*5.1. Pluripotent Stem Cells in Cardiovascular Disease Modeling*

The use of PSCs to model cardiac disease in vitro has become highly attractive, especially after the introduction of iPSCs [107]. This is mainly because of inadequacies of other models in terms of sampling, propagation, and maintenance, as for human primary cardiomyocytes, or their ability to fully recapitulate physiological properties of human CMs, as in rodent models. Considering the relative difficulty in cloning and genetically modifying human ESCs, most established models of CVDs are iPSC-based [107]. The feasibility in sampling and propagation of iPSCs, as well as advances in reprogramming protocols, which later adopted nonintegrating genomic approaches to deliver the reprogramming factors, has greatly increased their popularity [159]. Patient-specific iPSC-CMs have enabled the study of genetic variants underlying several CVDs and establish a phenotype–genotype understanding of not only monogenic, but rather complex and difficult-to-model genetic variants (e.g., chromosomal deletions or translocations), and, most important, model congenital

heart disease (CHDs) in newborns [160,161]. As a result, several patient-derived iPSCs lines have been developed to model CVDs. The first of such was reported by Carvajal-Vergara et al. in 2010 for LEOPARD syndrome, an autosomal dominant developmental disorder characterized by hypertrophic cardiomyopathy [162]. Ever since, several other cell lines have been reported, mainly modeling cardiac channelopathies, (e.g., long QT syndromes), cardiomyopathies of wide etiology spectrum (e.g., dilated, hypertrophic, arrhythmogenic, Barth syndrome, and Pompe-disease-associated), and infectious myocarditis [163–177].

Despite the previously discussed disparities in structural and electrophysiological characters of iPSC-derived and adult CMs, these studies have shown that patient-specific iPSC-CMs recapitulate their corresponding disease phenotypes. For example, whole-cell patch-clamp analyses of different long QT syndrome (LQTS) patient-derived iPSC-CMs showed typically prolonged APs, decreased rectifier potassium currents IK, increased late sodium currents INaL, and impaired voltage-dependent inactivation of the L-type channels (LTC), due to malfunctions in corresponding proteins of potassium (KCNQ1, KCNH2 in LQTS1 and 2), sodium (SCN5A in LQTS3), and calcium (CaV1.2 in LQTS8 or Timothy syndrome) channels, respectively. Moreover, these patient-specific models demonstrated great utility for pharmacological screening of several drugs with disease-modifying abilities, leading to both novel and/or personalized therapeutic strategies (reviewed in [160]).

Finally, patient-specific iPSC-derived non-CMs were also generated, for example, of SMCs or endothelial cells. A more recent example is an elegant publication by Gu et al., utilizing iPSC-derived endothelial cells from patients with autosomal-dominant mutations in *BMPR2* associated with familial pulmonary arterial hypertension (FPAH) [178]. In their study, comparing symptomatic patients with unaffected carriers highlighted important modifiers of the BMP-receptor pathway, as well as differentially expressed genes, which imparted protection against FPAH. Their findings were of great importance as to the identification of multiple genetic factors affecting disease penetrance, which could be therapeutically targeted to modify disease progression and severity.

Importantly, the previous example behooves an important consideration when conducting studies on patient-specific iPSCs for CVD modeling, which pertains to the identification and/or the availability of proper control lines. This is because, even among patient-matched donor cohorts, genetic variability can still confound the analysis of the disease phenotype, especially in the presence of disease modifiers, or when the genotype–phenotype is less conspicuous [169,179]. In such cases, it is possible to rely on more than one control cell line—albeit a laborious approach. Alternatively, the patient's iPSC-CMs can be compared to those from a healthy sibling, thus limiting genetic variability [171]. However, recently developed computational in silico models of iPSC-CMs and their optimization by Paci and colleagues have provided an unprecedented approach to this issue, enabling simulation and calibration of over a thousand diseased or control iPSC-CM models [180–182]. Finally, in case of monogenetic diseases, an isogenic cell line created by correction of the disease-causing mutation in the patient iPSCs by means of gene-editing approaches can serve as the best control cell line (discussed below). An elegant example was reported in a study by Bellin and colleagues, where they used iPSC-CMs from LQTS2 patients with a distinct mutation in potassium channel KCNH2, and compared it to an isogenic control upon correction of the genetic mutation [183]. Furthermore, they reproduced the study model in human ESC-CMs, where they introduced the same mutation, and recapitulated the disease phenotype, thus generating two genetically distinct isogenic pairs of LQTS2 and control lines.

