Stem Cell Therapy of Ischemic Heart Disease

Ischemic heart disease (IHD) accelerates death of cardiomyocytes and leads to the onset of cardiac failure. Due to the application of stem cells, there exists a potential for the regeneration of a damaged myocardium. Here we present a brief review of the modern data on the application of various types of stem cells for the IHD therapy. We consider different types of stem cells, which are most preferable for the clinical application, including mesenchymal stem cells, cardiac stem cells, embryonic stem cells, iPS cells and others. In particular, we discuss their advantages and strategies which can be applied in order to boost their regenerative potential, as well as optimization of their delivery. Besides, our review refers to the contemporary achievements in the field of tissue engineering of heart, using both polymer scaffolds and scaffold-free constructs. We also discuss the most prominent known clinical trials on stem cell therapy of ischemic heart disease.


Introduction
Ischemic heart disease (IHD), or coronary artery disease, is a condition in which the blood flow to the cardiac muscle is reduced, potentially leading to a heart attack.Atherosclerosis is the main cause of IHD, with an atherosclerotic plaque's rupture being the culprit for coronary artery narrowing or blockage resulting in a severe angina and a heart attack.As a result of a heart attack, the cardiac muscle remains permanently damaged by the cut-off of the blood supply.
According to the Mayo Clinic (www.mayoclinic.org),the most common symptom of IHD is a typically leftsided chest pressure or pain (angina pectoris).Angina may also be presented as a painful feeling in a shoulder, arms, neck, back, or jaw.Other common symptoms include a faster heartbeat, palpitations, shortness of breath, dizziness or weakness, sweating, nausea and vomiting.
IHD-induced myocardial infarction (MI) is the most common cause of mortality and disability in the Western society [1].In MI, necrosis and apoptosis of cardiomyocytes accelerate, resulting in an extremal heart loading and finally in appearance of heart failure.Progressing loss of cardiomyocytes occurs not only due to an ischemic condition, but also as an adverse effect of chronic hypertension [2].On the contrary, suppressing apoptosis of cardiomyocytes leads to the heart function improvement in animal models [3].Pharmaceutical drugs, medical interventions and heart transplants, being common methods of therapy for patients with cardiovascular diseases, have shown their limitedness.To overcome these limits, various new techniques for IHD treatment have been developed as a result of scientific research of the last decades.In particular, active studies have been performed on the possibility of applications of various types of stem cells for IHD therapy since 2000s [4].It was believed earlier that cardiomyocytes could not proliferate into the adult human heart.However, as it has been found recently, adult cardiomyocytes are still capable of division and proliferation, although their ability to independently regenerate the heart, damaged by ischemic heart disease, is not sufficient [5].A large number of experiments on animals have clearly demonstrated that the damaged heart function may be restored by administration of stem cells, which were preliminarily reproduced in a significant quantity in vitro.Some of the stem cells proliferated and differentiated into cardiomyocytes in the region of the heart damage [4].However, in the majority of cases such a differentiation was limited and did not have an essential effect on the observed functional improvement of the heart, which determined a necessity of a versatile analysis of the heart regeneration mechanisms caused by stem cells.Since the heart is a post-mitotic organ, cardiomyocytes exist in the condition of the cell cycle arrest.Nevertheless, certain experiments on newts and Danio rerio fishes have shown that a damaged heart can regenerate due to resident stem cells with ability to self-renewal and differentiation into cardiomyocytes, leading to the damage repair.
Cardiomyocytes do not proliferate in the normal conditions, but in the case of a heart damage they undergo division, thus restarting the DNA synthesis and propagation via the cell cycle [6] [7].In an adult human organism, a small ability of cardiomyocytes to renewal still exists; however, only about 1% or 0.4% of cardiomyocytes renew each year in humans of 20 and 75 years of age, respectively [8].Opposite to the above, in patients with MI about 4% of cardiomyocytes in the peri-infarct zone are positive by the Ki-67 marker of cell proliferation [9], and they also show an elevated expression of specific genes, which facilitate the cardiomyocytes division and their release form the state of the cell cycle arrest [10].
Not only stem cells have an ability to self-renewal, they are also multipotent, and they can differentiate into various types of cells, including cardiomyocytes.Especially interesting are the following types of stem cells, as applied to the study of the heart regeneration in IHD: embryonic stem cells (ESC); induced pluripotent stem cells (iPSC); bone marrow stem cells (BMSC), including hematopoietic stem cells(HSC) and endothelial progenitor cells (EPC); mesenchymal stem cells (MSC); skeletal myoblasts (SkM); resident cardiac stem cells (CSC) (Figure 1).

Embryonic Stem Cells (ESC)
ESC are stem cells prepared from the inner cell mass of a blastocyst and having a totipotency.Theoretically, ESC are the most attractive type of cells for regeneration, since they can differentiate into any cell types [12].ESC have been shown to differentiate into cardiomyocytes under specific culturing conditions, such as coculturing with cells of murine visceral endoderma, with different factors facilitating ESC differentiation via the paracrine regulation [13].Xue et al. showed that, during transplantation of human cardiomyocytes, prepared by differentiation from ESC, to experimental animals, the transplanted cells incorporated into the recipient's heart tissue and performed electric and functional interaction with resident cardiomyocytes of the host organism [14].Besides, such a transplantation played an important role in the regeneration of the ischemic region in the process of the MI treatment, showing a positive effect on the condition of the left ventricle, according to the hemodynamic analysis of the heart contraction, analysis of the local disruption of the heart wall kinetics and the diastolic function of the left ventricle [15].But in general, in spite of the obtained positive results, it has been shown that only a small fraction of cardiomyocytes prepared by ESC differentiation have an innate contractile ability [16].Besides, the mechanism of ESC differentiation into cardiomyocytes has not been studied in detail [17].Besides, the use of this type of stem cells is unfavorable from the ethical viewpoint, since ESC preparation involves destruction of a human embryo which has started to develop.Moreover, ESC transplantation may lead to a tumor (teratoma) growth or an immune rejection reaction.According to these reasons, no clinical studies have been conducted in which ESC would be used as a cell therapy agent for IHD patients.

