2.1. Induced Pluripotent Stem Cells (iPSCs)

In 2006, Yamanaka and colleagues reported the reprogramming of mouse and human fibroblast to an ESC-like pluripotent state by forced expression of Oct3/Oct4, Sox2, Klf4, and Myc genes. This reprogrammed cell was named induced pluripotent stem cells (iPSCs) [14]. ESCs and iPSCs are both pluripotent stem cells that can differentiate into all kinds of tissues in the body. However, the application of ESCs has been met with ethical concerns. In contrast, iPSCs, which can be induced from autogenous fibroblast of the patient, have gained great interest in regenerative applications.

The discovery of iPSCs offered a regenerative approach for damaged heart repair. In culture, iPSCs can be induced to generate large numbers of functional cardiomyocytes. By temporal modulation of the expression of regulators in the canonical Wnt signaling pathway, iPSCs can be induced to yield a high purity (80–98%) of cardiomyocytes in 14 days [15,16]. iPSC-derived cardiomyocytes (iPSC-CMs) were transplanted for treatment of MI in a rodent model [17,18,19,20,21]. After transplantation, engrafted iPSC-CMs improved ventricular function and reduced pathological remodeling [17,20,21]. Moreover, pretreatment with Y-27632, an inhibitor of Rho-associated protein kinase (ROCK) [18], or genetic overexpression of the cell cycle activator cyclin D2 (CCND2) [19], enhanced the regenerative potency of iPSC-CMs by stimulating proliferation, contraction, and suppressing apoptotic death [18,19]. In a clinically relevant non-human primate model of ischemia/reperfusion injury, direct intramyocardial (IM) injection of iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation. The grafted cells exhibited electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09 [22]. In addition, the incidence of ventricular tachycardia was detected in iPSC-CMs injected monkeys [22], and in another study, using human ESC-derived cardiomyocytes, post-transplant arrhythmias were also recorded [23]. Despite the replenishment of cardiomyocytes in infarcted myocardium with iPSC-CMs implantation, the engraftment rate is still low because of immune rejection. The concerns of ventricular arrhythmias have also been highlighted.

To address this problem of low engraftment and arrhythmia, Ye et al. [24] fabricated a cardiac muscle patch by combining iPSC-derived cardiovascular lineage cells (cardiomyocytes, endothelial cells, and smooth muscle cells). Four weeks after epicardial implantation, iPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomere structure. The engrafted endothelial cells and smooth muscle cells contributed to host vasculature. Most importantly, by using the epicardial cardiac patch, they achieved cardiac regeneration without inducing ventricular arrhythmias [24]. Furthermore, they engineered functional human cardiac muscle patches (hCMPs) with clinically relevant dimensions (4 cm × 2 cm × 1.25 mm), which were implanted to treat pig MI [25]. Four weeks after transplantation, measurements of cardiac function, infarct size, and wall stress were significantly improved with no increase in the occurrence of arrhythmogenic complications [25]. Patching up the heart has been proposed to be an efficient way to restore functional myocardium by using stem cell-based therapy [26,27,28]. However, the reported benefits of transplanted cells have been attributed to, paracrine effects in large part, instead of to the direct replacement of infarct scar by viable cardiomyocytes [29].

2.2. Cardiac Progenitor Cells (CPCs)

Even though the adult heart lacks regenerative abilities, cellular turnover occurs in the heart [30], and the existence of a cardiac progenitor cell population has been reported [31,32,33]. Different than terminally differentiated cardiomyocytes, CPCs are highly proliferative and have differentiation potential [31,34]. CPCs represent another kind of stem cell that is used for cardiac repair. Various approaches have been developed to increase CPC populations [35,36,37] due to their extremely low levels in the heart [37].

CPCs are identified by detection of specific markers, such as c-kit [38], Sca-1 [39], Mesp1 [40], and Flk-1 [31]; c-kit+ CPCs have been broadly employed for MI treatment [38,41,42]. Although c-kit+ CPCs cannot differentiate into cardiomyocytes [43,44,45,46], transplantation of c-kit+ CPCs has been reproducibly shown to be beneficial in both preclinical and clinical studies for heart disease treatment [42,47,48,49,50]. These reports imply that c-kit+ CPCs are working via paracrine effects. Stem cell antigen-1 (Sca-1) positive cells are also defined as CPCs [51,52,53,54]. When implanted into ischemia-damaged mice myocardium, Sca-1+ CPCs differentiated into endothelial-like cells and promoted cardiac reparation [51,53]. By using a genetic lineage tracing method, Ronald J. Vagnozzi et al. [55] reported that Sca-1+ cells reveal endothelial but not myogenic contribution to the murine heart. In response to pathological stress, Sca-1+ CPCs expand and preferentially contribute to vasculature. In other studies, the absence of cardiomyocytes, differentiated from Sca-1+ cells, has also been reported [56,57]. Nevertheless, other than the differentiation of vascular cells, Sca-1+ CPCs can also exert cardioprotective roles by secreting bioactive factors [58].

