**1. Cardiac Regeneration—A Problem to Solve or A Solution with Promise?**

With morbidity rates associated with cardiovascular diseases in the decline in the developed world from improved treatments and pharmacological intervention, scientists and clinicians have been approaching therapies recently for these diseases with vigor. However, there is still no reliable therapy for acute cardiac conditions like myocardial infarction (MI), which account for nearly half of all cardiovascular deaths in the industrialized world [1,2]. Regenerative medicine-based strategies for infarcted myocardium have shown promise in preclinical animal models as well as early clinical trials [3]. Whilst these have demonstrated some physiological improvements in ventricular function, they were associated with very low cell retention after some weeks, suggesting a paracrine effect of transplanted cells rather than functional integration within the damaged tissue [4].

The heart was long viewed as a post-mitotic or terminally differentiated organ with no ability to regenerate or repair, a dogma that has been challenged abundantly in recent years [5,6]. Cardiac regeneration, following injury, is still an unresolved debate over whether it is attributed to dedifferentiation and proliferation of resident cardiomyocytes or from an inherent trigger in differentiation of cardiac stem or progenitor cells in putative cell niches within the heart [7–11]. The turnover of cardiomyocytes in the adult heart is around 1% per year which is insufficient to counter the loss caused by MI that can lead to loss of around 1 billion cardiomyocytes [12]. Therefore, the only

long-term solution relies on heart transplantation, but this does not come without its own issues such as insufficient number of donors coupled with the requirement for a life-long immunosuppressive therapy. This catapulted research towards cell-based therapies for cardiac regeneration [13]. Cardiomyocytes are the main cardiac cell type that is lost in cardiovascular disorders, like heart failure, myocardial infarction, and ischemia, and therefore, replacing these cells could potentially restore heart function. However, transplanting cardiomyocytes to repair diseased hearts has shown to yield only transient responses as most cells are eventually lost in the host environment [14,15]. This is because cardiomyocytes have very limited proliferative ability and as a result, they are unable to repopulate and replenish the damaged tissue efficiently [16,17]. Furthermore, other cell types like smooth muscle cells, and endothelial cells can suffer from collateral damage and their functional renewal is vital for effective heart regeneration [18]. This puts emphasis on the role of a precursor cell type capable of extensive expansion and differentiation into the key cell players of cardiac regeneration.

Even though some level of cell turnover has been observed in the adult heart, cells with self-renewal or potency capabilities are generally considered lacking in this tissue [19]. Nevertheless, several studies report the evidence of a progenitor population from resident cardiac stem cells (CSCs) in the heart, called Cardiac Progenitor Cells (CPCs) [20–23]. In contrast to terminally differentiated cardiomyocytes, CPCs are highly proliferative and can theoretically differentiate into all the necessary cardiac cell types for effective reconstitution of damaged cardiac tissue and promoting its neovascularization [14,18,20, 21,24–31]. Therefore, CPCs present a more effective cell source than cardiomyocytes for cell-based regenerative strategies. However, the application of CPCs has not been straight-forward particularly in chronic infarcts, where CPCs are associated with senescence, decreased telomerase activity and increased apoptosis [7]. Cell therapy using CPCs generally involve transplantation of in vitro-expanded CPC populations which in turn yield mild improvements in cardiac function [32]. However, long term prognosis with such therapies are poor owing to reduced cell viability and inefficient engraftment into the host tissue. This is compounded by the somewhat hostile microenvironment created by MI, from scar formation and associated inflammatory or tissue alterations, which compromises the effectiveness of such therapies [33–35]. There are also reports that the administration of CPCs predisposed the risk of cardiac arrhythmias and teratoma formation [36]. Therefore, better understanding of the CPC cell behavior in dynamic pathophysiological microenvironments could aid in developing strategies to optimize their contribution to cardiac repair.

Various approaches have been developed to generate CPCs ex vivo, in the hope of obtaining reliable source of cells that can trigger mechanisms of cardiac regeneration. For example, CPCs from the heart tissue (also known as putative CPCs) can be isolated and expanded in vitro [27,37–40]. However, such cells are hard to access and are present in low numbers in the tissue, making them extremely challenging to harvest and realize their potential [41]. Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are thought to be a superior alternative cell source since they could potentially provide an unlimited supply of cardiac progenitor cells. However, ESC-based therapy faces several challenges like immunogenicity, high risk of tumor formation and the characteristic ethical concerns, which have prevented their clinical application [42,43]. On the other hand, iPSCs avoid the ethical issues associated with ESCs and allows for the development of patient-specific derived CPCs, which represents an advantage over other cell sources in the creation of immune-compatible cardiac therapies [44,45]. However, with issues surrounding the safety of iPSC-based therapies, in terms of the potential risk of tumor formation associated with such therapies or immune rejection of iPS-derived cells from a common donor, scientists are looking at reprogramming from a different perspective [46–48]. Reprogramming patient somatic cells into other cell types, bypassing the step of stem cell generation, can potentially overcome issues with translating iPSC technology. This process is known as direct cellular reprogramming or transdifferentiation, and might represent a more robust approach to rapidly generate sufficient numbers of CPCs from somatic cells for therapy [49].

This review focuses on the ongoing progress and limitations in generating CPCs from iPSCs and through direct reprogramming. It will start by providing a concise introduction about the various cardiac progenitor cells identified in embryonic and adult heart tissues. The review will then move towards discussing reprogramming approaches that were successful in generating CPCs and the functionality of these CPC-derived cells. Strategies to improve efficiencies of current protocols and tissue engineering advances to mimic CPC microenvironment and in vivo applications of CPCs will also be evaluated. Finally, the review will finish with a summary of existing challenges and limitations and future directions for CPC research, hopefully convincing readers it is a promising strategy for cardiac regeneration (Figure 1).

**Figure 1.** The interplay between genetics and biomaterials for understanding Cardiac Progenitor Cells (CPCs) biology, function, and its regenerative applications. eCPCs (endogenous CPCs), rCPCs (reprogrammed CPCs), iPSCs (induced Pluripotent Stem Cells), SPs (Side Population-derived CPCs), CSs/CDCs (Cardiospheres/Cardiosphere-Derived Cells), EDPCs (Epicardium-derived CPCs), FHF/SHF (First Heart Field-/Second Heart Field-derived CPCs) CMs (Cardiomyocytes), SMCs (Smooth Muscle Cells), ECs (Endothelial Cells).

#### **2. Cardiac Progenitor Cells (CPCs) In Vivo**

Progenitor cells are distinct from embryonic stem cells as they have a predetermined differentiation fate and therefore, their ability to self-renew and differentiate into other cell types is restricted [19]. CPCs generate cells of the three cardiac lineages: cardiomyocytes, smooth muscle cells and endothelial

cells. These cells are also responsible for the maintenance of cardiac homeostasis under physiological and pathological conditions [50]. Several studies have identified and isolated multiple CPC populations from distinct stages of cardiac development and heart locations. These cells are collectively characterized according to their cell surface and genetic marker expression profiles. The various CPCs reported to date are described below and their specific features are summarized in Table 1.