Induced Pluripotent Stem Cells (iPSC)
In 2007, Yamanaka et al. obtained artificially created cells, similar to ESC by their plutipotency, but prepared from mature diploid somatic cells with the use of the following transcription factors-Oct3/4, Sox2, Klf4 and c-Myc, which were called "induced pluripotent stem cells" (iPSC) by the authors [18].iPSC, similar to ESC, can differentiate into various types of cells, including cardiomyocytes [19], but they are not unfavorable from the ethical point of view.Nelson et al. have reported that cell therapy for the murine IHD model applying murine iPSCs differentiated towards cardiomyocytes, induced by human transcription factors, restores the impaired heart functions [20].
As in the ESC case, in spite of the positive results, the clinical use of iPSC is presumed disadvantageous, first, because of the low yield of iPSC prepared using the 4 gene types, second, because of the low efficiency of their differentiation into cardiomyocytes, third, because of the fact that the differentiated cells represent a heterogeneous mixture of different cell types, including those which are not cardiomyocytes [21].Moreover, the therapeutic application of iPSC creates the same problems as that of ESC, namely, growth of a teratoma and a potential rejection reaction after the transplantation.The iPSC studies are currently at the pre-clinnical stage, the techniques of their preparation and application are gradually perfected.

Induced Cardiomyocytes and Direct Fibroblasts Reprogramming into Cardiomyocytes
Recently performed studies have shown that postnatal cardiac or dermal fibroblasts may be directly reprogrammed into cells similar to cardiomyocytes by using three cardiac growth transcription factors: Gata4, Mef2c and Tbx5 (GMT) [22].Moreover, fibroblasts may be easily reprogrammed into cardiomyocytes if using exogenously expressed genes of pluripotency, such as Oct4, Sox2 and Klf4 [23].Indeed, Qian et al. demonstrated that, after local application of the GMT-retrovirus in a murine model of MI, genetically labeled myocyte-like cells infiltrated into the peri-infarct zone; it was confirmed that these cells were the offspring of cardiac fibroblasts [24].It was also shown that the GMT delivery in vivo reduced the size of the infarction region and somewhat relieved the heart dysfunction for up to 3 months after the coronary artery ligation.In spite of the impressive results, the gene manipulations were performed with the use of viral vectors which is undesirable in the clinical practice from the safety viewpoint.

Bone Marrow Stem Cells: Hematopoietic Stem Cells and Endothelial Progenitor Cells
Bone marrow stem cells (BMSC) are the most well-characterized stem cells, both by the characteristics of sur-face antigens and by the growth properties in vitro and in vivo.Numerous studies have shown a positive effect of BMSC on the restoration of the heart function in the therapy of cardiovascular diseases [25]- [27].A recently performed analysis of the results of 33 randomized clinical studies (1765 MI patients in total) has demonstrated that mortality and disability of the patients after the MBSC therapy only insignificantly differ from those of the patients treated with a standard therapy.At the same time, the BMSC therapy improved the indices of the left ventricular ejection fraction (LVEF) for a long period after the transplantation (12 -61 months) [28].It should be noted that BMSC are not a homogeneous cell population, they include cells of various origin, including hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC).It has been shown, that c-kit + /Lin − and c-kit + /Sca-1 + /Lin − populations of HSC improve the heart function [19] [29].Moreover, the results of the clinical studies (COMPARE-AMI) have demonstrated that CD133 + HSC also improve the left ventricular function after transplantation [30].In spite of the obtained results, the discussion on the potential differentiation of this stem cell type into cardiomyocytes is still going on [31] [32].EPC are progenitor cells capable of differentiation into endothelial cells.In a number of studies, a positive effect of EPC transplantation on the damaged heart function has been found [33] [34], although this type of stem cells does not differentiate into cardiomyocytes after transplantation [35].The observed positive effect is most likely explained by the EPC role in the stimulation of angiogenesis and sufficient recipient's cardiomyocytes supply with nutrients and oxygen, which are necessary for their survival and division, as well as for cardiomyocytes renewal from the endogenous cardiac stem cells and enhancement of the newly formed cardomyocytes' viability via the paracrine effect.It should be noted that the EPC definition remains not ultimately established, since a clear identification of markers for their separation is absent, hence, additional experiments are necessary for this problem solution.

