ESCs are pluripotent cells extracted from the inner cell mass of human blastocysts. They exhibit the capacity for indefinite symmetrical division; and for asymmetric division into progenitors of mesoderm, ectoderm and endoderm germ layers [48]. Owing to their pluripotency, ESCs are strong candidates for the repair of various adult tissue types including ischaemic myocardium. Several methods have been employed to induce cardiomyogenic differentiation from a pool of ESCs with the resulting cells termed embryonic stem cell-derived cardiomyocytes (ESC-CMs) [49]. ESC-CMs express early cardiac-specific transcription factors including homeobox protein Nkx2.5, GATA binding protein 4 (GATA-4), myocyte enhancer factor 2C (MEF2C) and T-box transcription factors Tbx-5 and Tbx-20 [50]. Furthermore, ESCs give rise to progenitor cells with temporal regulation of foetal liver kinase 1 (Flk1), Islet-1 (Isl-1) and Brachyury (a T-box transcription factor), demonstrating its capacity for differentiation into cardiomyocytes, endothelial cells and vascular smooth muscle cells in vitro [51, 52].

In a seminal study, Caspi et al. [53] transplanted undifferentiated ESCs into infarcted rat hearts, as it was thought the in vivo cardiac environment may be sufficient to induce cardiomyocyte (CM) differentiation. This proved unsuccessful, and teratomas consisting of cells from all three germ layers were observed, highlighting the importance of inducing CM differentiation and ensuring homogeneity of the cell population prior to transplantation, reducing the risk of tumorigenesis. In a follow-up study, ESC-CMs were grafted into the infarcted area 7–10 days following left anterior descending artery (LAD) ligation in a rat model. Improvements were noted in both scar remodelling and left ventricular function, with transplanted cells able to form gap junctions with host cells as assessed by the expression of the left ventricular gap junctional protein connexin-43. However, ESC-CMs exhibited an immature phenotype, which has been identified in subsequent studies from independent laboratories [54, 55]. ESC-CMs are observed as being smaller in size, having a slower action potential upstroke, a less extensive network of T-tubules and poor sarcomeric organisation among other issues [54, 55]. These differences to adult cardiomyocytes may impair the ability of ESC-CMs to effectively integrate within host myocardium, increasing the risk of arrythmia generation due to the difference in electrophysiology. Promising pre-clinical studies including the recent work of Liu et al. [56] have demonstrated that transplantation of ESC-CMs into infarcted myocardium in a non-human primate model improves left ventricular ejection fraction up to 3 months post-transplantation, however this was associated with an increased incidence of ventricular arrythmias.

To reduce the incidence of these events, current studies are aimed at enhancing the maturity of ESC-CMs in vitro before transplantation. Methods of improving maturation include inhibiting hypoxia-inducible factor 1-alpha (HIF-1$\alpha$) and lactate dehydrogenase A (LDHA)—targeting metabolic maturity, along with bioreactor systems employing pulsatile flow, cyclic strain and extended culture time [57, 58].

In addition, transplantation of ESC-CMs into the recipient heart is allogenous, requiring lifelong immune system modulation [59]. Further, legal and ethical concerns exist as the traditional method of isolating ESCs for in vitro expansion results in the destruction of the human embryo [48]. While alternative, non-destructive approaches are being explored to isolate ESCs, these legal issues effectively rule out the use of ESCs as a viable treatment in many parts of the world.

Recently, the Transplantation of Human ESC Derived Progenitors in Severe Heart Failure (ESCORT) trial was completed (NCT02057900)—assessing the safety of ESC derived cardiac progenitor cells (CPCs) when engrafted into human patients [60]. Despite the knowledge that ESC-CMs exhibit an immature phenotype, the team behind this trial conducted several pre-clinical trials using these ESC derived CPCs, in which no arrythmia or teratoma formation was observed [61]. Six patients with severe ischaemic left ventricle (LV) dysfunction received ESC derived CPCs delivered as epicardial injection during coronary artery bypass graft (CABG) surgery and were followed up for a median of 18 months. All patients who were followed up had an uneventful recovery with no observed arrhythmia development. Importantly, a significant increase in systolic motion was observed in patients receiving ESC-CPCs. These results were encouraging despite a small sample size and warrant further investigation to confirm the efficacy of ESC derived CPCs in human populations. The on-going HECTOR study (NCT05068674) is a phase I trial evaluating the safety of administering varying doses of ESC-CMs to patients with LV dysfunction secondary to MI. This study aims to recruit 18 patients and will provide further insight into the safety profile of ESC-CM transplantation for cardiac repair.

