Cardiac Stem Cell Therapy, Resident Progenitor Cells and the role of Cellular Signalling; a Review

Paracrine cell signalling involving exosomes plays a key role in the interaction between donor and recipient cells upon implantation [16]. This form of intercellular communication allows horizontal transfer of genetic information between stem and resident adult cells via extracellular vesicles. Several studies have demonstrated that the exosomes of cardiac cells may be vital to their capacity for renewal [6,17].

Recent studies have shown that CPC exosomes (CPCe) and bone marrow derived mesenchymal stem cell exosomes (BMCe) can both promote angiogenesis [6,18]. Injection of CPCe into the heart following induction of myocardial infarction in rats resulted in a larger capacity for healing and improved function when compared with BMCe or control. It is hypothesised that BMCe offer less protection against oxidative stress than CPCe, which are also associated with higher levels of proteins such as PAPP-A, a protease which releases IGF-1 (known to have a cardioprotective role) [6]. Important to note is the finding that exosomal cargo can be manipulated by changing parental cellular conditions, for example, inducing hypoxia.

A recent study suggests that retinoic acid (RA), as well as being crucial for guiding correct development of the embryo via intercellular signalling, may also play a role in cardiac repair. Retinoic acid receptor (RAR) agonists were shown to be potent proliferators of CPCs [19]. Importantly, new CPCs formed maintained their progenitor phenotype. All-Trans Retinoic Acid (ATRA) and AM580 were 2 RAR agonists identified and shown to increase proliferation of induced pluripotent stem cell derived - CPCs. Additionally, the ATRA compounds did not increase proliferation of terminal cardiac cell types [19]. Whether ATRA and AM580 hold therapeutic value in future treatments remains to be investigated. A previous study demonstrated the importance of RA in heart health by noting that blocking signalling of this molecule in chick embryo myocardium inhibited cardiomyocyte proliferation. It was suggested that erythropoietin and retinoic acid work in tandem to regulate signalling in the developing heart [20].

Low blood levels of IGF-1 have been shown to correlate with poor heart health and death of CPCs. IGF1 treatment was shown to improve cardiomyopathy, decreasing apoptosis, restoring heart metabolism and cardiac gap junctions [21]. It is believed that by upregulation of the Akt pathway (shown experimentally by the pAkt/Akt ratio), IGF-1 is cardioprotective and promotes survival and differentiation of CPCs. In vitro, treatment of pig CPCs with IGF-1 had potential to reduce adverse cardiac remodelling and promote regeneration of the myocardium [22].

Suppressing cell cycle inhibitors may promote cardiomyocyte proliferation potential. Specifically, downregulating Retinoblastoma1 (Rb1) and Meis Homeobox 2 (Meis2), via small interfering (si)RNA’s, induced proliferation of CMs in rats and human cell models and had a positive effect on heart health and improved wound healing [23].

The in vivo experimental methods involved siR-NA-infused hydrogel injection to the myocardium following ischaemic heart injury in rats. Interestingly, separate inhibition of either of these genes did not result in significant proliferation. Additionally, control injections of hydrogel alone without siRNAs still offered a small degree of cardio-protection, suggesting that intracardiac injection of biological materials following acute myocardial injury may have some small therapeutic benefits. The results of RT-PCR, Western Blot and immunohistochemical analysis together suggested a site-specific induction of CM cell cycle re-entry, keeping the potential low for off-site pathogenic effects such as unintended hyperplasia [23]. Previous attempts to induce re-entry of CMs into the cell cycle have resulted in heart dysfunction. Notably, the hydrogel technology holds promise as a new delivery method for donor stem cells [24].

Although the results of this experiment highlight a potential treatment for ischaemia, it is important to note that the number of new cardiomyocytes formed is still too low to effectively replace damaged myocardium.

Four cell cycle regulators (CDK1:CCNB and CD-K4:CCND complexes) and subsequent intramyocardial delivery induced cardiomyocyte proliferation in vivo with an efficiency of over 15%. Similarly, cardiomyocytes’ stimulated re-entry into the cell cycle promoted mitosis and increased cardiac health significantly. The same study suggests that use of Wee1 and TGFβ inhibitors may prevent inactivation of endogenous CDK1:CCNB complexes and thus remove the need of delivery of these compounds [25].

MicroRNAs are a class of non-coding RNA found to regulate gene expression post-transcriptionally. The cluster miR 302-367, previously shown to be connected to regulation of CM proliferation in the adult heart, was demonstrated to significantly increase CM proliferation after delivery via hydrogel injection. Overall heart health increased when compared with controls demonstrating that delivery of relevant biomaterials, without implantation of stem cells, could play a key role in future therapeutic options [26,27].

Hypoxia was shown in 2012 to upregulate Pax7 and downregulate MyoD, encouraging self-renewal and reducing differentiation of muscle progenitor cells. As a result, these satellite cells are maintained in an undifferentiated state [28]. As previously mentioned, hypoxic injury to cardiomyocyte cells may trigger paracrine factors to induce proliferation of CMs.