#### *5.2. Pluripotent Stem Cells in Pharmaceutical Screenings*

Since their first introduction, iPSC-CMs have become attractive for drug testing, antiquating the hERG test, which utilizes cell lines that stably express the human ether-a-go-go-related gene (hERG) *KCNH2* encoding the IKr channel involved in cardiac repolarization. Whole-cell patch-clamp screening for compounds that block the IKr current serves as a good marker of cardiotoxicity, as such blockade leads to the prolongation of the QT interval, i.e., ventricular repolarization, resulting in potentially fatal ventricular tachycardia called Torsade de Pointes [184]. Since the actual risk for cardiac toxicity is not confined to a certain channel and/or mechanism, iPSC-CMs are hence more representative in typifying cardiac toxicity to drugs. Furthermore, recent introduction of automated patch-clamp (APC) devices, all-optical cardiac electrophysiology with novel optogenetic actuation, and video microscopy have all revolutionized drug screening in iPSC-CMs and tissue constructs, enabling high-throughput testing platforms for hundreds of samples and/or drugs, thus creating a wealth of information in short time [185–188]. Furthermore, comprehensive in vitro proarrhythmic Assay (CIPA) has recently emerged as a powerful model to predict cardiac toxicity by integrating the knowledge from both in vitro and recently developed in silico computational models (http://cipaproject.org/about-cipa/) [189]. However, as discussing this is beyond the scope of this review, we refer the reader to the cited work by Paci et al.

#### *5.3. Genetic Modification of Pluripotent Stem Cells*

The advent of genome-editing methods has incited great progress in PSC research. Exploiting the cell's inherent DNA-repair mechanisms, such as nonhomologous end-joining (NHEG) or homologous recombination (HR), has long been used to introduce small but disruptive mutations to target genes, either by insertion or deletions of base pairs, also known as "Indels". The discovery and later advances of nucleases that can more specifically target desired sequences, such as zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), have enabled the study of several disease causing mutations [190–192]. Many PSC-lines have been generated by using this technology for both disease modeling and even clinical applications [193–196]. Vector-mediated delivery of sequence-specific nucleases along with a homologous DNA template to patient-derived iPSCs leads to the excision of targeted locus and, by virtue of cellular homology directed repair (HDR) system, can be corrected by the homologous template with the desired genetic modification. A prominent example is the combination of ZFNs and piggyBac technology which could achieve a biallelic correction of a disease-causing mutation in human iPSCs [197]. In a recent study by Karakikis et al., they reported the use of TALENs to correct gene mutations in patients with hereditary heart failure [198]. These patients harbor an amino acid deletion mutation (R14del) in the coding region of the phospholamban (PLN) gene, which is an important regulator of cardiac calcium cycling in the sarcoplasmic reticulum (SR). They display a phenotype of dilated cardiomyopathy, hypertrophy, episodic ventricular arrhythmia, and overt HF by middle age [199,200]. Skin-derived iPSCs from these patients were isolated, edited, and CM-differentiated, where further analyses showed reversal of the disease's phenotype. Nevertheless, engineering of sequence-specific ZFNs or TALENs, as well as achieving their robust delivery for this purpose, can be laborious and technically challenging, let alone high in cost [192,194,201].

In recent years, CRISPR/Cas9 has emerged as the new horsepower of genome-editing technology, overshadowing ZFNs and TALENs [202]. The system, first described in prokaryotes as part of their adaptive immune system, relies on an RNA-guided endonuclease (Cas9) that localizes to complementary DNA sequences, where it creates double-strand break amenable for correction by the cell's endogenous HR. Provided that a homologous sequence is available, desired gene modifications can be introduced [202]. Indeed, CRISPR/Cas9 has been zealously received by cell biologists as an attractive tool for SC research [203]. In cardiovascular biology, CRISPR/Cas9 was successfully applied to patient-derived iPSC to target disease-causing mutations of CVDs [204–206]. A recent study demonstrated the utility of CRISPR/Cas9 in phenotypic characterization of iPSC-CMs from patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) [207]. In this patient cohort, mutations in the *SCN5A* encoding the Nav1.5 sodium channel protein led to the phenotype, which could be reversed in this study upon editing with CRISPR/Cas9.

A study published earlier this year by Seeger and colleagues made use of genome-editing techniques to create isogenic iPSC lines from patients with heterozygous mutations in the myosin-binding protein C3 (*MYBPC3*), which is deemed as the underlying cause of hypertrophic cardiomyopathy (HCM) [208]. Their results refuted previous hypotheses of either MZBPC3 haploinsufficiency or truncated poison peptide as the underlying cause of HCM. However, they were able to provide evidence for chronic activation of the nonsense-mediated decay (NMD) as the initial pathogenic trigger that leads to dysregulated gene expression and aberrant calcium signaling upon MYBPC3 mutations.