In spite of these recent clinical trials, there are issues that need to be solved regarding the use of ESC derivatives for repair of the ischaemic heart. These include improving long-term engraftment rates, deciding the stage of differentiation for transplantation, increasing in vitro maturity of ESC-CMs and establishing the optimal dosage and safety profile. Further investigations are also required to accurately determine the nature and underlying mechanisms of the beneficial effects observed on ischaemic myocardium—whether this stems from remuscularisation of the heart or through paracrine mediated mechanisms.

In 2006, Takahashi et al. [62] generated pluripotent cells from adult fibroblasts and coined the term iPSCs, a discovery which would go on to win the Nobel Prize in Physiology or Medicine in 2012. As iPSCs are generated from somatic cells, they are not associated with the same ethical and legal issues as ESCs. Furthermore, patient-matched autologous cells can be created—thereby reducing the possibility of immune rejection. The most common method of iPSC production is the viral transduction of genes encoding the transcription factors octamer-binding protein 3/4 (Oct-3/4), sex-determining region Y box 2 (SOX2), Myc proto-oncogene protein (c-Myc) and Kruppel-like factor 4 (KLF4) [62]. Several studies have documented the ability to differentiate iPSCs into cardiomyocytes (iPSC-CMs), with the differentiation efficacy being comparable to that of ESCs, with ESC-CMs and iPSC-CMs sharing near identical transcriptional profiles [63, 64, 65]. In recent years, methods for the derivation and purification of cardiomyocytes from iPSCs have improved, with some studies reporting a purity of the differentiated cell pool for cardiomyocytes of around 95%, with 100 cardiomyocytes generated per input cell [66, 67].

Despite these advantages, differentiation of iPSCs poses some risks. Viral transduction carries with it the inherent risk of genome insertion at unwanted locations, with the potential to promote oncogenesis and disrupt cellular function [68]. As a result, non-viral vectors (e.g., plasmids) have been developed for iPSC production along with RNA and protein delivery. However, these alternate methods tend to be less efficient in iPSC production, as summarised in a review by Rao et al. [69]. While the ability for autologous transplantation using iPSCs is an advantage, this process can be costly and time consuming, with recent studies instead attempting to reduce the immunogenicity of allogenic iPSCs from healthy donors for clinical application [70]. Differentiation of iPSCs into CMs in vitro is achieved through similar protocols to those established for ESC differentiation, with the resulting CMs sharing an immature phenotype and therefore also carrying the risk of arrythmia and teratoma development [55]. This was highlighted in a recent pre-clinical trial conducted by Shiba et al. [71], in which iPSC-CMs were delivered by intra-myocardial injection into the infarcted hearts of non-human primates. Significant improvements in contractile function were reported, however with an increased incidence of ventricular tachycardia in the treatment group.

