In healthy human hearts, only 10-20% of the total cells are contractile cardiomyocytes and, at the age of 25 years, no more than 1% of them are annually substituted by progenitor cells, this percentage reducing to less than 0.5% at the age of 75. In total, less than 50% of cardiomyocytes are renewed during a normal human life span . For this reason, the topic of cardiac repair is among the major challenges for the tissue engineers worldwide. In fact, cardiac diseases are a predominant cause of mortality and morbidity in industrialized countries, despite the recent advancements achieved in pharmacological treatment and interventional cardiology procedures. Nonetheless, end-stage heart failure management still relies on organ transplantation as unique approach, and, notwithstanding the use of massive immunosuppressive drugs, still a percentage falling within 20%-40% of patients encounters immune rejection during the first year post-transplant . Among the patients not facing severe immune rejection, almost 70% is forced to retire or reduce their working activity, their survival rate falling below 70% during the first five years post organ transplantation . Last, but not least, the economic impact of cardiovascular diseases and stroke has been estimated in 2010 at $503.2 billion .
Currently, post-infarction myocardial revascularization protocols include the administration of raw bone marrow stem cells, while a number of clinical trials have been performed or are currently in progress in which different cell subsets are implanted in the damaged tissue by means of surgical techniques. The results of such trials are still controversial. In fact, when autologous skeletal myoblasts were injected into the heart of patients suffering from ischemic cardiomyopathy, the modest functional improvement obtained was impaired by the arising of arrhythmia events, thus requiring the adoption of a pacemaker . On the other side, intracoronary administration of bone marrow mesenchymal stem cells resulted in minimal improvements in cardiac contractile function in patients with dilated cardiomyopathy . These mild results were mostly ascribed to a paracrine effect exerted on host tissue, rather than to a direct contribution of stem cells to the contractile activity.
Thus, among the criticisms to be challenged before efficient cell therapy protocols for cardiac diseases can be setup, the choice of the appropriate cell subset to generate new vessels and contractile cardiomyocytes, as well as the route of cell delivery remain key steps. The solution of such problems requires additional efforts in basic research to clarify the processes leading to stem cell differentiation as well as technological advancements to setup efficient protocols to implant the cells.
In principle, adult stem cells could be extracted from patient’s own tissues and expanded in culture by means of well-known techniques (Figure 1).
Nonetheless, a number of issues should be challenged before safe procedures to manipulate stem cells
The employment of autologous stem cells would avoid the problem of immune rejection and the need for immune-suppressive drugs, while, in the treatment of pathologies for which a genetic basis is suspected the use of autologous cells is hampered. As far as the use of autologous cells is concerned, the possibility that a significant patient-to-patient variability in stem cell quality exists should be taken into account . Finally, the use of cellular and tissue-based products in human disease therapy is subjected to regulations issued by the European Union and Food and Drug Administration (FDA) aimed at establishing classification criteria for advanced therapy medicinal products (ATMP). In particular, the European Regulation states that human cells to be used in cell therapy have to comply with the principles of Good Manufacturing Practice (GMP) protocols [11, 12].
2. Adult stem cells for cardiac repair
A number of stem cells and progenitors have been so far proposed for cardiac repair, due to the inability of cardiomyocytes to proliferate after birth . Among the cell sources challenged for the possibility to produce new cardiomyocytes, skeletal myoblasts have proven to be able to acquire a contractile phenotype
The role of hematopoietic stem cells (HSC) in cardiac repair has been investigated by several research groups and their contribution to cardiac regeneration
The presence of a small reservoir of cardiac resident progenitor cells (CPC or CSC) has been recently demonstrated in human as well as in other mammals’ heart . Such tissue-resident cells participate in myocardial homeostasis and retain a limited regenerative capacity throughout organism lifespan . All the subsets so far identified through the expression of stemness markers (c-kit+, Sca-1+, Islet-1+) demonstrated the ability to give birth to new contractile cells
3. Stem cell delivery to the injured heart
As previously said, cell route of delivery to damaged heart represents the major topic in the setup of efficient, minimally invasive techniques to treat cardiac pathologies. Recently, a number of techniques to deliver stem cells to the injured site have been proposed but questions remain regarding the optimal approach able to favor high cell retention, differentiation rate and clinically relevant improvement in cardiac performance.