The aforementioned examples give a great promise to SC therapy of CVDs. One might also envisage the possibility of autologous cell transplantation of iPSC-derived CMs with rectified mutations to ameliorate or even cure disease conditions. However, great challenges remain as to the validation of these technologies, let alone deciding on a safe and effective clinical setting for PSCs delivery to treat CVDs. The next chapter outlines recent advances in preclinical research on SC-based therapy for CVDs.

#### **6. Translational Potential of PSCs in Cardiovascular Regenerative Therapy**

Harnessing the multifaceted potential of SCs for effective therapeutic purposes to treat CVDs is the ultimate goal of the above-introduced laborious efforts of scientists over the past decades. Provided that SC-derived CMs are sufficiently propagated, differentiated, and maturated, their application to the diseased myocardium spans a wide spectrum of delivery methods, from intravenous administration to direct myocardial injection. Nevertheless, several factors are to be considered with regard to engraftment of transplanted cells and integration, as well as functional contribution to host myocardium, electromechanical coupling between graft and host CMs, and long-term survival. The aforementioned limitations have long been challenges to preclinical and translational applications of SC therapy in general, and in cardiac regenerative therapy in particular. The following summarizes advances made in the realm of preclinical and translational research with PSCs over the past decades, in light of examples from small and large animal models and up until the first clinical initiatives.

#### *6.1. Pluripotent Stem Cells in Rodent Models*

Earlier studies attempted to engraft human ESC-derived cardiomyocytes in rodent models and reported transient functional improvement in cardiac parameters [209,210]. However, poor engraftment and survival of transplanted cells has been a challenge in these settings. Laflamme et al. utilized pro-survival factor cocktail to limit CM death upon engraftment in infarcted rat heart and reported positive outcomes [108]. To overcome poor engraftment and survival issues, Masumoto and colleagues developed a layered-sheet assembly of three cardiovascular cell populations, namely CMs, endothelial cells, and vascular mural cells, differentiated from mouse ESCs and transplanted into nude-rat model of MI. The transplanted sheets were reported to ameliorate infarct size and improve cardiac function; however, such benefits were shown to be attributed to paracrine-mediated neovascularization and not to actual contribution of transplanted cells [211]. Despite these results, the same group of scientists from Kyoto continued to optimize the stacked-sheet approach, and they recently reported successful long-term survival of engrafted cells through insertion of gelatin hydrogel microspheres between each cardiovascular cell sheet [212]. Analogously, human iPSC-CMs have recently demonstrated favorable therapeutic outcomes when injected in infarcted myocardia of mice. Interestingly, however, the engraftability and survival of those cells depended heavily on the maturation stage [142].

Importantly, electromechanical coupling between the graft and host myocytes is a rather crucial consideration to avoid ventricular arrhythmia. In a Guinea pig model, Shiba et al. reported successful engraftment of human ESC-derived CMs with a 1:1 electrophysiological coupling and improved mechanical function of injured hearts [213].

#### *6.2. Pluripotent Stem Cells in Large-Animal Models*

#### 6.2.1. Porcine Models

Swine models were featured in the earliest attempts of cell therapy for heart disease. A number of features make the pig an attractive translational model. These include a heart weight-to-body ratio that is equal to a human's and a similar sinus rate (~90 bpm) [214]. ESC-derived cardiomyocytes have been functionally tested in a swine model of complete atrioventricular block as biologic pacemaker for the treatment of bradycardia [215]. Hereof, Kehat et al. reported survival and functional integration of the transplanted cells, which were able to pace the porcine ventricle with complete heart block [215]. In another porcine model of acute MI, Ye and colleagues used a mixture of cardiovascular cell populations—this time from human iPSCs origin—loaded on a three-dimensional fibrin patch containing IGF-1 (insulin-like growth factor 1) and reported functional integration and significant improvements of several cardiovascular parameters [216]. More recently, Kawamura et al. took a rather unprecedented approach to enhance survival and engraftment of transplanted human iPSC-CMs by combining cell-sheets with pedicle omentum flap as a source of angiogenic factors and reported enhanced engraftment, survival, and therapeutic outcome in a porcine model of ischemic cardiomyopathy [217,218]. The aforementioned tissue sheet technology from Kyoto was also recently applied to a porcine model of MI, where a heterogeneous mixture of cardiovascular cell populations differentiated from human iPSCs and reported functional restoration of the infarcted hearts and attenuated remodeling [219]. Another study from 2018 by Gao and colleagues reported the application of human iPSC-derived fabricated cardiac muscle patches (hCMPs) composed of CMs, smooth muscle and endothelial cells, reprogramed from cardiac fibroblasts and maturated in dynamic culture conditions [220]. They transplanted these patches in infarcted pig hearts and demonstrated significant improvements upon histological and functional analyses. Altogether, these results highlight the importance of co-administration of nonmyocyte cardiac cells, which provide paracrine and angiogenic support equally important to both host and graft tissue. Furthermore, they highlight the superiority of modern tissue engineered scaffolds over direct application of cells. Finally, these studies corroborate the utility of swine models for translational cardiovascular research.