In a recently published first-in-human clinical trial in Japan, a 51-year-old male with severe ischaemic cardiomyopathy underwent transplantation of three allogenic iPSC-CM patches onto the ischaemic myocardium [72]. These patches were constructed of clinical grade iPSC-CMs which underwent screening for tumorigenesis and arrhythmogenesis risk. Following transplantation, no tumour development, arrythmias or effects related to immunosuppressive treatment were observed. Furthermore, the patient experienced increased quality of life, systolic motion, reduced LV global wall stress (attenuation of fibrosis) and an increase in the coronary flow reserve after 6 months and one year of follow up. The investigators hypothesized the observed benefit was primarily mediated through paracrine stimulation of angiogenesis. In support of this, a pre-clinical study conducted by Tachibana et al. [73] provided evidence that iPSC-CMs release high levels of interleukin-8 (IL-8), granulocyte colony stimulating factor (GCSF) and vascular endothelial growth factor (VEGF) promoting angiogenesis in the ischaemic heart. While the first-in-human clinical trial is promising, the extent of mechanical contribution of the engrafted iPSC-CMs to cardiac contractility and the percentage of cell retention necessitates further investigation. While no randomised clinical trials of iPSC-CM efficacy have been completed, one trial registered in China (NCT03763136) aims to assess the safety, feasibility, and efficacy of intramyocardial delivery of allogenic iPSC-CMs at the time of CABG surgery in patients with CHF. Another registered clinical trial (NCT03759405) will determine changes in quality of life and cardiac function following intravenous injection of iPSC-CMs in three patients with CHF. The recruitment has not started yet, with the trial expected to be completed in 2024. Although early results from case-studies are encouraging, further research needs to be carried out to standardize the protocol for developing mature cardiomyocytes from iPSCs that can electromechanically couple with endogenous cardiomyocytes for its clinical translation to be successful.

SkMs are multipotent stem cells located between the basal lamina and sarcolemma layers of mature skeletal muscle, becoming activated in response to muscle damage and degeneration [74]. SkMs were one of the earliest adult stem cell types explored as a candidate for cardiac repair due to their ability to form mature myofibers, resistance to ischaemia (owing to their skeletal muscle origin), relative ease of harvest and the potential for subsequent autologous transplantation [75, 76].

Pre-clinical murine studies including the pioneering work of Taylor et al. [77] demonstrated beneficial effects of SkM transplantation in improving cardiac contractility, preventing left ventricular remodelling, and decreasing diastolic pressures [76, 77, 78]. However, SkMs are committed to a myogenic lineage and therefore cannot form fully functioning cardiomyocytes, instead differentiating into muscle fibres called myotubes in vivo [79]. Cardiomyocytes behave as an electrical syncytium due to the presence of intercalated discs, more specifically gap junctions between neighbouring cells [80]. Myotubes derived from SkMs fail to form these gap junctions owing to a decreased expression of the intracellular adhesion molecules N-cadherin and connexin-43, therefore remaining electromechanically isolated from the host myocardium [79]. Thus, myotubes do not contract in synchrony with the surrounding myocardium, predisposing the transplant recipient to arrythmia development. Studies using skeletal myoblasts genetically modified to overexpress connexin-43 demonstrated improved myocardial integration and synchronous contractility stemming from an increase in the number of gap junctions formed with host cardiomyocytes [81, 82, 83]. However, subsequent studies have concluded this is insufficient in preventing the development of arrhythmias [84]. Furthermore, the Myoblast Autologous Grafting in Ischaemic Cardiomyopathy (MAGIC) phase II clinical trial showed no improvement in left ventricular ejection fraction (LVEF) in patients treated with skeletal myoblasts compared with the control group, with a higher number of arrhythmic events observed in the SkM-treated group [85]. Due to these major issues, skeletal myoblasts have not progressed as a candidate for cardiac repair.

