 2014). Compared to small animal models (mice and rats that have higher heart rates) (Laflamme et al., 2007; Shiba et al., 2012), large animals (non-human primates and swines) may provide a better model system to predict human response to hiPSC-CM transplantation to restore cardiac function in infarcted heart (Chong et al., 2014; Liu et al., 2018). Although cardiomyocytes generated from hiPSCs or hESCs are heterogeneous, subtype-specific cardiomyocytes, such as atrial-like(Devalla et al., 2015), ventricular-like(Kehat et al., 2004), nodal-like(Protze et al., 2017) and pacemaker-like(Chauveau et al., 2017) hPSC-CMs have been described. Further research on method development to generate subtype-specific cardiac cells from hiPSCs may lead to the discovery of specific functional cardiomyocytes based on electrophysiological characteristics. For example, ventricular-like cells are transplanted into infarcted myocardial region to improve contractility and promote cardiac regeneration, whereas nodal-like or pacemaker-like cells can be used for the treatment of rhythm disorders or as a biologic alternative to electronic pacemakers to replace implantable pacing devices (Jung et al., 2014). Furthermore, ischemic-induced microenvironmental changes may contribute to abnormal automaticity. Compared to ESC-CM co-culturing with MSCs, ESC-CM co-culturing with adult cardiac fibroblasts alter electrophysiology of ESC-CMs, potentially inducing abnormal automaticity and proarrhythmic changes (Trieschmann et al., 2016). The paracrine factors from adult cardiac fibroblasts contribute to proarrhythmic changes of ESC-CMs (Trieschmann et al., 2016). Therefore, to avoid arrythmias post-transplantion, dissociated PSC-CMs should not be transplanted into scars that are rich in fibroblasts (Trieschmann et al., 2016). Optimal transplant route/time and site should be chosen according to the specific disease.

Figure 3.

Nature Hierarchy of Pacemakers

5. Problems with transplantation techniques:

Direct intramyocardial injection may elicit transient inflammation and in the long term could cause injection route fibrosis, leading to conduction anomalies or trigger other harmful activities around this area. Injection of a large quantity of cells via the coronary artery might cause myocardial edema. These technical factors can increase the occurrence of arrhythmia and hence the methods of transplantation need to be evaluated. Gerbin et al. compared two methods of engineered cardiac tissue delivery and found that contrary to the intramyocardial injection of micro-tissue particles, the engineered cardiac tissue patch of hESC-CMs implanted on the epicardium cannot form gap junctions and is not electrically coupled with the cells of host tissue, whereas intramyocardial injection of micro-tissue particles of hESC-CMs and suspended hESC-CMs results in electrical coupling to the host heart at spontaneous rate (Gerbin et al., 2015).

Conclusion

After decades of incessant research, multiple types of stem/progenitor cells have been utilized to treat heart diseases, and the outcomes are reassuring, some even having entered late clinical trials. There are many challenges to be overcome (Table 1) in order to achieve a significant clinical benefit. Arrhythmogenicity associated with stem cell therapy is one of the challenges. No matter which type of stem cells, their potential and predictable arrhythmogenic risks should be thoroughly evaluated and analyzed before they are applied in clinical settings. Electrophysiological changes in transplanted cells can be induced to prevent arrhythmia (Gnecchi et al., 2017). Sophisticated projects like stem cell transplantation need to integrate the knowledge of cell biologists and medical professionals to bring the stem cell therapy into a new era to relieve the pain of the suffering.

Table 1.

Challenges of Cariac Cell Therapies and Solutions to Overcome

CHALLENGES

SOLUTIONS

Ineffective gap junction formation among transplanted cells can impair electrical coupling Low excitability of stem cells can cause conduction block Shorter AP duration than of normal adult cardiomyocytes leads to biological heterogeneity and cause VF Presence of cells other than iCMs in culture Transplantation techniques may cause complications like myocardial edema, imflammation and injection route fibrosis

Evaluation of electrophysiological properties of cells to be tranplanted Genetic modifications and pre-treatments to control expression level of gap junctions and ion channel Develop methods and programs to predict APD, conduction velocity and vulnerability to re-entry Segregate and purify the cells before transplantation Introduce paracine factors to change electrophysiological properties

AP, action potential; VF, ventral fibrilation; iCMs, induced cardiomyoctes; APD, action potential duration