**2. Adult Stem Cells**

The body's regenerative capacity is a well-ingrained piece of knowledge from ancient times. Modern science attributes this phenomenon to the presence of resident SC niches in different organs and tissue, i.e., adult SCs. These cells are undifferentiated, but they are capable of self-renewal and differentiation to one or more cell type, which sets them apart on a potency spectrum, e.g., multipotent SCs. Adult SCs' regenerative potential becomes even more conspicuous in organs and/or tissues with high turnover rates, but, more importantly, as a response to tissue injury. A wealth of knowledge is now available on different adult SC populations, and efforts have been made to reap the benefits of these cells to treat CVDs. We highlight below a few examples of adult SCs, which declared themselves as powerful research targets for cardiovascular medicine and made their way to the clinic.

#### *2.1. Skeletal Myoblasts*

Intuitively, due to embryonic and morphologic commonalities between skeletal and cardiac muscle tissues, skeletal myoblasts have been among the early attractive research targets for cardiac regeneration. Skeletal myoblasts (SM) constitute a group of satellite cell-derivatives residing within skeletal muscle fibers, which are activated upon injurious insults to migrate, proliferate, and differentiate, forming new muscle fibers, i.e., myogenesis [11]. Facilitated by their being readily accessible from autologous muscle biopsies, rapid in vitro expansion, ischemic tolerability, and low risk of tumorigenicity, the cardiac regenerative potential of SMs has been the subject of several preclinical investigations in both small and large animal models of CVDs [12–17]. Indeed, results from these studies have demonstrated positive outcomes by reducing infarct size, as well as myocardial fibrosis, thwarting ventricular remodeling and improving overall cardiac function. Consequently, several clinical trials were initiated to verify their efficacy [18–22]. Despite initially reported improvements in cardiac parameters of patients transplanted with SMs, many have experienced ventricular arrhythmias, which were later attributed to the lack of electromechanical coupling between the transplanted SM-derived myotubules and resident cardiomyocytes where they failed to form gap junctions [23–26]. Furthermore, larger randomized, placebo-controlled, double-blinded clinical studies not only failed to show any therapeutic benefits of SMs in patients with severe ischemic heart disease at both short- and/or long-term follow-up, but also reported postoperative arrhythmic events even upon prophylactic pharmacological treatment [27–31]. As a result, SMs have lost their popularity as SCs for cardiac applications.

#### *2.2. Bone-Marrow-Derived SCs*

Since the mid-20th century, the BM has long been praised for its SC abundance. BM transplantation has been a clinical practice since the mid to late 1960s, intended for correction hematologic, as well as immune disorders. However, reports from the late 1990s first demonstrating the ability of BM-derived cells to migrate to injured tissues and support regeneration have instigated a wave of research on their therapeutic potentials for CVDs [32,33]. Indeed, early studies in animal models of MI corroborated the aforementioned expectations. The first tentative clinical translation of this finding was reported in 2001 in Düsseldorf, Germany, where a MI patient received autologous BM-derived nucleated cells upon catheter angioplasty and reported positive outcomes [34]. This was followed by several controlled clinical studies, albeit with inconsistent findings [35,36].

Generally speaking, BM-derived SCs can be sub-grouped into two large cell populations; hematopoietic (HSCs) and nonhematopoietic SCs. HSCs give rise to all blood-cell types and include a subpopulation of pro-vasculogenic endothelial progenitor cells (EPCs), which can be found in the circulating blood among others [37]. Of the nonhematopoietic BM-derived SCs, mesenchymal stromal/stem cells (MSCs) are the most studied, due to their greater multipotency, manifested in their ability to differentiate into osteoblasts, adipocyte, and chondrocytes under defined in vitro conditions, adding to their reported immune-modulatory and anti-inflammatory properties [38]. With better characterization of these cells based on surface-marker expression, studies were led, examining the therapeutic potential of each BM-derived SC type.