BMCs are isolated from bone marrow throughout the body and can be further divided into HSCs and BdMSCs. HSCs exhibit myeloid and lymphoid differentiation, whereas BdMSCs differentiate into bone, cartilage and adipose tissue lineages in vivo [86, 87]. Unfortunately, many clinical trials using BMCs have not specified which sub-population was used, and definitions of these cells remain controversial. This section will focus on studies reportedly using HSCs, and more generally BMCs collectively. BdMSCs will be described in further detail in subsequent sections. One of the earliest studies involving BMC transplantation for repair of the ischaemic heart was conducted by Orlic et al. [88], who injected BMCs enriched for Lin${}^{-}$c-kit${}^{\text{POS}}$ cells (used to distinguish HSCs) into the infarcted left ventricle of mice. The group reported direct transdifferentiation of BMCs into cardiomyocytes, endothelial cells and smooth muscle cells. The same group provided evidence that the administration of stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) induced mobilisation of BMCs to the infarct area, improving post-MI survival and supporting myocardial repair in a mice model [89]. While these findings were encouraging, landmark contradictory studies conducted by Balsam et al. [90] and Murry et al. [91] demonstrated that when injected into the myocardium of mice post-MI, HSCs did not improve survival or reduce infarct size. Furthermore, no cases of transdifferentiation into cardiac lineages were observed [91, 90]. The commonly accepted mechanism of repair is that BMCs exert their beneficial effects on ischaemic myocardium through the activation of endogenous progenitor cells, and the release of paracrine mediators including VEGF, basic-fibroblast growth factor (bFGF) and angiopoietin-1 (Ang-1) [92, 93]. Preclinical studies demonstrating the efficacy of BMCs for cardiac repair prompted quick clinical translation into randomised controlled trials in human populations [88, 89, 92, 93, 94].

The Reinfusion of Enriched Progenitor Cells and Infarct Remodelling in Acute Myocardial Infarction (REPAIR-AMI) study was a placebo-controlled phase III trial assessing the efficacy of BMCs delivered via intracoronary infusion following acute MI. This study demonstrated a significant improvement in left ventricular ejection fraction (LVEF) in the treatment group [95]. The PreSERVE-AMI study was a phase II study in which autologous purified CD34${}^{+}$ cells (a subset of BMCs) or a control were administered via intra-coronary infusion to STEMI patients [96]. After one year, while adjusting for the duration of ischaemia a dose-dependent improvement was observed in LVEF, infarct size and survival—suggesting purified CD34${}^{+}$ cells may have beneficial effects on the ischaemic heart. Although these two trials reached positive conclusions, several other trials have produced contradictory findings demonstrating little or no beneficial effect [97, 98, 99, 100]. BMCs have been extensively studied in human trials for cardiac regeneration over the last two decades, with around 100 randomised controlled trials (RCTs) produced. Meta-analyses of these RCTs has proven difficult due to the heterogeneity of methods used for cell production, administration, and measurements of cardiac performance [101, 102]. Despite this, BMCs remain an attractive candidate for cardiac repair. To draw robust conclusions, a greater number of well performed RCTs are required with consistent methodology and clearly defined cell populations.

Mesenchymal stromal cells (MSCs) are multipotent with potential for differentiation into mesenchymal lineages (osteoblasts, chondrocytes, myocytes, adipocytes and fibroblasts), as defined by the International Society for Cell & Gene Therapy [103]. MSCs are attractive candidates for therapeutic cell transplantation due to their low immunogenicity and immunomodulatory capacity resulting from low major histocompatibility complex (MHC) II expression and the secretion of several anti-inflammatory cytokines. This increases the feasibility of allogenic MSC transplantation as an attractive option for large scale clinical implementation [104]. MSCs isolated from several sources have been investigated for their use in cardiac repair. The following sections will focus on bone-marrow derived mesenchymal stem cells along with MSCs derived from adipose tissue and the umbilical cord.

EPCs exhibit the capacity for both direct endothelial differentiation and maintenance of existing vasculature through paracrine mediated mechanisms [139, 140]. However, substantial debate exists in the literature regarding the classification and origin of EPC sub-populations. Early studies suggested that EPCs originate from the bone marrow, which has recently been challenged [141, 142]. Although the exact developmental origin of EPCs remains unknown, major sources include both peripheral blood and umbilical cord blood [143].

Two distinct EPC populations have been identified and termed ‘early’ and ‘late’ EPCs [140]. Early EPCs refer to cells of a haemopoietic origin, now termed myeloid angiogenic cells (MACs). These cells express CD45, CD14 and CD31 while being negative for CD146 and CD133 [144]. The major mechanism of endothelial repair from MACs is the release of paracrine mediators promoting angiogenesis including VEGF [145]. Late EPCs are now referred to as endothelial colony forming cells (ECFCs) and are considered the ‘true’ endothelial progenitors. ECFCs display an endothelial phenotype, and are identified by their expression of CD146, CD31 and CD105 while being negative for CD45 and CD14 [144]. It is thought that ECFCs primarily exert their beneficial effects on the vasculature by direct differentiation into endothelial cells, profoundly contributing to de novo blood vessel formation and angiogenesis [145, 146]. The heterogeneity of definitions and the lack of a clear unambiguous marker of EPCs has made it increasingly difficult to draw valid conclusions from trials.

