**2. Cardiomyoplasty**

at the age of 75 [31]. This regenerative capacity is achieved thanks to a small population of cardiac stem cells [32]. Nevertheless, their regenerative capacity is limited and in any case it is not enough to regenerate the heart if it suffers severe damage, like the one provoked by a myocardial infarction. New therapies under development like cell therapy or tissue engineer‐ ing, aim to boost this limited regenerative potential of the native tissue by employing cells,

The aim of cardiac cell therapy is to heal the damaged infarcted tissue by the implantation of cells into or onto the pathologic myocardium by different techniques (figure 1 a). In tissue engineering strategies, different types of cells have been combined with materials and with bioactive molecules if necessary to again try to recover the injured tissue. The employed materials will support cells, provide them 3D organization, protect them, stimulate and guide its growth, maintain them in the site of interest, etc.; in sum, they will act as an artificial extracellular matrix during the regeneration process. But the use of materials either injectable, or *ex vivo* conformed (gels –patches- or scaffolds) (figure 1 b) has an additional and important effect: the implantation of a material in the scarred ventricular wall, increases its thickness and by Laplace's law, this increase leads to a reduction in the wall stress. This side-effect could be by itself very positive, even although regeneration did not arrive to happen, to limit ventricular

**Figure 1.** (a) Classical cell therapy in the heart (*freely inspired* in Strauer BE, Kornowski R, *Circulation* 2003; 107: 929-934). (b) Tissue engineering approaches with cell sheets, scaffolds or injectable materials (*freely inspired in* Masu‐

da S *et al*, *Adv. Drug Del. Revs* 2008; 60(2): 277-85). (c) Ventricular restrain device.

remodeling and improve the quality of life of cardiac patients [29].

drugs, factors or patches.

278 Regenerative Medicine and Tissue Engineering

#### **2.1. Need for cell cardiomyoplasty**

Cardiomyoplasty has evolved from "dynamic" to "cellular cardiomyoplasty". The term dynamic cardiomyoplasty is referred to a surgical procedure developed in 1987 [33] to wrap the heart with the latissimus dorsi muscle, aiming to support the heart beating and limit the remodeling. Nevertheless, the obtained results were not as good as expected. With the advances in cell therapy, cellular cardiomyoplasty appeared as a promising therapeutical approach. This name encloses the therapies that use the injection of cells, from different origins, directly into the heart to try to obtain an improvement in the reduced heart function after an ischemic insult (figure 1 a).

The injected cells are envisaged to induce angiogenesis, inhibit apoptosis, help to recover hibernating myocardium, activate endogenous repair mechanisms, and create new contractile tissue that will replace the damaged one. Also they are expected to reverse the remodeling process that provoked ventricular dilation [34]. Many cells have been employed and the initial promising results obtained in animal models made this technique moved very fast to clinical trials, even if the mechanisms involved in the observed improvements were unknown. Unfortunately, the results obtained from the clinical trials were not as good as expected, and some were contradictory between them. One possible contributing cause to this discrepancy is that studies are carried out in young healthy animals, while patients susceptible to receive these treatments normally are aged people and in many cases with other co-morbidities [35].

Different ways to deliver cells into the damaged heart have been explored: intracoronary infusion (with the hope that cells will migrate through the vessels and be hosted in the infarcted area) or directly into the infarcted area either by intramyocardial or endocardial injection [36], as shown in figure 1 a. The advantage of injecting them directly into the infarcted area is that this will ensure that the cells are delivered in the site of interest.

#### **2.2. Related problematic**

Many different cell types have been employed in the numerous studies that have been done. Autologous cell sources are interesting because they do not require immunosuppression treatment of the patient and there is no risk of illness transmission. On the contrary, allogenic cells could be ready to use whenever a patient needs them, but would require immunosup‐ pressive therapy after their implantation, and there is always a remaining risk of illness transmission. Another disadvantage is that prior to implantation cells need to be extracted and expanded. This whole process in some cases may take several weeks, limiting its application in the acute state. Besides, autologous cells coming from patients that suffer other conditions like diabetes or are simply aged, may have limited proliferation and attachment [37].