Mesenchymal Stem Cells (MSC)
MSC are one of the most convenient for use types of stem cells of adult organism, since, on the one hand, they can be easily isolated from various tissues: bone marrow, adipose tissue, umbilical and peripheral blood and some other sources, and, on the other hand, they can be rather easily reproduced in a cell culture and, if necessary, differentiated into different cell types: adipocytes, osteoblasts, chondrocytes, cells of muscle (myoblasts and cardiomyocyte) and nervous tissues [36]- [38].Moreover, MSC are immune-privileged cells [39], hence, allogeneic MSC may be successfully used along with autologous MSC.
In 1999, Makino et al. prepared cardiomyogenic cell line from the murine bone marrow MSC by the MSC treatment with 5-azacytidine in vitro.It was shown that in that case about 30% of cells differentiated into cardiomyocytes which expressed a number of specific genes and had a phenotype similar to that of fetal ventricular cardiomyocytes [40].The same results were demonstrated by Tomita et al. (1999), who provoked a myocardial cryo-damage by administration of liquid nitrogen [41].Other researchers (Davani et al., 2003) showed that MSC, along with formation of cardiomyocytes, also formed smooth muscle and endothelial cells which also promoted the heart function improvement [42].Wang et al. launched a hypothesis that the myocardial microenvironment creates certain conditions and signals for cardiomyogenic MSC differentiation [43].
Thus, MSC carry a number of properties which make them favorable for transplantation to patients suffering from IHD: in local or systemic administration MSC are capable of homing in damaged fragments of the heart muscle, MSC are immune-privileged, the apoptosis of transplanted MSC is lower than that for other stem cell types; MSC can differentiate into cardiomyocytes; between the MSC-derived cardiomyocytes, intercalated discs are present with gap junctions allowing action potentials to spread from the host's cardiomyocytes to the transplanted cells; transplanted MSC have a strong paracrine effect.
Locally or systemically transplanted MSC into the infarcted heart provide a powerful cardiac repair action (Figure 2).
The therapeutic effect of MSC in the IHD treatment was initially associated with their ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells [41].According to [42], MSC, cultured for 15 days, expressed α-smooth muscle actin and β-actin filaments, characteristic of smooth muscle cells and nonmuscle cells, correspondingly, but no CD31 expression was observed.After injection into infarcted heart, MSC demonstrated either a smooth muscle cell (αSM actin + ) or an endothelial cell (CD31 + ) phenotype, as was shown by immunofluorescence studies.
An increased vascular density was also found in a dog model of chronic IHD [45], for which a colocalization of engrafted MSC with the markers of endothelial cells and smooth muscle cells was evidenced by immunohistological studies.
Although, in MSC transplantation the degree of their spontaneous differentiation into cardiomyocytes is low [46].Recent studies have revealed that the number of cells created by MSC differentiation is insufficient for serious functional improvements, and the paracrine mechanisms of MSC action probably play the key role in the cardiac repair in IHD [47].
Immunomodulation is at the top place among various paracrine mechanisms of MSC therapeutic effect in IHD.Since most injuries are associated with inflammation, the MSC ability to quench inflammatory processes at the site of injury appears highly likely to underlie their reparative effect in the damage of cardiac tissue.Monocyte and macrophage immunomodulation by MSC have recently been shown to play an important role.MSC were shown to inhibit production of macrophage pro-inflammatory cytokines (TNF-a, IL-6 and IFN-g), in the studies by Maggini et al. and other researchers [48]- [50].
At the same time, MSC were found to stimulate production of anti-inflammatory cytokines IL-10 and IL-12p40 [48] [49].By doing so, MSC restrict the local inflammation and minimize the cardiac tissue damage, as well as facilitate clearing the site of injury by enhancing apoptotic cell phagocytosis.Monocyte-derived dendritic cell differentiation is also controlled by MSC [51].PGE2 secretion, induced by MSC, appears to be responsible for these positive effects due to its direct action on macrophage activation.
Reduction of fibrosis and scar tissue formation [52] is another reason for the therapeutic efficiency of MSC.The studies performed on a rat model of myocardial infarction demonstrated that transplanted MSC regulated remodeling of left ventricle via decreasing mRNA expression and protein levels of TGF-β, collagens of type I and type III, as well as of tissue inhibitor of metalloproteinase (TIMP)-1 [53].
Adipose-derived MSC were shown to increase both capillary and arteriole density in infarcted areas, as a result of paracrine signaling [61].Other studies of adult stem cells in the treatment of myocardial infarction sup-port this mechanism [59] [62] [63].
A correlation between the increase in capillary density and mRNA and protein levels of VEGF was found in [56] after a treatment with adipose-derived MSC.
There exists an interesting hypothesis on the paracrine mechanisms of MSC action which is based upon endogenous cardiac regeneration provided by activation of resident cardiac stem cells and stimulation of cardiomyocytes replication by MSC transplantation.In [66], MSC were injected into infarcted pig hearts, and the newly created cardiomyocytes were found to stain positive either for c-kit or for Ki67.Transplanted BMSC may also control the host cardiomyocytes proliferation; a direct evidence of BMSC-induced proliferation and endogenous CSC differentiation was recently obtained in [67].
To date, the MSC delivery in the therapy of acute or chronic MI has been achieved mainly by intracoronary administration, sometimes combined with EPC [68]- [71].Recently, an IM MSC infusion using a catheter has been performed [72].In both cases, inversion of the left cardiac ventricle remodeling was observed, along with reduction of the infarction region size and enhancement of the regional contractile function of the myocardium (the patients' observations were conducted for 1 year).A possibility of rapid preparation of cells in the sufficient amount for transplantation, and a possibility of application of both autologous and allogeneic MSC for elderly patients with comorbidities make MSC exceptionally attractive for the cell therapy.Creation of a donor bank of MSC from young healthy donors makes it possible to provide an appropriate system of the cells' quality and consistency of their preparation.An example is randomized, double blind, placebo-controlled study of 53 patients after acute MI, in which the patients were injected allogeneic MSC (the cell drug Prochymal, Osiris Therapeutics, Inc., Baltimore, Maryland) [73].It should be noted that, due to the rate of MSC reproduction, it is possible to obtain up to 5000 ready-to-use doses of cells from one donor.MSC were infused intravenously for 10 days.Allogeneic product was well tolerated by the patients after systemic administration [74].6 months post-infusion, the LVEF increase and inversion of remodeling and, moreover, reduction of arrhythmia were observed.Similar results were obtained in Russia in the studies conducted at the Medical Radiological Research Center of the Russian Ministry of Health (MRRC, the city of Obninsk) [75].In those studies, systemic (intravenous) transplantation of (150 -200) × 10 6 autologous bone marrow MSC was applied to patients with a severe chronic heart failure (including that after MI).It has been shown that such a type of cell therapy is minimally invasive, does not cause any complications (allergic reactions, life-threatening arrhythmias, embolism, severe hemodynamic disorders etc.) and does not lead to worsening of the condition after the therapy.In the first 3 -6 months after the systemic transplantation of such cardiomyocyte progenitors, the patients experienced improvement in the myocardium contractile ability and perfusion, which manifested itself clinically as a reduction of the heart failure degree with the subsequent prolonged stable compensation (1-2 and more years of observation).
At the University Hospital, Touluse, a clinical trial "Mesenchymal Stem Cells and Myocardial Ischemia (MESAMI)" Clinical Trial Registration: NCT01076920 is conducted, which has been reported to produce rather impressive results [76].The study objective is to confirm the safety and prospects of transendocardial injections of autologous bone marrow MSC guided by 3-dimensional NogaStar XP mapping, for treatment of chronic myocardial ischemia and left ventricular dysfunction.This bicentric phase 1 study recruited 10 patients with chronic myocardial ischemia, with the following inclusion criteria: NYHA Class II-IV and/or angina pectoris CCS Class III or IV, chronic ischemic cardiomyopathy with LVEF ≤ 35%.MSC were cultured for 17 days, from the iliac crest-aspirated autologous bone marrow.14 -16 MSC transendocardial injections of the 16.8 ± 2.0 ml volume, containing 61.5 × 10 6 MSC, were performed into the intact muscle in the left ventricular scar border regions.According to the 1 month post-procedure follow-up, all patients tolerated the procedure well, no adverse events were registered.Thus, the primary end-point showed both safety and feasibility of the procedure.At 3 months post-procedure, the secondary endpoint showed that the summed stress score measured by SPECT essentially decreased from 34.1 ± 8 to 25.3 ± 9.5 (p < 0.05), although the effect did not last (32.1 ± 8.0, NS, at 12 months).The LVEF by echocardiography at 3 months was enhanced from 29.4% ± 6.5% to 35.9% ± 6.7% (6%, p < 0.001) and remained the same (35.7%± 8.1%; p < 0.001) until the 12 months time point.The other assessed parameters were NYHA, 6-minute walk test, VO 2 peak, and Quality of life.All of them improved at 3 months and lasted until 12 months time point (except for the Quality of life).Thus, the MESAMI clinical trial has shown the safety and feasibility of transendocardial injections of autologous bone marrow MSC.MESAMI 2, a randomized, double blind, multicenter, placebo-controlled trial, Registration No. NCT01076920 is meant to assess the procedure efficiency in a larger group of patients.