In one of the only completed clinical trials investigating EPCs for repair of the ischaemic heart in humans, Zhu et al. [147] studied the safety and efficacy of EPCs pre-treated with thymosin beta-4 in patients with acute ST segment elevation MI. After six months of follow-up, patients treated with EPCs exhibited increased walking distance and significant improvement in cardiac function compared to the control group. Interestingly, despite these positive results, the use of EPC in the clinical setting for IHD has not advanced further. The primary issues holding back the clinical investigation of EPC therapy include the aforementioned lack of clear and consistent phenotypic classification, extended duration of in vitro expansion due to their low occurrence, and high immunogenicity of the cells [139, 144].

The longstanding dogma that adult mammalian heart was traditionally viewed as a post-mitotic organ with little capacity for self-renewal was challenged in the early 2000s, when a group of researchers identified a population of cells in the myocardium of rats exhibiting classical features of stem cells [148]. These cells were identified as expressing the tyrosine kinase receptor c-kit while being negative for common haemopoietic lineage markers such as CD34. Although a number of early studies demonstrated the efficacy of c-kit${}^{+}$ cells to differentiate into cardiomyocytes, this was challenged by later studies due to the inability to reproduce earlier results, and several early papers have been retracted [149, 150, 151, 152, 153, 154, 155]. It has since been established that transplanted c-kit${}^{+}$ cells mediate their beneficial effects on ischaemic host myocardium through the release of paracrine factors, with one study proposing that c-kit${}^{+}$ cells are a cardiac endothelial cell population rather than an endogenous CPC population [153, 156, 157, 158].

In parallel to research focusing on c-kit${}^{+}$ cells, research has also progressed in determining the efficacy of cardiosphere derived cells (CDCs) for the treatment of ischemic heart disease. Cardiosphere-forming cells are isolated from the explant outgrowth through enzymatic digestion. CDCs are subsequently derived from cardiospheres [159]. They are characterised by their unanimous expression of the surface marker CD105 (part of the TGF$\beta$ receptor complex) and CD90, while being negative for the haemopoietic marker CD45 [160, 161]. CDCs exhibit the capacity for differentiation into endothelial cells, cardiomyocytes and smooth muscle cells in vitro, but are thought to exert their beneficial effects on ischaemic myocardium primarily through the release of paracrine mediators promoting angiogenesis, activating EPCs and encouraging cardiomyocyte proliferation while suppressing LV remodelling, apoptosis and inflammation [162, 163]. The phase I, randomised dose-escalation Cardiosphere-Derived Autologous Stem Cells to Reverse Ventricular Dysfunction (CADUCEUS) trial assessed the safety and efficacy of autologous intracoronary CDCs infusion to HFrEF patients 2–4 weeks post-MI. Patients treated with CDCs showed a significant reduction in infarct size along with both improved viable heart mass and regional contractility with no increase in adverse events relative to the control group. However, changes in end diastolic volume (EDV), end systolic volume (ESV) and LVEF did not differ between treatment and control groups after six months [164]. Due to the encouraging results of the CADACEUS trial, this was followed by a large multicentre randomized, double-blinded placebo-controlled phase I/II trial - Allogeneic Heart Stem Cells to Achieve Myocardial Regeneration (ALLSTAR) [165]. This study used allogenic CDCs as they are a more viable and cost-effective treatment option. After one year of follow-up no safety concerns were raised. Furthermore, patients treated with CDCs exhibited a decreased infarct size, increased viable myocardial mass and improved regional function of the ischaemic myocardium. These positive results warrant continued investigation into their efficacy of CDCs for the treatment of IHD.