Flk1+ cells and Mesp1+ cells represent another two types of CPCs identified in heart [33,59,60]. Both of them direct cardiovascular genesis during embryonic development. Flk1 is expressed in multipotent mesodermal progenitor cells, which is specified to form myocardial and endothelial lineages during development [61]. Through genetic fate mapping, researchers have demonstrated that Flk1+ cell populations contribute to progenitors that have differentiation potentials to cardiomyocyte, endothelial, and vascular smooth muscle cell [31], but there was no proof for preclinical and clinical regenerative efficacy. Mesp1 is a transcription factor that drives cardiovascular progenitor cell specification. Transient expression of Mesp1 increases the number of cardiac progenitor cells in heart [40,62,63]. Mesp1 can directly bind to the promoter region of the transcription factors known to direct cardiomyocyte differentiation. These transcription factors include Hand2, Gata4, Nkx2.5, Myocardin, FoxH1, and FoxC1 [40]. After in vitro induction from ESCs, Mesp1+ cells were injected into hearts of MI mice. The results showed that Mesp1+ CPCs emerged de novo into terminally differentiated cardiac myocytes, smooth muscle cells, and vascular endothelial cells, accompanied by significant functional improvement in mice [60].

In addition to the above intrinsic types, CPCs can be induced to generate from iPSCs and ESCs. These cells are named induced cardiac progenitor cells (iCPCs) [35,36]. In contrast to the direct implantation of iPSCs and ESCs, which pose the risks of tumorgenesis or teratomas [64], iCPCs are safer for application because of their restricted differentiation potential. Moreover, iCPCs are expandable (can be passaged more than 18 times), which limits cost and opens additional avenues for clinical translational use of patient-specific cell sources [65].

2.3. Cardiosphere-Derived Cells (CDCs)

Given the notion that tissue-derived cells hold potential for regeneration and repair [66], patient cardiac tissues can be acquired and cultured, and then yield spherical multicellular clusters called cardiospheres [67,68]. Culturing cardiospheres produces cardiosphere-derived cells (CDCs), which are used for regenerative therapy for MI [69,70,71,72,73]. In culture, CDCs showed myogenic differentiation and angiogenic potential [74]. After intramyocardial injection, CDCs preserved ventricular ejection function and attenuated cardiac remodeling [71,75]. In 2012, the first clinical trial of CADUCEUS (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction) [76,77] explored the safety and efficacy of intracoronary infusion of autologous CDCs. Preliminary indications included decreased scar size, increased viable myocardium, and improved regional function of infarcted myocardium [77]. These studies showed attractive benefits of using CDCs for MI treatment. However, CDCs cannot differentiate toward cardiomyocytes. The underlying therapeutic effects of CDCs have been attributed to paracrine factors [74,78].

2.4. Mesenchymal Stem Cells (MSCs)

MSCs are a population of stromal cells that are isolated from various kinds of tissues, such as adipose, placenta, and bone marrow. In culture, MSCs can be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic cell lineages [79]. These intrinsic differentiation properties are usually used for the identification or characterization of MSCs [80]. Moreover, MSCs can be identified by detecting the expression of surface marker CD73, CD90, and CD105, and the lack of expression of hematopoietic lineage markers CD45, CD34, CD11b, CD79, and HLA-DR [81].

Because of the wide distribution of MSCs throughout the body and the multipotent differentiation actions, MSCs are the most commonly used cell type in regenerative medicine for the treatment of various types of diseases, including myocardial infarction [82]. In vitro studies have demonstrated the differentiation potential of MSCs into cardiac lineage cells when media is supplemented with additional factors such as 5-azacytidine, bone morphogenetic protein-2 (BMP-2), angiotensin-II, dimethyl sulfoxide, and fibroblast growth factor-4 [83,84]. In addition, co-culture with MSCs protects cardiomyocytes from oxidative stress [85]. In preclinical studies [86,87,88,89,90], in vivo delivery of MSCs contributed to vascular regeneration and inflammation resolution in the infarcted myocardium, as well as improvement of left ventricular ejection function. Mounting data acquired from basic research has driven the implementation of clinical trials [91,92]. In a randomized, double-blind, placebo-controlled, dose-escalation, parallel-assigned clinical trial, the safety and efficacy of intravenous infusion of bone marrow MSCs for the treatment of acute MI was shown [86]. Furthermore, intravenous injection of human umbilical cord MSCs have been shown to be safe and effective for improving cardiac perfusion and function [93,94].