Skeletal Myoblasts (SkM)
Chrolologically, autologous skeletal myoblasts (SkM) were the first type of stem cells used in limited clinical studies for IHD therapy.While pluripotent stem cells, such as BMSC, can potentially differentiate into fibroblasts after transplantation into the post-infarction fibrosis region, SkM, being direct myocytes' precursors, can differentiate only into myocytes [77]- [79].Their preparation and application pose no complications of ethical or immunological character.
SkM manifested a pronounced resistance under the ischemia conditions [78].In the animal experiments, it was demonstrated that SkM transplantation led to improvement of the heart function, as judging by the LVEF enhancement [77] [79].Unfortunately, in spite of the positive effect, it has been convincingly proven that the transplanted SkM are incapable of transdifferentiation into cardiomyocytes [80].Moreover, the joint clinical studies taken place in several European clinics, MAGIC (Myoblast Autologous Grafting in Ischemic Cardiomyopathy) have shown that SkM, transplanted to IHD patients, caused serious adverse effects, namely, arrythmia [81].

Resident Cardiac Stem Cells (CSC)
Several research groups have identified a number of populations of resident cardiac stem cells (CSC), based on different markers characteristic of adult stem cells.

Sca-1 + (Stem Cell Antigen-1) Cells
Sca-1 is an antigen used for HSC isolation.However, certain results were obtained showing that Sca-1 + cells may be isolated from an adult heart and may undergo transdifferentiation into beating cardiomyocytes when cultured under specific conditions [82] [83].Transplanted Sca-1 + manifest homing into the peri-infarct zone, where they differentiate into cardiomyocytes expressing specific cardiac markers, such as Nkx2-5 и GATA4.According to the results of Wang and coauthors, transplantation of the Sca-1 + /CD31 − population of CSC led to improvement of the left ventricular function in the murine MI model due to angiogenesis stimulation [84].

c-kit + Cells
c-kit + cells can differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro, and they contribute to the myocardium regeneration after transplantation in vivo [85].The results of the SCIPIO clinical trial (Stem Cell Infusion in Patients with Ischaemic cardiOmyopathy) have shown that transplantation of c-kit + cells improved the left ventricular function [86].Zaruba et al. analyzed the cardiomyogenic potential of the c-kit + CSC isolated from a normal neonatal, normal adult and infarct adult murine heart [87].In the neonatal group, only the c-kit + /CD45 − subpopulation of cells demonstrated a notable cardiomyogenic activity, but not c-kit + /CD45 + or c-kit − /CD45 + cells subpopulations from bone marrow.It should be noted that c-kit + cells from a normal adult heart did not undergo cardiomyogenic differentiation either when co-cultured with fetal cardiomyocytes, or when transplantated into a normal or infarcted heart of an adult mouse.The obtained data testify that, in the process of an organism development, resident c-kit + cardiac cells lose their ability of acquiring the cardiomyogenic phenotype.These results differ from numerous earlier data on the c-kit + cells isolated from the rat or human heart.The co-culture of these CSC with cardiomyocytes or their injection into a damaged myocardium resulted in a more powerful cardiomyogenic differentiation [88] [89].
It is worth attention that the genesis of the c-kit + cells in the heart still remains not entirely known.In particular, experiments of Fazel and coauthors [90] on the bone marrow transplantation suggest that many of the c-kit + cells in the adult heart are of a bone marrow origin, indeed, the c-kit is expressed not only in CSC, but also in the bone marrow cell populations [30].

Isl-1 + Cells
Isl-1 play an important role in the development of different organs, including the heart [91].Laugwitz et al. first identified Isl-1 + CSC in a mouse, rat and human postnatal myocardium [92].The further investigations showed that Isl-1 were absent in an adult heart [93]- [96].It has been recently demonstrated that Isl-1 + cells participate in the formation of all the basic types of a murine heart cells [91] [92] [97] [98].Isolated Isl-1 + cells proliferate and differentiate into cardiomyocytes, endothelial cells and smooth muscle cells after transplantation [98].In contrast to Sca-1 + CSC and c-kit + CSC, Isl-1 + CSC do not express other markers of cell surface.