For example, BM-derived CD133- and/or CD34-positive HSCs were utilized for phase I and II clinical trials, where patients of MI received intramyocardial transplantation or intracoronary injections of these cells. Despite short-term follow-ups showing positive outcomes, characterized by enhanced left-ventricular ejection fraction (LVEF) along enhanced myocardial perfusion, these studies failed to show any long-term benefits [39,40]. Most recently, results from randomized, placebo-controlled, double-blinded phase III clinical trials also showed a congruent trajectory [41].

On the other hand, MSCs (CD73-, CD105-, and CD90-positive) have been a subject of greater scrutiny in both basic and translational research. Adding to their paracrine- and exosome-mediated immunosuppressive properties, MSCs are unique in their ability to evade the immune system [42]. This is largely due to their moderate levels of HLA class I expression, while lacking the expression of HLA class II, B7, and CD40 ligand conferring privilege to the immune system of their host, thus enabling allogenic transplantation without the need of concomitant immunosuppression [42,43]. Indeed, studies in large animals have shown improvements in LVEF upon MSC therapy in the setting of myocardial ischemia. Nevertheless, results from translational attempts of these findings in clinical studies fall into a wide spectrum of significance with regard to their benefits, notwithstanding their mode of transplantation (i.e., autologous vs. allogenic) [44]. Despite some showing significant improvements in patients with acute MI, other randomized controlled studies concluded no significant differences [45–48]. Nonetheless, two randomized pilot studies were conducted in 2012 and 2017 in patients with ischemic cardiomyopathy (ICM) and nonischemic dilated cardiomyopathy (NIDCM), respectively, comparing autologous to allogenic MSC therapy [49,50]. Results from these studies, also known as POSEIDON, alluded to the efficacy of MSC therapy in these patient cohorts, with superiority given to allogenic transplantation. However, these studies were limited to the small sample size and lack of a placebo control group.

#### *2.3. Cardiac Progenitor Cells and Stem Cell Niches*

Indeed, the heart's endogenous regenerative capacity has been an area of extensive research over the past decades. Contrary to the long-held dogma of being a postmitotic organ, studies have challenged this notion, claiming that the mammalian heart is indeed capable of self-regeneration, albeit exiguously. Studies using mitotic index, as well as DNA labeling, have conveyed the finding that cardiomyocytes can self-renew during adulthood. However, debates have flared as to what extent this self-renewal takes place, and even to the reliability of the methods used to quantify it. Herein, nuclear labeling is not reliable, due to the characteristic polyploidy that human CMs undergo during growth or disease [51–54]. Radiocarbon (14C) dating, on the other hand, has provided more accurate estimates of cardiomyocyte turnover in the adult heart [55]. Interestingly, studies have shown a significant increase in cardiomyocyte count and/or ploidy in neonatal and preadolescent life in both rodents and humans, which contributed to heart growth [56,57].

Furthermore, the existence of SC niches harboring cardiac progenitor cells (CPCs) has also been reported and highlighted by research as evidence of the heart's regenerative capacity, notwithstanding another yet-unresolved debate [51,58]. CPCs are multipotent as was shown by their ability to differentiate to cardiac cell lineages, including cardiomyocytes; they were claimed to confer cardiac tissue repair and regeneration. As a heterogeneous population of cells, they are each identified by expression of distinct markers. Of these cells, c-kit-, Isl1-, or epicardial Tbx18-positive (also WT1-positive) cells are three heavily studied cell populations due to their cardiomyogenic differentiation potential attributed during development, neonatal life, and even in adult hearts.