The abovementioned data recapitulate the therapeutic applications of MSCs for treatment of MI. However, there are also concerns regarding the therapeutic efficacy. The diverse sources of MSCs have resulted in different efficacy yields in similar studies [95]. Meanwhile, MSCs cannot form cardiomyocytes after implantation. As with other stem cell types, the potential mechanisms underlying MSCs therapy are broadly accepted as paracrine effects [95].

3.1. Promotion of Angiogenesis

Angiogenesis, also known as neovascularization, is the process by which new vasculature is generated from pre-existing blood vessels in response to pathophysiological stimuli [102]. In the setting of MI, ischemic insult destroys intrinsic blood vessels, and subsequent formation of new capillaries has been shown to promote cardiac repair [103]. In recent advances of stem cell-based therapy, promotion of angiogenesis is a key goal. Notably, MSCs are propagates to promote angiogenesis and restoration of ischemic tissues [104]. MSCs can participate in angiogenesis via direct differentiation, cellular contact interaction, and secreting pro-angiogenic factors, of which paracrine actions are considered to be the principal mechanism [105]. By analyzing the cytokine profile in secretome of umbilical cord MSCs, a series of pro-angiogenic factors, including VEGF, IGF-1, and IL-8 have been identified [106]. Moreover, MSCs have been found to stimulate angiogenic activities in endothelial cells by the activation of VEGF-A signaling pathway via secreting endothelin-1, IL-8, platelet-derived growth factor-AA (PDGF-AA), and IGF-2 [107].

In addition to the secretion of proangiogenic factors, exosome transfer from MSCs to target cells can induce angiogenesis-related cellular activities. Exosomes are extracellular vesicles with a size ranging from 40 to 160 nm in diameter [108]. They carry nucleic acids, proteins, lipids, and metabolites, and are mediators of intercellular communication. MSC exosomes promote angiogenesis by delivering microRNAs and protein factors. Various microRNAs, including microRNA-21 (miR-21), miR-126, miR-155, and let-7a, have been characterized in MSC exosomes that mediate capillary-formation behavior in endothelial cells [109,110]. MiR-21 induces tube-forming capacity of primary bovine retinal endothelial cells by highlighting the expression of VEGF [111] and miR-126 benefits vascular integrity and regulate angiogenic signaling in endothelial cells [112]. Expression oflet-7a promotes endothelial cell proliferation and motility [113]. Taken together, the abovementioned research shows the pro-angiogenic activities of MSCs mediated by paracrine effects.

3.2. Suppression of Inflammation

Numerous studies have confirmed the immunosuppressive effects of MSCs in promoting inflammatory resolution [114,115]. PGE2 is a lipid signaling molecule that prevents the maturation of dendritic cells and inhibits the proliferation of cytotoxic T cells [116]. Moreover, PGE2 participates in the repair of tissue injury by stimulating angiogenesis [117]. It has been reported that MSCs maintain their immunoprivilege by secreting high levels of PGE2, which suppresses cytotoxic T-cell proliferation and promotes the production of regulatory T-cell by inducing CCL12 and CCL5 secretion [118]. TSG-6 has been reported to play a positive role in treatment of MI with MSCs [119]. TSG-6 is highly expressed in MSCs in response to injury, which subsequently leads to increased extracellular secretion of TSG-6 protein. By activating alpha-1, a serine protease inhibitor, TSG-6 reduces cardiac inflammation [120].

Macrophages modulate the inflammatory microenvironment during injury. In the first phase of MI, infiltration of macrophage (M1 macrophage) elicits a release of proinflammatory cytokines that exacerbate the ischemic injury [121]. Following the clearance of necrotic debris, the macrophage changes its phenotype into the pro-reparative, and becomes an M2 macrophage [121,122]. Accelerating the transition of macrophage from M1 to M2 has been reported to be cardiac protective [122]. Several cytokines that are reported to promote the transition of macrophage phenotypes can be secreted by MSCs, such as IL-10 and TGF-β [123]. IL-10 is an anti-inflammatory cytokine that plays a critical role in the control of immune responses [124]. Most importantly, IL-10 has been reported to trigger changes in macrophage phenotype by promoting anti-inflammatory gene (Arg1, Mrc1 and Tgfb1) expression, which facilitates heart wound healing and improves cardiac performance [125].