Side Population Cells (SP)
SP cells were isolated using a vital dye (Hoechst 33342 or Rhodamine 123), by sorting out the cells mostly "excluding" the dye and thus obtaining so-called "side population cells".SP cells were found in different organs, including bone marrow, skeletal muscle, adipose tissue, and the heart [99] [100].SP cells express Sca-1 and can be transdifferentiate into cardiomyocytes with leads to improvement of the left ventricular function after transplantation [101].These stem cells express Nkx2.5, GATA4 and Mef2c after differentiation into cardiomyocytes.SP cells can migrate to the damaged heart fragment after appearance of the damage.In general, SP cells are promising for cell therapy, but the use of a toxic dye for their preparation is currently limiting their application by only experimental animal models.

Cardiosphere-Derived Cardiac Cells (CDC)
CSC populations have a tendency to clonogenicity and can form spheroid aggregates in culture, so-called cardiospheres [102].CDC cells-cells, isolated from cardiospheres-can differentiate into endothelial, smooth muscle cells, as well as into cardiomyocytes [103].Various studies have shown that CDC cells improve the left ventricular function [89] [104] [105].Unfortunately, in contrast to other CSC populations, CDC cells preparation requires a rather long time because of the slow growth of cardiospheres.Besides, CDC are frequently contaminated with other types of cells, such as with heart fibroblasts.

In Vitro Modification of Stem Cells
Stem cells modification in vitro is used to improve their biological functions playing a major role in their transplantation in vivo.Among those are: their capability of homing, i.e. migration into a target tissue, survival in the ischemia conditions, proliferation, synthesis of paracrine factors, ability to transdifferentiation.Here we consider the techniques for stem cells modification, by the example of MSC.

MSC Preconditioning
In the course of MI, the blood flow to a part of myocardium is interrupted, hence, cardiomyocytes are undersupplied with oxygen, the level of which drops to 0.2% [106].Soon after that, the combination of residual tissue hypoxia, oxidative stress during "ischemia-reperfusion", death of cardiomyocytes and inflow of inflammatory cells create an unfriendly medium for stem cells potentially transplanted into the damaged myocardium.Since MSC naturally exist in the bone marrow in the conditions of low oxygenation (~2%) [107], they are presumably rather tolerant to the ischemic conditions in infarcted myocardium.However, there exist possibilities for even better enhancement of the inherent MSC stability, if they are undergone pre-conditioning (prior to transplantation and incubation) with different factors.Indeed, it has been shown in in vitro and in vivo stidues that MSC subjected to hypoxia produce an elevated amount of transcription and growth factors, enhancing the cells' survival, including VEGF, HIF-1α, survivin and Bcl-2, as compared to MSC incubated in normal conditions [108].The enhanced survival and functional advantages of MSC, preconditioned in the hypoxia conditions, have been demonstrated in their transplantation in MI models [106] and in diabetic cardiomyopathy models [109].
In particular, hypoxic pre-conditioning may provide advantages for enhancing the cells' survival via the induction and stabilization of intracellular HIF-1α, which undergoes nuclear translocation to bind several important promoters.The subsequent target it can affect is glucose-6-phosphate transporter serving to increase the glucose concentration in cells via enhancement of gluconeogenesis.Besides, MSC can be pre-conditioned with agents causing formation of nitrogen oxide [110], with hydrogen peroxide or diazoxide [109] to boost their survival and paracrine effect.
Diazoxide is known as a stimulator for opening of mitochondrial ATP-sensitive potassium channels (Mito-KATP), which is believed to provide a cell's protection in the ischemic stress.A brief incubation of MSC with diazoxide has been shown to strengthen the cell resistance due to increased expression of factors facilitating the survival, as well as of signal factors (VEGF, HGF, NF-κB, Akt factors of signal pathway, microRNA-146a etc.) [111] [112].It has been known for several decades that heat shock proteins (HSP) play a key role in a cell's protection from a number of ecological stress factors.HSP transcription may be induced in cultured cells under hyperthermia (e.g., incubation at 42˚C for 1 hour).This approach was applied to skeletal myoblasts pre-conditioning to increase their survival inside myocardium in vivo [113], while MSC were genetically modified for an elevated expression of various HSP before transplantation [114] [115].Although the specific mechanisms of the HSP effect on MSC remain unclear, the experimental data testify that these proteins inhibit regulators of necrosis and apoptosis pathways and finally activate Akt.Moreover, they also may enhance the trophic properties of MSC via increasing the secretion of various soluble factors, such as VEGF, FGF-2 and IGF-1 [114].A wide spectrum of pharmaceutical drugs have been investigated either in combination with MSC, or as an additional therapy during cell transplantation (e.g., statins [116] [117], sildenafil [118]), or as drugs for cells pre-conditioning.The last examples include application of phosphodiesterase inhibitors [119], modulators of angiotensin signal pathways [120] and neuropeptide Y [121].These agents are believed to have different mechanisms of action.It has been recently suggested that a block of angiotensin II may enhance human MSC both in experimental and clinical conditions.However, the MSC nature is rather complicated, especially regarding their translocation from a controlled microenvironment in a culture into a dynamic and unpredictable microenvironment of a damaged heart.One of the primary tasks in the field of transdifferentiation towards cardiomyocytes [120].A great attraction of preconditionining is related to its simplicity cell therapy optimization is deciphering the most important signal pathways involved in the processes of cell survival and accomplishment of the cell's repair function after transplantation, thus the strategy of preconditioning should be directed towards the highest benefit in this context.