An important aspect of this technique is the low engraftment into the heart tissue of the supplied cells. The retention of the cells in the heart seems to be determined by the cell type and delivery route [38]. It has been estimated that in humans 50-75 min after intracoronary injection of bone marrow cells only 1,3-2,6% of the injected cells remain in the myocardium [39]; after 2 hours less than 10% of the injected cells survive [32]. Many causes can be advanced: the heart beats, so cells can easily be pumped out of the heart; the solution in which cells are injected has a low viscosity, so cells can be washed away; the mechanical loss of the cells through the injection hole left by the needle, etc [40]. A different contributing cause to the low cell engraftment is that the injured heart is not a cell-friendly environment, type I collagen fibers have been substituted by type III, which has worse properties in terms of adhesion and promoting angiogenesis, what can induce anoikis [4]. Another problem is cell survival itself. The conditions in the infarcted myocardium are very hostile for the cells: hypoxic conditions (studies show that the survival of injected cells decreases towards the center of the scar), cytokines, inflammatory factors, etc., are present in the damaged myocardium, and can negatively affect the survival of the injected cells. Immunological rejection can be another cause reducing cell survival [41].

An interesting approach is to train cells prior to their implantation for them to resist the hostile conditions they will find in the implantation site. For instance, the resistance to hypoxic conditions is key and needs to be improved even for skeletal myoblasts (which are the cells that have better resistance to lack of oxygen). Privation of glutamine re‐ duces the oxygen consumption rate, what has been proved to improve survival of myoblasts when implanted [42].

The fact that most of the cells did not graft into the host myocardium in the studies performed to date, that there is a very limited transdifferentiation of implanted cells into beating cardio‐ myocytes (the differentiation reported in animals may have been fusion events between native cardiomyocytes and injected cells [41]), and that a wide range of non-myogenic cells also induce an improvement of the ventricular function [36], suggests that the mechanism leading to this enhancement cannot be only myogenesis regenerating the myocardium. The pathways through which cell implantation induces improvements in cardiac function remain to be elucidated, but different events that can take place simultaneously have been proposed. The most remarkable are the induction of angiogenesis (formation of new vessels) and the improvement in the myocardial perfusion, the reduction of the wall stress because of the increase in cell mass [43] and the paracrine effect of the injected cells [32].

#### **2.3. Cell types investigated**

As previously said, many cell types from different origins have been employed: embryonic stem cells, mesenchymal stem cells, bone marrow cells, induced pluripotent stem cells, cardiac stem cells, skeletal myoblasts, umbilical cord blood cells and amniotic fluid stem cells, among others. In what follows the use of these cell types is discussed, with the advantages and disadvantages that each one presents for its application in heart regeneration.

#### *Embryonic Stem Cells (ESC)*

ESC can be obtained from the inner mass of an embryo in the blastocyst stage. These cells have the capacity of growing undifferentiated indefinitely, and when they differentiate they can form any cell from the three germ layers. But the use of ESC raises ethical issues, requires immunosuppression, and has the risk to form theratomes. Their use in clinical trials has been limited because of these ethical considerations and risks [36, 44].

A protocol for ESC differentiation into cardiomyocytes and improving their survival when implanted has been established; when these differentiated cells were implanted in rodent models the heart function was improved [45]. In another study in mice, ESC-derived cardio‐ myocytes implantation reduced the reactive collagen deposition in the ventricular septum, which is one of the remodeling process hallmarks. Nevertheless, the implanted cells were isolated from the host myocardium by scar tissue, although the implanted cardiomyocytes were able to couple functionally to each other [46].

#### *Induced Pluripotent Stem cells (IPS)*

Induced pluripotent stem cells are fibroblasts treated with viral factors to recover their pluripotency. Therefore, IPS do not raise the ethical concerns of the ESC. IPS are very inter‐ esting because they can be autologous pluripotent cells. However, their application in clinical trials has been limited precisely for the use of viral vectors that may promote malignancy and act as oncogenes [43], as well as for the intrinsic risk of theratomes inherent to their pluripo‐ tency [44].

#### *Adult stem cells*

[39]; after 2 hours less than 10% of the injected cells survive [32]. Many causes can be advanced: the heart beats, so cells can easily be pumped out of the heart; the solution in which cells are injected has a low viscosity, so cells can be washed away; the mechanical loss of the cells through the injection hole left by the needle, etc [40]. A different contributing cause to the low cell engraftment is that the injured heart is not a cell-friendly environment, type I collagen fibers have been substituted by type III, which has worse properties in terms of adhesion and promoting angiogenesis, what can induce anoikis [4]. Another problem is cell survival itself. The conditions in the infarcted myocardium are very hostile for the cells: hypoxic conditions (studies show that the survival of injected cells decreases towards the center of the scar), cytokines, inflammatory factors, etc., are present in the damaged myocardium, and can negatively affect the survival of the injected cells. Immunological rejection can be another cause