Hypoxia is known to reduce CM proliferation in the hearts of developing rats [29,30]. A population of hypoxic cardiomyocytes in transgenic mice improved CM proliferation in the adult heart, suggesting that hypoxic cell signalling plays an important role in CM cell cycling [31]. It is possible that induction of CM proliferation may be based on developmental stage of cells [30,32,33]. The changing effects of hypoxia on CMs across developmental stages may be linked to changes in structure and composition from embryo to adult [33]. By reducing presence of reactive oxygen species, hypoxia may benefit CM proliferation [32].

Hypoxic mesenchymal stem cells, upon implantation, had a reversal on cardiac fibrosis via prevention of fibroblast to myofibroblast transition [34].

Cardiac fibroblasts (CF) constitute a cell population known to play a role in repair and regeneration of cardiomyocytes, for example, in myocardial remodelling following injury. CF are the most populous cell type in the heart, surrounding myocardial layers and providing support to cardiac development, function, and structure via synthesis of extracellular matrix components.

Contrary to CMs, CFs have large capacity for proliferation and thus play a key role in the repair process of the heart, mounting an inflammatory response along with immune cells and subsequent repair via fibrosis. Because CMs hold such low potential for self-renewal, the remodelling of myocardial tissue by fibrosis following injury is inadequate to maintain heart health.

Although resident cardiac immune cells have been shown to contribute to inflammation and fibrosis, in general they are believed to have a minimally inflammatory role and promote angiogenesis [35]. Better understanding of the interactions between CMs, CFs and immune cells following acute injury is important to guide future research [36].

Because cardiac fibroblasts represent a heterogenous population with no specific cell markers, they are among the least characterised cell types relevant to heart health [37]. Although adult CFs promote cardiomyocyte hypertrophy, it has been shown that embryonic CFs contribute to proliferation of CMs through paracrine signalling [38].

Previous experiments demonstrate that overexpression of specific transcription factors, (Gata4, Mef2c and Tbx5) reprogram cardiac fibroblasts into mature cardiomyocytes [39,40]. These 3 transcription factors co-operate in activating cardiac gene expression and promoting CM differentiation.

Twist transcription factors act as regulators of cell fate across organisms. In mice, a population of cells expressing Twist2 (TEC) were shown to contribute to the development of new cardiomyocytes, endothelial cells and fibroblasts using lineage tracing techniques. Notably, this cell type may be able to induce proliferation by fusion with existing cardiomyocytes [41]. It is believed that this TEC cell type may play a pivotal role in maintenance of cardiac function, although more research in the area is necessary.

Annual cardiomyocyte turnover in the adult heart is reported to reduce from 1% at age 25 to 0.45% at age 75 [43]. Before treatments involving inducing CM proliferation can be implemented, a comparative analysis of the potential of siRNAs, miRNAs and transcription factors to induce proliferation must be gathered [44]. Rate of proliferation induced must be accurate to avoid possibility of heart dysfunction, taking into account the nature of the particular heart (ie. an adolescent’s heart vs. a 75-year old’s heart).

An understanding of the effects of inducing hypoxia in the heart to stimulate proliferation is required. The effects of hypoxia on the myocardium are known to be linked to the developmental stages of the organism. Further study is required to uncover potential of a possible therapeutic treatment [30,33].

A standardized/optimal method of isolating and purifying exosomes from cells of interest is yet to be achieved [7,45]. It has been suggested that the capacity for CPCs to heal the damaged myocardium may be related to exosomal signalling [17,46]. Manipulation of these extracellular vesicles may offer an alternative, or complementary treatment to implantation. Exosomes may also serve an important use as biomarkers of disease [47].

Identification of intracellular markers of CPCs and CMs is required to study and determine the nature of cell lineage and proliferation of CMs in the adult heart.A summary of discovered progenitor cell types associated with the heart and their properties is presented in table 1.

There are ongoing debates related to definitions of cell lineage in cardiac stem cells. Originally believed to have no capacity for self-renewal due to mature cardiomyocytes’ exit from the cell cycle, the heart is now believed to be residence to several different populations of stem or progenitor cells, with the potential for cardiac repair [48]. These cells are speculated to originate from a single cardiac stem cell type, and the different populations to represent different physiological stages of the parent stem cell [49]. However, the exact source of these resident progenitor cells have not been discovered.

Recently, doubt has been cast over the possibility of a single cardiac parent stem cell type [13]. The potential of CPCs to differentiate into new cardiomyocytes is limited to early development of the embryo and is minimal in the adult heart, suggesting that any new cardiomyocytes formed in the adult heart are derived from other cardiomyocytes [8].

To summarise, optimising treatments for CVD to maximise angiogenesis and cardiomyocyte proliferation, minimise fibrosis and diverse myocardial remodelling, may involve cell types including CPCs, CMs, CFs and resident immune cells, as well as small signalling molecules such as exosomes and growth factors. Further study into the interactions and signalling mechanisms of these cell types and molecules is necessary.