Enhancing the Effect of Paracrine Factors
As described above, a significant part of the MSC reparative potential in IHD is related to production of a wide spectrum of soluble paracrine factors.Essential efforts have been directed towards the support of this paracrine potential with the use of either transfer of appropriate genes, or pre-incubation with agents which cause an elevated expression of cytokines or growth factors.
VEGF (vascular endothelial growth factor) is an important regulator of MSC-induced vasculogenesis and angiogenesis а [122] and can be excessively expressed in MSC via the induction of HIF-1α.MSC pre-incubation with TGF-β [123], SDF-1α [124] and lipopolysaccharides [125] has also been shown to increase the VEGF cell production accompanied by advantages for cell survival after transplantation and enhancement of angiogenesis and myocardium regeneration in rodent MI models.Adenoviral rat MSC transfection with the human VEGF165 gene was also a successful "hybrid" strategy for transplantation of cells and genetic therapy to improve the therapeutic angiogenesis after MI [126].
IGF-1 (insulin-like growth factor-1) possesses a pleiotropic activity, as well, which affects the growth, proliferation and survival of cells, predominantly via the activation of signal pathways of Akt and MAP-kinase.MSC preconditioning with IGF-1 enhanced homing and survival of cells in the myocardium after a systemic administration and led to functional advantages, as compared to common MSC [127].
An important component of the MSC paracrine effect is their ability to attract the corresponding cells, including hematopoietic, endothelial or cardiac progenitor cells, into the region of myocardium damage.SDF-1α and its G-protein transmembrane receptor (CXCR4) plays a key role in the cell attraction from the bone marrow into the region of infarcted myocardium [128].Immediately after MI, the SDF-1α level in the blood serum and in the myocardium rapidly grows and reaches a peak in 48 and 72 hours, respectively, then it returns to the basic level.This facilitates homing of progenitor cells in the region of myocardium damage, including circulating, pro-angiogenic CD34 + cells which promote neovascularization.Besides, SDF-1α may also have a direct proangiogenic effect, inducing expression of HIF-1α and VEGF in cells [129].In models of cardiovascular diseases, mechanisms of SDF-1α/CXCR4 were manipulated in different ways to enhance the myocardium function and perfusion, as well as the reparative properties of transplanted MSC.In one of the studies on a rodent model of MI, SDF-1α was administered directly into myocardium as an addition to an intravenous MSC injection which resulted in a better homing and survival of those exogenous cells in the recipient's heart, the effect being neutralized if a functional blocking antibody was added [130].MSC pre-incubation with SDF-1α also demonstrated pro-mitogenic and anti-apoptotic effects in the course of incubation in vitro with hydrogen peroxide and after transplantation into an infarcted myocardium [124].In the latter case, it was accompanied by a decrease in the size of the infarction and fibrosis region, as well as by improvement in the heart function, as compared to the common MSC.A similar improvement of the MSC properties was observed in the cells transduction for a stably elevated SDF-1α expression [131].After transplantation into the heart, transduced MSC were more resistant to apoptosis and better survived than unmodified MSC which led to a decrease in the collagen deposition and to expression of matrix proteinases.In other preclinical studies, MSC were used with hyperexpression of HGF [132] and other factors (e.g.CCR1 [133]) for boosting their therapeutic potential.Additional advantages were obtained when performing a double transfection of MSC with VEGF and SDF-1α genes [134].

Activation of Cytoprotector Pathways
Cytokines, described above, accomplish sophisticated interactions and functions with different cell and tissue substrates.A significant part of their ability to protect cells from death is regulated via the effect on mechanisms of the Akt signaling pathway (Figure 3).
The Akt gene codes serine-threonine protein kinase which stimulates further signal pathways (e.g., PI3K/ mTOR) under activation and finally, as a result, inhibits BAD, a protein of the Bcl-2 family and, vice versa, activates NFκB [135].This leads to inhibition of apoptosis and transcription of genes activating the cells' survival, respectively [136].The activation of the Akt gene expression is also known to stimulate angiogenesis and overcoming the cell cycle arrest [137].Correspondingly, manipulations with mechanisms of the Akt signal pathway in stem cells are considered positively as a way of survival optimization for both transplanted cells and cardiac and vascular cells of the host organism.In a series of highly cited articles on fundamental and preclinical studies, Dzau et al. have demonstrated that not only the reparative MSC properties are mostly provided by their paracrine effect, but also these properties can be notably improved via the genetic modification of MSC for Akt [138] [139].The advantages of Akt-transfected MSC vs. intact cells have been demonstrated in vivo not later than 72 hours after transplantation into an infarcted rat myocardium.In particular, the cardiac cells apoptosis was essentially reduced, the MSC survival was enhanced, and formation of VEGF, HGF and IGF-1 in the myocardium was stimulated.Impressing results were obtained even when using only the conditioning medium from cultures of Akt-transfected MSC which emphasized the paracrine basis of their cytoprotector and reparative properties.It has also been demonstrated very recently that the cell therapy of MI with the use of Akt-transfected MSC is also accompanied by restoration of the normal metabolic function of the heart, with a limited use of highly energetic phosphates and normalization of the myocardium pH and glucose metabolism [140].The genomic analysis has Figure 3. Activation of PI3K/Akt signaling pathway in MSC for cytoprotection [135].identified that Akt-transfected MSC have an elevated content of the Sfrp2 protein, a key mediator of the cytoprotector paracrine properties of these cells [141].
Another strategy used for enhancement of the MSC cytoprotector properties is their modification for hyperexpression of integrin-linked kinase (ILK) [142] and heme cyclooxygenase-1 (HO-1) [143].Heme cyclooxygenase-1 enzymatically destroys heme to bilirubin, carbon monoxide and free iron and has an anti-inflammatory, antioxidant, anti-apoptotic and pro-angiogenic effect.Elevated HO-1 expression in MSC enhances the survival of transplanted cells and improves the left ventricular function of a porcine heart in the "ischemia-reperfusion" model [143].Thus, the studies described above demonstrate good opportunities for boosting the trophic potential of MSC, their resistance to stress and apoptosis, as well as their ability to the heart repair.Besides, they help to disclose many of the critically important molecules and signal pathways, regulating the adult stem cells functions (survival, proliferation and migration).
To date, a development of technologies for the clinical use of the above strategic methods is in need.Although the genetic modification of MSC and other cell types creates a number of formal questions concerning the safety of their application, some results have already been obtained.Currently, the ENACT-AMI clinical trial is going on (clinicaltrials.govNCT00936819), in which the patients with severe MI undergo administration of circulating mononuclear cells (MNC), transfected with the gene of human endothelial NO-synthase (eNOS) [144].