3.3. Promotion of Survival and Proliferation

Numerous studies have repeatedly demonstrated the decrease of apoptotic cell counts in injury after treatment with MSCs or MSCs-conditioned medium (MSC-CM) [126,127]. The PI3K/Akt signaling pathway is crucial for regulating cell cycling, mitosis, and proliferation while suppressing apoptosis. In a mouse model of renal injury, treatment with MSC-CM increased the phosphorylation of Akt [127], which led to the dissociation of Bcl-2-associated cell death promoter (BAD) proteins and eventually suppressed apoptosis. Transplantation of hypoxia-pretreated MSCs enhanced morphological and functional improvements in infarcted hearts by increasing expression of Bcl-2 and its receptor Bcl-xL. Upregulation of Bcl-2 and Bcl-xL have been shown to prevent cell death and apoptosis under hypoxic conditions [128]. Conditioned medium from hypoxia-pretreated Akt-modified MSCs (Akt-MSCs) markedly inhibited hypoxia-induced apoptosis of adult rat cardiomyocytes. After in vivo infusion, Akt-MSCs significantly limited infarct size and improved cardiac performance as compared with controls [129]. Moreover, conditioned medium, from heat shock protein (HSP)-20, overexpressing MSCs, protected adult rat cardiomyocytes against oxidative stress via enhanced activation of Akt and increased secretion of growth factors VEGF, FGF-2, and IGF-1 [130]. To summarize, the abovementioned studies show the protective roles of MSCs on preventing apoptotic cell death through modulation of the PI3K/Akt and MAPK signaling pathways via paracrine effects [131]. Nevertheless, more studies are still required to elucidate the detailed mechanisms.

Coinciding with the activation of anti-apoptotic pathways, cellular proliferation machinery can be activated [127]. Co-culture of MSCs with cisplatin-treated renal epithelial cells has been shown to significantly increase the expression of growth factors such as FGF, EPGF, VEGF, and HGF [132]. These growth factors promoted proliferation of intrinsic endothelial cells and fibroblasts. Moreover, MSCs recruit endogenous stem cell homing to an injury site by aid of SDF-1/CXCL4 axis. On the one hand, endogenous MSCs sense the SDF-1 gradient, proliferate, and migrate towards damage [133,134]. On the other hand, MSCs secret SDF-1 to recruit endogenous cardiac stem cells [135]. These actions further accelerate the shift of the microenvironment from damaged to reparative prone.

5.1. Bioengineered Fabrication of Stem Cell Mimics

Nanomedicine is one important aspect of biomedical engineering. Nanoparticles (NPs) have attracted lots of interest in biomedical studies because of their targeting and loading properties. Secretome is the sum of all the products secreted by cells. It can be easily acquired by lyophilizing conditioned medium. To achieve high-efficient delivery, it is reasonable to develop a paradigm to fill secretome into capsule-like materials. Poly(lactic-co-glycolic acid) (PLGA) is an FDA approved biodegradable polymer that has been widely used for nanomedicine preparation. By using PLGA as the carrier, secretome derived from MSCs have been loaded to make therapeutic particles [147] (Figure 1). After infusion, these particles promoted cardiac repair. Moreover, this strategy was also used to fabricate cell-mimicking microparticles (CMMPs), in which cardiac stem cell (CSC)-derived secretome was recoated with CSCs membrane [148]. After intramyocardially injection, CMMPs contribute to the preservation of viable myocardium and augmentation of cardiac functions. The therapeutic efficacy was similar to cardiac stem cell therapy [146]. In addition to the optimization of therapeutic efficacy, construction of the secretome-containing cell mimics increased the stability of paracrine products. By fabrication of an artificial cardiac patch that is loaded with synthetic CSCs (synCSCs), Huang et al. [149] provided a clinically feasible, easy-to-store, off-the-shelf, and cell-free alternative to myocardial repair. In a rat model of acute MI, treatment with synCSCs enhanced cardiac recovery by reducing scarring, promoting angiogenesis, and boosting cardiac function. In a clinically relevant porcine model of MI, the safety and efficacy synCSCs delivery for cardiac repair was confirmed [149].