a) Direct injection
Stem cell direct
Stem cells can be delivered
Finally, an interesting attempt with stem cells being injected into the
b) Injectable scaffolds
Injectable scaffolds are defined as materials offering the unique solution of replacing damaged myocardial ECM and/or delivering cells directly to the infarcted region while holding the potential for minimally invasive delivery . Such scaffolds can be composed of biocompatible microspheres or in situ gelling materials having reasonable dimensions as to surpass capillary barrier. They are considered a promising tool for stem cell delivery to damaged myocardium. In situ gelling materials are generally made of components of extracellular matrix (ECM), which are induced to a transition after being implanted
The use of injectable, synthetic microspheres has already been proven promising in the treatment of neurological diseases
c) Scaffold-based technology
The possibility of using biocompatible scaffolds to deliver stem cells to the injured heart has been explored by a number of independent research groups so far. The scaffolds proposed are natural of synthetic but when designing cardiac-specific constructs, a number of requirements should be fulfilled. For example, it cannot be neglected that myocardial contractile function relies on the transmission of electrical and mechanical forces throughout a functional syncytium. So, the integrity of the tissue has to be preserved. For this reason, a cardiac-specific scaffold should comply with tissue architecture and thus be deformable enough to indulge and, if possible sustain cardiac contraction. Moreover, as far as stem cell engraftment is concerned, scaffolds should be able to start at least cell alignment and commitment to favor stem cell electromechanical coupling with host tissue. In this respect, the work of Mandoli and collaborators using Cerium Oxyde nanoparticles to affect poly-lactic acid film surface and obtain a controlled nanorugosity appears intriguing . In fact, far from being a noxious compound for stem cells, ceria was able to induce cardiac stem cell alignment and growth. Nonetheless, cardiac tissue is extremely complex and highly demanding in terms of blood supply and catabolite removal, so that porous scaffolds that could allow microvascular branches formation and oxygen perfusion are to be preferred. To fulfill such requirements, the first attempts were performed by the group of Thomas Eschenhagen. Neonatal cardiomyocytes were seeded in Collagen I + Matrigel to produce Engineered Heart Tissue (EHT). Continuous contractile activity up to 1 week
d) Preparation of thick cardiac substitutes by Scaffold-free technology
To overcome the problem of poor cell retention reported in cell injection experiments in the heart  and avoid the release of possibly harmful scaffold byproducts, scaffold-free technology has been developed, in which cells are grown in a monolayer onto thermo-responsive surfaces and easily detached in the form of cell sheet by lowering the temperature . Such technology takes advantage of the ability of polymers like poly-N-isopropylacrylamide (PNIPAAm) to shift between hydrophobic and hydrophilic status when the temperature ranges from 37ºC to 32ºC. Cell sheets can be serially stacked to obtain multilayered scaffoldless constructs (Figure 3). Such an approach has already been applied to obtain cell sheets composed of rodent [48, 49] and human  cells. Given the need for thick cardiac substitutes suited to comply with cardiac muscle continuous contractility, thermo-responsive technology has been envisaged as a possible answer to the lack of heart donors. Pre-clinical trials performed onto experimentally infarcted animals demonstrated that when a murine adipose-derived monolayer sheet is leant onto injured myocardium, it can be retained and help tissue repair . Similarly, striking results are obtained when a Sca-1+ cardiac progenitor cell-derived sheet is used . Finally, an interesting approach has been recently proposed to deliver cardiac stem cells cultured in the form of cardiospheres to the injured heart: cardiospheres were embedded into a cardiac stromal cell-derived sheet obtained by using poly-lysine/ collagen IV-coated dishes . The formation of mature vessels as well as new cardiomyocytes
4. Clinical trials
In the attempt to transfer bench experience to bedside, a number of clinical trials in which different stem cell or progenitor subsets are used have been approved (see http://www.clinicaltrials.gov). Most of them are still in the recruitment phase while some already gave indications and preliminary results. Since most of the ongoing trials are based on the injection of raw stem cell preparations (mostly bone marrow-derived cells), the time and route of cell application remain the key problems to be addressed before proceeding to routine clinical practice. In this respect, recent animal experiments demonstrated that the acute phase of myocardial infarction is probably not suitable for stem cell engraftment and differentiation . Therefore, the right moment in which stem cells should be delivered is to be studied. An overview on some of the ongoing clinical trials is given below.