Manipulation of the MSC Cardiomyogenic Potential
Efforts are also applied to increase the MSC potential towards their cardiomyogenic differentiation.As was mentioned before, the initial attempts to verify and then to manipulate the cardiomyogenic potential of MSC were focused on the use of 5-azacytidine, a DNA-methyl transferase inhibitor [40].Although this non-specific agent may promote a moderate MSC transdifferentiation into cardiomyocytes, its action was less reproduclible in human MSC, than that in murine MSC [40].Besides, pre-conditioning with 5-azacytidine may have a genotoxic effect on cells with consequences for their safe transplantation in vivo.Preconditioned MSC also appear in the state of the cell cycle arrest, thus losing their replication potential required for cardiomyocytes repopulation.
Another strategy consists in a directed MSC cardiopoiesis.Cardiopoietic programming was developed as a way of ESC modification, in which the cells are subjected to exposure to a combination of specific recombinant factors usually present in embryonic medium, in order to boost their cardiomyogenic regenerative potential [145].Important mechanisms of differentiation into cardiomyocytes have been discovered, where the members of TNF-α, TGF-β and FGF families act as essential regulators of stem cells cardiopoiesis, which, in combination with each other, can reprogram ESC into cardiomyocytes without the carcinogenesis risk [146].
Recently, the principle of directed cardiopoiesis has been spread to MSC from the human bone marrow [147].As a result of different IHD patients screening, a rare patients' subpopulation was identified, whose MSC demonstrated a spontaneous ability to improve the myocardium contractile function, along with a high expression of de novo early and late cardiac transcription factors (e.g., Nkx2.5, TBX5, MESP1, MEFC2).These cells underwent the action of a combination of recombinant factors consisting of TGF-β1, BMP4, activin A, retinoic acid, IGF-1, FGF-2, alpha-thrombin и interleukin-6 which consolidated the cardiospecific MSC potential.In a murine MI model, transferred into the chronic phase of the heart muscle damage, the delivery of cardiopoietic MSC allowed stable functional and structural benefits without adverse consequences, as compared to intact MSC.It was related to a higher degree of the MSC capture within the myocardium, their more efficient transformation into cardiomyocytes in vivo and in a higher paracrine stimulation of endogenous c-kit + cardiac stem cells (CSC).
This work was continued in the form of clinical studies in Europe, in which cardiopoietic MSC were used for the therapy of patients with chronic ischemic cardiomyopathy [148].Cardiopoietic MSC (0.6 -1.2 × 10 9 ) were transendocardially transplanted to 21 patients into a viable dysfunctional myocardium of the left ventricle.At the average, in slightly less than a year, the left ventricular ejection fraction increased more notably in MSC recipients than it did in the control group (absolute increase by 5.2% vs. 1% in the control group), besides, an improvement of the functional state was observed (by the results of the 6-min walking test) and reduction of the arrhythmia episodes.

MicroRNA and Cardiomyogenic Differentiation of MSC
As was found in the last years, microRNAs play an active role in the processes of MSC renewal and differentia-tion [149] [150].MicroRNAs (miRNAs) are endogenous, non-coding RNA molecules, of 20 -23 nucleotides of length, negatively regulating gene expression in different biological and pathological processes, including cell differentiation, proliferation, apoptosis in cardiac diseases, neurological diseases and cancer in humans [151]- [155].MicroRNAs participate in MSC regulation at different stages of this cell type development.In general, the mechanisms of microRNA expression differ for intact MSC and for completely differentiated cells, e.g.osteoblasts, adipocytes and chondrocytes, which suggests that microRNAs are important at the stage of MSC differentiation decision.Indeed, a low, or, on the contrary, a high level of a certain microRNA expression may be a preliminary determinant condition for MSC commitment and differentiation into specific cell lines, as was demonstrated in the studies by Guo and coauthors [156].For instance, it was demonstrated that the reduced level of miR-138 expression was associated with osteogenic [157] and adipogenic [158] differentiation of human MSC.Other microRNAs, miR-204/211 and miR-637 control the balance between MSC differentiation into adipocytes and osteoblasts [159] [160].It is extremely interesting that such microRNAs as miR-1, -206, -24 and -181 may induce MSC differentiation into cardiomyocytes in vitro.In the presence of 5-azacytidine, miR-143 and miR-181 are expressed, while indirect co-culture of MSC with neonatal rat myocytes leads to an elevated expression of miR-143, -206, -208 and -181 [161].AT the same time, miR-133 may block this differentiation [161].Moreover, Liu et al. (2012) have recemtly shown that miR-16 is involved into myogenic differentiation of human MSC in the cardiac microenvironment (a niche of resident cardiac stem cells) [162].
Hyperexpression of miR-16 significantly increased the delay of human MSC in the G1-phase of the cell cycle and enhanced expression of the cardiac tissue marker genes: GATA4, Nkx2-5, MEF2C and TNNI3.Finally, it induced MSC differentiation into cardiomyocytes in the cardiac microenvironment.The use of miR-16 may represent a promising approach to MSC pre-modification before their transplantation to IHD patients.

Optimization of MSC Delivery
An imperative requirement for efficient myocardium repair is the sufficient number of viable MSC to reach the corresponding target sites soon after transplantation and remain there for a long time, with appropriate survival, proliferation and functioning.
The cells can be administered to the heart: 1) via a systemic intravenous injection, 2) regionally, via an infusion into a coronary artery or vein, 3) locally, via a direct transepicardial, transendocardial or intrapericardial implantation.Unfortunately, in spite of some differences in the efficiency of these various ways of cell delivery, the MSC preservation and their survival in the myocardium remains imperfect in all the cases [163].
Fluoroscopically guided intracoronary infusions is one of the most wide-spread techniques applied in the clinical cell therapy.This technique is used to distribute the cells in the affected ischemic artery region [164]- [166].Its advantages include a low cost, minimal invasiveness, relatively short duration and a possibility of immediate accomplishment via the primary transcutaneous coronary intervention in the acute MI therapy.But the use of this technique is associated with a potential risk of adhesive MSC aggregation inside the coronary microvessels [167]- [169].MSC administration via an IM injection (open transepicardial or transendocardial, transcutaneous using a catheter) delivers MSC into the corresponding myocardial regions more specifically.Such a direct injection probably has advantages related to cardiac cells preservation [170], capture of non-resident cells [168] and the general therapeutic effect [171], as compared to systemic or intracoronary delivery.There is some potential risk of creation of foci with electrophysiological heterogeneity and arrhythmia.However, it is not characteristic of MSC and mainly specific for skeletal myoblasts [172].Although the cells' preservation and their distribution is similar for transendocardial and transepicardial injections [173], transcutaneous delivery via a catheter is less invasive and thus presents wider possibilities for the clinical use [174].