#### 6.2.2. Non-Human Primate Models

The utility of non-human primates (NHPs) in regenerative medicine has long been appreciated, especially in transplantation medicine [221]. In this regard, certain macaque species (e.g., *Macaca fascicularis* or Mauritian Cynomolgus macaque) are a valuable preclinical model to study allogenic transplantation of iPSCs [222]. This is because they exhibit limited diversity in their major histocompatibility complex (MHC) genes, which are distributed only among seven haplotypes and are structurally identical to those in humans [223,224]. Indeed, allogenic transplantation of iPSC-CM among MHC-matched Cynomolgus monkeys was shown in a study by Shiba et al. to be immune-tolerable, and improved cardiac contractile function upon MI [224]. Matching MHC antigens between donors and recipients was shown by others to reduce immunogenicity upon allogenic transplantation of iPSC-CM in the Cynomolgus macaque [225].

NHPs continue to provide unmatched insights to PSC-therapy of CVDs in late-translational studies. In a recently published elegant work by Chong et al., human ESC-CMs were utilized in the pigtail macaque (*Macaca nemestrina*) as a model of ischemia-reperfusion injury [226]. They reported significant re-muscularization of the infarcted areas, structural and functional integration of the grafted cells via establishment of adherent junctions, and electromechanical coupling, typified by synchronous calcium transients. Such promising results were slightly relegated by the presence of arrhythmia in the grafted animals—albeit nonfatal.

#### **7. Pluripotent Stem Cells in First Human Trials**

The aforementioned successes in late-translational studies with large animal models, as well as the advances made in tissue engineering and grafting techniques, paved the way to the first clinical application of PSC therapy in cardiac settings, which was recently reported in a case study by Menasche et al., using ESC-derived cardiac progenitors [227]. They used the ESC I6 line, which was enriched in vitro by culturing on clinical-grade irradiated human foreskin fibroblasts as feeder cells. Cardiac commitment was then achieved by bone morphogenic protein-2 (BMP-2) and a specific tyrosine kinase inhibitor of the fibroblast growth factor receptor (FGFR), and then confirmed by the

expression of the cardiac specific transcription factor Isl-1, as well as the stage-specific embryonic antigen-1 (SSEA-1), which was used for cell purification by immunomagnetic sorting. The cells were embedded in a fibrin scaffold patch and surgically implanted in the infarcted area of a 68-year-old woman patient with severe heart failure. The three-month follow-up showed functional integration of the patch, evident by electrocardiography, and overall symptomatic improvement marked by enhanced left-ventricular ejection fraction (LVEF), with no complications of arrhythmia, tumors, or immunosuppression-related adverse events. The results were encouraging, and the Parisian group conducted a larger-scale study, wherein six patients received cellularized patches of ESC-derived committed cardiac progenitors [228]. Their one-year follow-up demonstrated safety and tolerability of the grafted cells, with no detected tumors. Moreover, they reported modest symptomatic improvements, as well as in different cardiac parameters.

As for iPSCs, a group of scientists from Osaka have reported their granted permission to pursue with their clinical application last year and the results are yet to be reported [229].

#### **8. Conclusions and Remarks**

Stem cells are a novel source of cells which might be used as a screening tool for pharmaceutical developments. Here, single cells on iPSC status, as well as differentiated cardiomyocyte progenitors, might be used. In addition, engineered heart tissue displays a second model situation for screening of novel therapeutic options, before applying in animal experiments or clinical trials. Since these systems are based on human sources of cells, testing in these model situations might enhance safety and side effect prediction in novel approaches of cardiovascular therapies. Utilization of stem cells in patients suffering from cardiovascular disease is a second interesting field with great potential. The allogenic transplantation of stem cells requires only a modest immunosuppression and might improve cardiac function and thereby survival, as well as quality of life, in patients suffering from cardiac conditions, e.g., heart failure. However, the clinical potentials, as well as the potential side effects, need to be investigated in clinical trials before establishing stem-cell-based therapy as a standard of care in cardiovascular patients. In summary, stem cells, especially induced pluripotent stem cells, have wide therapeutic potential, but need to be characterized and investigated in more detail in preclinical, as well as clinical, trials to understand in more detail their potentials and risks.

**Author Contributions:** M.S., writing—original draft and visualization; R.H., writing—review and editing.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflicts of interest.

### **Abbreviations**


#### **References**

1. WHO. Cardiovascular Diseases (CVDs). Available online: http://www.who.int/en/news-room/fact-sheets/ detail/cardiovascular-diseases- (accessed on 1 June 2019).


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*