1.MAGIC (Myoblast Autologous Grafting in Ischemic Cardiomyopathy). In one of the first phase II clinical trials setup to study the possibility to use stem cells to treat cardiac pathologies, ninety-seven (97) patients undergoing coronary artery bypass grafting (CABG) were enrolled. 400-800 X 106 autologous myoblasts harvested from patient muscle biopsy were implanted in the akinetic area of ventricular wall 21 days after
2.TOPCARE-CHD, -AMI, -DCM (Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction, Chronic Stable Ischemic Heart disease or Dilated Cardiomyopathy). In this complex clinical trial, a total of 346 patients were classified to CHD, AMI or DCM pathologies and infused either with bone marrow cells (BMCs), blood-derived stem cells, or no infusion. In TOPCARE-CHD, 121 patients (mean age: 59) with chronic stable ischemic heart disease (CHD) were treated. Although complications occurred in 21% of the patients during 3 months follow-up, BMC intracoronary administration was related with a reduction of both brain and atrial natriuretic peptide (NTP) serum levels (indicators of LV remodelling process) in the remaining population (79%), especially in patients with higher NTP levels at baseline and receiving a greater BMC number with a high functional capacity. Moreover, these results were also correlated with a left ventricular ejection fraction (LVEF) increase and better survival during the further follow-up, suggesting that cell therapy could be associated with cardiac function enhancements in patients with advanced chronic post-infarction heart failure . Similarly, two hundred and four (204) patients were treated using bone-marrow-derived progenitor cells directly into the infarct artery three to seven days after an acute myocardial infarction (AMI). A statistically significant 2.5% improvement in left ventricular ejection fraction at four months was reported for patients randomized to the bone marrow injection . Finally, intracoronary infusion of bone marrow cells was performed in 33 patients with dilated cardiomyopathy (DCM) by using an over-the-wire balloon catheter. Three month follow-up demonstrated an improvement in left ventricular pump function while a modest improvement in Brain Natriuretic Peptide (BNP) levels was reported after 1 year . Importantly, the conditions chosen in the present clinical trial were representative of different conditions (acute, chronic phase) encountered in the clinic. Unfortunately, no clear indication on stem cell characterization or on their actual ability to regenerate contractile cells is available.
3.TRACIA STUDY (Intracoronary Autologous Stem Cell Transplantation in ST Elevation Myocardial Infarction). The phase II/ III clinical trial aimed at evaluating the effects of intracoronary administration of adult stem cells on LV ejection fraction and major adverse cardiovascular events (MACE) after 6 months follow-up. For this reason, 1-2 million CD34+ cells were injected through the infarct-related artery few days after post-infarct angioplasty using an "over-the-wire" catheter in 80 patients aging from 20 to 75 years. The results of this study are still to be published.
4.Combined CABG and Stem-Cell Transplantation for Heart Failure. Intramyocardial delivery of autologous bone marrow cells extracted from iliac crest and purified by Ficoll centrifugation, during cardiac surgery for CABG intervention in 30 patients, as compared to 30 patients undergoing CABG without cell infusion. Although information on the number and characteristics of cells to be injected has not been given, the trial is currently ongoing and the follow-up is scheduled in 6-12 months (http://clinicaltrials.gov).