Cardiac Tissue Engineering
Cardiac tissue engineering is a complex of new technologies based on application of stem cells, biocompatible natural or synthetic matrices, and also, in certain cases, growth factors, differentiation and pro-angiogenic factors, inducing the processes of the cardiac tissue regeneration.A perfect artificial cardiac tissue will reproduce structural, mechanical and electrophysiological properties of the native cardiac tissue, which provide the viability of transplanted stem cells, as well as stimulate vasculogenesis in the implanted tissue.
Stem cell culturing in temperature-sensitive polymer plaques facilitates separation of cell monolayers without the need of using enzymes [175].Such a construct adheres to the ischemic zone providing intra-cardiac implan-tation of the monolayer cells.Besides, formation of new blood vessels and a general functional improvement are observed as a result of combined implantation of various MSC layers, derived from adipose tissue, in a murine model of chronic MI [62].Moreover, the use of neonatal cardiomyocyte monolayers generated the processes of intercellular communication activating the myocardium contractile function and signal transfer inside the construct [176].Besides, the intercalation of endothelial cell monolayers promotes formation of new blood vessels in the ischemia region.
Another approach is based on creation of natural hydrogels, such as Matrigel™ (laminin, type IV collagen and heparin sulphate) [177], collagen [178] or fibrin, in which stem cells are embedded for the subsequent intra-myocardial injection.Although the hydrogel effect was positive for the cells fixation [177]- [179], the injection pressure necessary for its delivery is too high and causes a notable cell death which finally decreases the therapeutic effect.Besides, it is difficult to sterilize and purify natural hydrogels [180].As an alternative, synthetic hydrogels have been developed (polyethylene glycol, polylactic acid, polylactic acid-co-glycolic acid, polycaprolactone, polyacrilamide and polyurethane), in which such disadvantages are minimized [181], the US Food and Drug Administration having approved clinical application of polyethylene glycol, polylactic acid and polylactic acid-co-glycolic acid.
There exists an alternative to IM injections of hydrogel based on preparation of a new tissue ex vivo from stem cells incorporated into the hydrogel.Two recent studies report the contractile ability of prepared in vitro constructs involving embryonic cardiomyocytes [182] or neonatal rat cardiomyocytes [183].The 3D environment favors the intercellular communication and prevents the cell death [184], and finally the prepared constructs form the extracellular matrix on their own [185].In the clinical trial MAGNUM (Myocardial Assistance by Grafting a New Bioartificial Upgraded Myocardium) a type I collagen matrix is used, with a size sufficient to completely cover the myocardium scar.In this trial, a common cardiomyoplastics was compared with combined cardiomyoplastics and tissue engineering, and the conclusion was made that the new alternative suggests better results from the viewpoint of functional restoration and ventricular remodeling [186].
For the development of cardiac tissue engineered constructs based on innovative materials (an elastomeric skeleton and hydrogel-PuraMatrix™), a European consortium has been recently opened (RECATABI, REgeneration of CArdiac Tissue Assisted by Bioactive Implants) [187].The preliminary results have shown a cardiomyogenic differentiation of implanted cells and formation of vascular connections between the construct and native myocardium.
A promising material for tissue engineering is extracellular matrix.Extracellular matrix consists of functional and structural proteins, such as collagen, elastin, laminin, fibronectin, proteoglycans and many others [188] [189].Since the matrix participates in many cellular processes, including proliferation, differentiation and migration [190], it is potentially attractive for cardiac tissue engineering, both due to favoring implantation of cells which repair the damaged myocardium, and due to a possibility of the damaged tissue replacement with the matrix itself.Extracellular matrix lamina was successfully isolated from different tissues, including native cardiac valves [191]- [193], and blood vessels [194] [195].A possibility of application of animal matrix, in particular, porcine matrix, is also very promising in the human tissue regeneration [196].

Conclusion
The use of stem cell therapy is an exciting and dynamic field of research, with a great potential for improvement in the health of patients with cardiovascular diseases, being the main cause of death in developed countries.While the advantages of this therapy for the cardiac function improvement after MI and in heart failure have been extensively demonstrated in animal studies, the results of clinical studies have sometimes been disappointing.However, the results of the clinical trials, conducted in the last decade, give hope for a rather rapid creation and wide use of a safe and efficient medical technology based on transplantation of stem cells to cardiology patients, i.e. for realization of methods and approaches of regenerative medicine.The accumulated data allow expecting good prospectives for therapeutic use of stem cells, especially, autologous and allogeneic MSC, as most studied and safe cells among all the stem cell types.It is evident that the MSC application needs additional perfection of techniques for MSC isolation and identification, study of molecular mechanisms for their paracrine effect, as well as of possibility of survival, proliferation and differentiation of cells in the myocardium, use of accompanying pharmaceutical drugs for stem cells transplantation and other means of enhancing the therapeutic effect of stem cell therapy.Cardiac tissue engineering provides an excellent prospective in this field, since ar-tificial cardiac tissue will be able to maintain structural, mechanical and contractile function of the native cardiac tissue, as well as viability of transplanted stem cells and vasculogenesis in the implanted tissue.

Figure 1 .
Figure 1.Stem and progenitor cells which are being studied for a potential cell therapy of IHD, and the ways of their delivery onto the heart [11].