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Figure 1

Fabrication and characterization of synthetic mesenchymal stem cell (synMSC). (A) Schematic illustration of the fabrication process of synMSC. Microparticles (MP) were fabricated by treating mesenchymal stem cells conditioned media with poly(lactic-co-glycolic acid) (PLGA). Synthetic mesenchymal stem cells (synMSC) were formed by coating the MP with MSC membranes. After that, we tested the therapeutic effects of synMSC injection in mice with acute myocardial infarction; (B) Scanning electron microscopy images (left) and fluorescent images (right) of the structure of MP and synMSC. MP were labeled with Texas Red succinimidyl ester (red) and synMSC, as cell membranes were labeled with green fluorescent DiO (red particle with green coat). Reproduced with permission [147].

5.2. Bioengineered Exosomes

In addition to secretome, exosomes can also be biomedically modified to have enhanced targeting properties. Exosomes are small vesicles that are released by cells to mediated cell–cell communication. Even though the application of exosomes for cardiac therapy have achieved massive advances, it is worthwhile to note that exosomes are mostly distributed in the liver and kidney after intravenous infusion (i.v.), which may result in undesired side effects and lower therapeutic efficacy. To overcome the drawbacks, modification of exosomes with cardiac homing peptides achieved infarct-targeted accumulation of exosomes after i.v. injection [146]. With the targeted delivery, significant improvement of cardiac function in MI rats was disclosed [146] (Figure 2).

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Figure 2

Cardiac-homing exosomes. Myocardium-targeting exosomes were produced by reacting DOPE-NHS to cardiac homing peptide (CHP). Then, the lipophilic tails of the DOPE-CHP spontaneously insert into the exosomal membrane, coating the exosome in CHP peptide. The exosomes were, then, intravenously injected into rats following I/R injury. Reproduced with permission [146].

5.3. Bioengineered Construction of microRNA Mimics

During the exploration of mechanisms underlying the therapeutic effects of exosomes, microRNAs are identified as a population of bioactive molecules that mediate gene expression and modulate cellular activities. MicroRNAs belong to the class of small non-coding RNAs that are involved in the regulation of gene expression. Because the nucleic acid sequence of a known microRNA can be defined, it has increased accessibility of microRNA in pharmaceutic application [150]. Nucleic acids have difficulty crossing the cell membrane to enter the cell. To favor transmembrane delivery, agents such as liposomes, cell penetrating peptide (CPP), and cholesterol [151] are used to construct carriers for microRNAs delivery (Figure 3). Liposomes are widely used for microRNA delivery. When preparing liposomes, functionalized groups can be chemically conjugated to improve the targeting and release behaviors of liposomes. Wang et al. [152] modified miR-302 with cholesterol, a lipid soluble macromolecule. This modification enhanced the internalization of miR-302 by cardiomyocytes and led to improved outcomes after myocardial infarction.

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Figure 3

Schematic illustration of the construction of cholesterol-conjugated miRNA. (A) Fully chemically stabilized hsiRNA with either docosahexaenoic acid (DHA) or cholesterol (Chol) bioconjugates at the 3′-end of the sense strand; (B) Solid-phase synthesis of DHA-conjugated hsiRNA. Htt, Huntingtin; (C) Intracellular uptake of Cy3-DHA-hsiRNA and Cy3-Chol-hsiRNA; (D) Gene expression detection after hsiRNA incubation; (E) High-performance liquid chromatography (HPLC) traces of DHA-hsiRNAHTT and Chol-hsiRNAHTT following reverse phase chromatography. Reproduced with permission [151].

5.4. Bioengineered Antibodies for Recruitment of Endogenous Stem Cells

As an alternative to delivery of exogenous stem cells, the recruitment of endogenous stem cells provides an efficient way to repair damaged heart. To target endogenous stem cells, Cheng et al. developed iron nanoparticles chemically modified with two different antibodies, one of which bound to CD34+ cells, and the other targeted cardiomyocytes. Following administration and with the use of an external magnet, the nanoparticles captured endogenous stem cells and transported them to the infarct site [153]. In addition to this strategy, platelet membrane was also used as an infarct-homing carrier [154]. Platelets modified with CD34 antibody on the membrane recruited CD34+ stem cells to the infarcted myocardium [155]. Moreover, it is feasible to synthesize bispecific antibodies (BsAb) to capture both platelet and endogenous stem cells [156].

We thank Blanca López de Juan Abad for her help in grammatical edits throughout the manuscript.