5.POSEIDON-Pilot Study (The Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis Pilot Study) Poseidon-pilot Study is a phase I/ II multi-center trial in which the trans-endocardial injection of autologous Mesenchymal Stem Cells (20-, 100-, 200 X 106) is compared to autologous non-purified bone marrow cells and to allogeneic human Mesenchymal Stem Cells. The implant is performed during cardiac catheterization using the Biocardia Helical Infusion Catheter in fifty (50) patients suffering from chronic ischemic left ventricular dysfunction secondary to myocardial infarction. The data collection is currently ongoing.
6.SCIPIO (Cardiac Stem Cell Infusion in Patients With Ischemic CardiOmyopathy). This phase I clinical trial is aimed at assessing the safety and effectiveness of intracoronary autologous cardiac stem cell therapy. As such, forty (40) patients suffering from ischemic cardiomyopathy are exposed to intracoronary injection of cardiac resident stem cells (CSC). Cardiac stem cells are harvested from right atrial appendages and selected for c-kit expression, cultured and expanded in vitro prior to injecting them via intracoronary route, three to five months after CABG surgery. The hypothesis is that CSC infused into nonviable myocardial segments will regenerate infarcted myocardium by differentiating into cardiomyocytes and vascular cells. The preliminary results are encouraging: in the nine patients treated at four months after CSC infusion, LVEF increased from 31.3 + 2.5 percent before CSC infusion to 38.8 + 3.2 percent four months after CSC infusion. Moreover, in the five patients in whom data are available at 12 months after stem cell infusion, the improvement in LVEF observed at four months was even greater, averaging 15% at 12 months. The follow-up is scheduled in 1,5 years.
7.ALCADIA (AutoLogous Human CArdiac-Derived Stem Cell to Treat Ischemic cArdiomyopathy). In this phase I, multicenter clinical trial, a rather different approach is followed. In fact, patients’ own cardiac stem cells obtained by endo-myocardial biopsies are delivered by a single intramyocardial injection. The cells injected are 0.5 million cells/kg (patient body weight) and their engraftment should be favored by the concomitant implantation of gelatin hydrogel sheet releasing human recombinant beta Fibroblast Growth Factor (bFGF), during CABG surgery. The study has been designed to treat refractory heart failure, ischemic cardiomyopathy or ventricular dysfunction cases. Importantly, this is the first clinical trial, to our knowledge, in which a human recombinant growth factor is used. Unfortunately, the number of enrolled patients is limited to six (6).
8.REGEN-IHD (Bone Marrow Derived Adult Stem Cells for Chronic Heart Failure). In this phase II/ III study, granulocyte-colony stimulating factor (G-CSF) is subcutaneously administered for 5 days to patients with heart failure secondary to ischemic heart disease to mobilize CD34+ bone marrow stem cells. A concomitant intracoronary or intramyocardial administration of bone marrow derived stem cells is performed. The number of enrolled patients is high (165) and the aim of the study is to compare the effects of G-CSF and autologous bone marrow progenitor cell infusion on the quality of life and left ventricular function in the patients. The follow-up timepoint is scheduled in 6-12 months.
A number of papers reporting statistical analyses and comparisons among the clinical trials in which stem and progenitor cells have been adopted are currently available. [For further information, please refer to www.clinicaltrials.gov].
The possibility to treat cardiac diseases by cell therapy techniques is an extraordinary promise. While a number of different approaches has been so far proposed to setup minimally invasive techniques for cardiac repair, few of them being already in the clinical experimental phase, basic questions still need to be addressed. In fact, the molecular processes leading to cardiac differentiation still need to be fully clarified, while the impact of novel, genetically modified cell types obtained from adult differentiated cells on cardiac microenvironment deserve further investigations. More importantly, the seek to identify suitable delivery systems (i.e. scaffolds) able to foster stem cell survival, growth and differentiation, while degrading without negative effects as the formation of new tissue occurs is still open. A look at the literature reveals that an impressive effort to translate the information obtained by