The c-kit-expressing cells are the most studied CPCs, however, with contradicting reports regarding their significance for cardiac cell repair the in adult postinjury [59–61]. Despite their demonstrated contribution to cardiac regeneration in the neonatal hearts, c-kit-positive CPCs' role in the adult setting of myocardial injury is largely debated [60,62,63]. A recent report alluded to the role of c-kit-positive cells in cardiac adaptation to injury, where c-kit was shown to be upregulated in response to pathological stress [64]. Furthermore, a RNA-sequencing study recently showed that c-kit-positive cells transiently adopt a cardiomyocyte-like pattern of gene expression upon myocardial infarction in vivo [65]. Contrary to these findings, more recent studies by Li and colleagues refuted the myogenic potential of these cells in the adult by using a new genetic-lineage tracing system [66]. Furthermore, the same group has shown that early segregation of myocytes and nonmyocytes during embryonic development (E10.5 to E11.5) is the cut-off line beyond which no contribution to new cardiomyocyte formation occurs, even during neonatal life [67]. Moreover, a study published earlier this year by Elhelaly and colleagues argued that c-kit-positive cells do not contribute to cardiomyogenesis, even during neonatal life [68]. Howbeit, the commonly agreed-upon consensus in the field is that CPCs are remnant SCs from developmental stages whose role in the adult heart, if any, confines to maintaining cardiac tissue homeostasis, and their cardiomyogenic potential in the context of injury is inexistent [69,70]. Importantly, however, the repercussions of the aforementioned findings instigated a wave of research endeavors to exploit the heart's endogenous regenerative capacity for novel therapeutic interventions. In summary, the field of cardiac progenitor cells is controversy discussed, and the regenerative potential (and existence) of the cells in the adult human heart need further investigations.

### **3. Pluripotent Stem Cells**

Despite the efforts that have been made with adult SCs, none of these cells could meet the expectations as a reliable treatment for CVDs. That is because not even the most potent adult SC could provide an appreciable source for myocardial tissue regeneration and/or functional compensation for the lost contractile element of the heart, e.g., as a result of infarction, let alone cardiomyopathies or congenital heart disease [71]. In this regard, the pursuit after functional CMs calls for a different type of SCs, i.e., pluripotent SCs (PSCs).

#### *3.1. Embryonic Stem Cells*

The ability of a cell to give rise of all three germ layers of the developing embryo, i.e., pluripotency, is the most vivid and sought-after character of SCs, not only in the context of regenerative medicine, but also for basic research purposes. Pluripotency of embryonic blastocyst inner mass cells was first shown in the mouse as early as 1981 by Evans et al. [72]. In 1998, Thomson et al. first reported the generation of pluripotent embryonic stem cells (ESCs, Figure 1) from human blastocysts, which are capable of self-renewal and differentiation to all three germ layers [73]. Nevertheless, ethical considerations have long hovered over human ESCs (hESCs), as their derivation entails destruction of an embryo. This has prompted legislative issues, in that many countries have imposed bans on their use and/or research funding [74,75]. To add insult to injury, ESCs' ability to form teratomas (tumors of mixed germ layers) when transplanted undifferentiated has further flared the argument against their clinical application, despite efforts to enhance differentiations and purifications protocols [76].

**Figure 1.** Differentiation of cardiomyocytes derived from pluripotent stem cells.

#### *3.2. Induced Pluripotent Stem Cells*

It was not long until the not-so-bright picture of SC research changed. Inspired by the preexisting knowledge of master regulator genes capable of imparting cellular identities, Takahashi and Yamanaka developed the first technique of somatic cell reprogramming in 2006 [77,78]. In their Nobel Prize experiment, induced pluripotent stem cells (iPSCs, Figure 1) could be generated from somatic cells, such as skin fibroblasts, by expression of four transcription factors that were found to be crucial for cellular reprograming to ESC-like inner mass cells, namely Oct3/4, Sox2, c-Myc, and Klf4 [78]. Ever since, scientist have raced to improve the reprogramming efficiencies of iPSCs by manipulating the transcription-factor cocktail and selecting for expression of other transcription factors, such as Nanog and Lin28 [79–81]. Generation of viable and tumor-free whole organisms with iPSCs that were capable of germ-line transmission was also made possible [82]. Unsurprisingly, human iPSCs (hiPSCs) were generated as soon as one year after their first generation in a mouse, and by the same pioneering group of scientists, as well as others [83,84].