In addition to c-kit${}^{+}$ and CDCs, few other CPCs populations such as islet-1 and cardiac side population cells have exhibited their potential to repair the ischemic heart in preclinical models, although further studies are required before they can be considered in human populations [166, 167, 168].

While the initial immune response to ischaemic injury is physiologically essential, a sustained inflammatory response is a direct contributor to adverse cardiac remodelling and progression to CHF [170, 171]. Both adult stem cells including MSCs, as well as ESC-CMs, secrete a plethora of anti-inflammatory cytokines which act to limit deleterious, sustained endogenous inflammation of the myocardium (Fig. 4). In particular, administration of these cells downregulates the expression of the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-$\alpha$), interleukin 1 beta (IL-1$\beta$), interleukin 6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1) which play a role in LV remodelling [172, 173, 174]. In contrast, EPCs are known to release pro-inflammatory cytokines including MCP-1 and IL-8 [175]. TNF-$\alpha$ acts through tumour necrosis factor (TNF) receptors present on all cell types within the myocardium. Once activated, this induces matrix metalloproteinases (MMP)—proteins which break down the extracellular matrix (ECM) and are implicated in maladaptive cardiac remodelling [176]. MSC administration has further been shown to influence various immune cell populations in the heart after ischaemic injury. MSCs inhibit the cytotoxic activity of CD8${}^{+}$ T cells and natural killer (NK) cells, prevent dendritic cell maturation and have the capacity to either enhance or inhibit plasma cell immunoglobulin G (IgG) production depending on the signal intensity [177, 178]. In addition, soluble factors produced by MSCs including prostaglandin E2 (PGE2) have been demonstrated to switch the phenotype of pro-inflammatory macrophages to regulatory, anti-inflammatory macrophages with the capacity to promote angiogenesis [179]. Furthermore, MSCs show decreased production of the pro-inflammatory IL-12, and an increased production of anti-inflammatory cytokines including interleukin 10 (IL-10) [180, 181]. Collectively, this immune system modulation is an important part of how stem cells mediate their beneficial effects within ischaemic myocardium following transplantation.

Cardiomyocyte apoptosis is a significant contributor to ischaemic injury and maladaptive cardiac remodelling [182]. Thus, protecting cardiomyocytes from apoptosis may attenuate ischaemic injury while promoting their proliferation. Cultured adult stem cells including BMCs, MSCs and c-kit${}^{+}$ CPCs have been shown to secrete factors including interleukin-11 (IL-11), VEGF, erythropoietin (EPO), fibroblast growth factor-2 (FGF-2), IGF-1, HGF and epidermal growth factor (EGF). These molecules activate several pro-survival pathways, improving cardiomyocyte survival in the hostile environment of ischaemic myocardium (Fig. 4) [156, 183, 184, 185].

Most of the above secreted factors function through the activation of pro-survival kinases Akt and ERK1/2, the downstream signalling cascade of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling respectively (PI3K/AKT, MAPK/ERK-1/2), collectively known as the reperfusion injury salvage kinase (RISK) pathway [186, 187, 188, 189]. Acute activation of this pathway mediates cardioprotection, however chronic activation can result in cardiac hypertrophy [190]. A study conducted by Noiseux et al. [169] provided evidence that in rats, activation of the Akt signalling pathway by BdMSCs conferred improved efficacy in enhancing cardiomyocyte survival and preventing apoptosis following myocardial infarction.

Other pathways involved in cardioprotection by stem cells include the surviving factor enhancement (SAFE) pathway and the protein kinase c epsilon (PKC$\epsilon$) pathway. These pathways are activated by IL-6 and IL-11, along with bFGF and EPO secreted from transplanted stem cells. The SAFE pathway leads to activation of signal transducer and activator of transcription 3 (STAT3) through Janus-kinase (JAK) signalling [191, 192, 193]. STAT3 contributes to cardioprotection by inhibiting the opening of the mitochondrial permeability transition pore and thus apoptosis [194]. Among other targets, PKC$\epsilon$ is known to activate aldehyde dehydrogenase 2 (ALDH2)—detoxifying reactive aldehydes, and further to interact with cytochrome-c oxidase, decreasing intracellular ROS, protecting against apoptosis [195].

Due to the imbalance between myocardial oxygen demand and supply in IHD, reperfusion of the ischaemic area is essential for improving clinical prognosis and halting the progression of IHD. Neovascularisation consists of angiogenesis (growth of endothelial sprouts from existing vessels), vasculogenesis (differentiation of angioblasts into endothelial cells) and arteriogenesis (smooth muscle migration, growth and remodelling) [196]. Secreted paracrine factors promoting neovascularization from BMCs, MSCs, c-kit${}^{+}$ CPCs, human iPSC-CMs and EPCs include VEGF, bFGF, Ang-1 and HGF among others (Fig. 4) [93, 184, 197, 198, 199]. Both VEGF and bFGF have direct mitogenic effects on endothelial cells, promoting angiogenesis [200]. Ang-1 mediates its beneficial effects on the vasculature through Tie2 receptors where it is primarily involved in endothelial stabilisation [201]. In a similar fashion to bFGF, HGF has also been demonstrated to work synergistically with VEGF to promote endothelial cell survival, proliferation and tubulogenesis [202]. The importance of VEGF for mediating the beneficial effects of MSCs after transplantation into ischaemic myocardium was highlighted by a study where the VEGF gene was ablated. Following ablation, MSCs exhibited a significantly impaired ability to promote functional recovery in the ischaemic heart [203]. Along with paracrine potentiation of angiogenesis, ECFCs (a subset of EPCs) participate in de novo vasculogenesis by direct differentiation into endothelial cells [146].

Recent studies have demonstrated the ability of transplanted exogenous stem cells to activate resident and circulating stem cells, enabling endogenous cardiac regeneration (Fig. 4) [204]. While the specific paracrine factors responsible are yet to be identified, Urbanek et al. [205] showed that c-Met/HGF and IGF-1 receptors expressed by CPCs were able to activate resident CPCs, forming de novo myocardium in a murine model. In another study from an independent laboratory, administration of HGF and IGF-1 to CPCs isolated from a porcine model promoted CPC proliferation, migration and the activation of downstream signalling pathways including phosphorylation of Akt. Interestingly, HGF and IGF-1 seem to act synergistically, as the observed effects were far greater in combination than either alone [206]. When applied to CPCs obtained from the neonatal rat heart, MSC conditioned medium was able to improve CPC proliferation and inhibit apoptosis [204]. In addition, MSCs also express bone morphogenetic proteins (BMPs), Wnt pathway modulators and FGF, all of which are involved in regulating CPC differentiation and commitment, suggesting these molecules may contribute to the regeneration of the myocardium through the activation of endogenous stem cells [207]. Along with activation of CPCs, circulating stem cells including MSCs, BMCs and HSCs home to ischaemic myocardium following insult from the bone marrow and circulation [208, 209]. Further, studies have suggested the ability of transplanted MSCs to recruit circulating EPCs, c-kit${}^{+}$ stem cells and CD34${}^{+}$ stem cells through the release of chemotactic homing factors stromal cell-derived factor 1 alpha (SDF-1$\alpha$), SCF and VEGF respectively [210, 211, 212].

Transplantation of stem cells into ischaemic myocardium exposes them to a hypoxic environment, triggering activation of transcription factors which have pro survival and proliferative potential [213, 214]. A key transcriptional factor overexpressed in MSCs among others is hypoxia-inducible factor-1$\alpha$ (HIF-1$\alpha$), playing a vital role in the cellular adaptation to ischaemia [213, 215]. Activation of HIF-1$\alpha$ leads to the downstream secretion of VEGF, platelet-derived growth factor (PDGF) and EPO which promote cell proliferation and angiogenesis, reducing apoptosis [216]. Interestingly, HIF-1$\alpha$ can also be stimulated by various hypoxia response pathways, including the PI-3K/AKT pathway [217].