An interesting approach is to train cells prior to their implantation for them to resist the hostile conditions they will find in the implantation site. For instance, the resistance to hypoxic conditions is key and needs to be improved even for skeletal myoblasts (which are the cells that have better resistance to lack of oxygen). Privation of glutamine re‐ duces the oxygen consumption rate, what has been proved to improve survival of

The fact that most of the cells did not graft into the host myocardium in the studies performed to date, that there is a very limited transdifferentiation of implanted cells into beating cardio‐ myocytes (the differentiation reported in animals may have been fusion events between native cardiomyocytes and injected cells [41]), and that a wide range of non-myogenic cells also induce an improvement of the ventricular function [36], suggests that the mechanism leading to this enhancement cannot be only myogenesis regenerating the myocardium. The pathways through which cell implantation induces improvements in cardiac function remain to be elucidated, but different events that can take place simultaneously have been proposed. The most remarkable are the induction of angiogenesis (formation of new vessels) and the improvement in the myocardial perfusion, the reduction of the wall stress because of the

As previously said, many cell types from different origins have been employed: embryonic stem cells, mesenchymal stem cells, bone marrow cells, induced pluripotent stem cells, cardiac stem cells, skeletal myoblasts, umbilical cord blood cells and amniotic fluid stem cells, among others. In what follows the use of these cell types is discussed, with the advantages and

ESC can be obtained from the inner mass of an embryo in the blastocyst stage. These cells have the capacity of growing undifferentiated indefinitely, and when they differentiate they can form any cell from the three germ layers. But the use of ESC raises ethical issues, requires

increase in cell mass [43] and the paracrine effect of the injected cells [32].

disadvantages that each one presents for its application in heart regeneration.

reducing cell survival [41].

280 Regenerative Medicine and Tissue Engineering

myoblasts when implanted [42].

**2.3. Cell types investigated**

*Embryonic Stem Cells (ESC)*

These cells have the advantage of being autologous and can be obtained from different sources like bone marrow or adipose tissue. In addition, they can be expanded *in vitro* and do not raise ethical or immunologic problems [47, 48].

Bone marrow cells (BMC) are easily accessible, can be obtained rapidly and have been reported to have certain plasticity. This property allows them to differentiate *in vivo* into cardiomyocytes [26] (although this fact remains controversial [42]). They can also differen‐ tiate into cardiomyocytes *in vitro* by supplementing the medium [49]. Studies in animal models demonstrate that the injection of these cells increases neovasculature improving heart function [42]. But the use of BMC is not exempt of risks: intracoronary administra‐ tion of them can cause microinfarctions due to their big size and irregular shape, making necessary the use of an alternative way of delivery [50]. In clinical trials, results indicat‐ ed only temporary benefits or no improvement after cell administration [38, 51]. A strategy to enhance the therapeutic efficacy of BMC is to precondition them: BMC treated with growth factors improve the therapeutic effect when implanted and show greater survival rate [52].

Adipose derived stem cells (ASC) can be obtained in great quantity without culturing them. These cells have been implanted in small animal models of AMI and left ventricular function was improved [48]. The underlying mechanisms are unclear, although the hypothesis of a paracrine effect is considered [53]. Clinical trials are ongoing for the implantation of ASCs: PRECISE and APOLLO [54]. These cells are also are under study at the moment in the RECATABI project [55] as part of a strategy that combines them within a three-dimensional polymer scaffold with a peptide gel filling, to lengthen their positive effect and serve as a mechanical support for the dilated ventricle.

#### *Cardiac Stem Cells (CSC)*

CSC are undifferentiated cells found in the heart that can become endothelial cells, smooth muscle cells, and functional cardiomyocytes [36]. In undamaged hearts, these cells seem to contribute to the normal self-renewal of the tissue. CSC can be isolated from biopsies and can be expanded *in vitro* [56], although there is a lack of availability from human origin as they are obtained from biopsies. Human CSC injected in mice hearts after infarction led to functional improvement and to support myocardial regeneration [57]. Currently, autologous cardio‐ sphere-derived cells are being evaluated in the CADUCEUS clinical trial [58].

#### *Skeletal Myoblasts (SM)*

SM are cells present in the basal membrane, where they remain in a quiescent state while there is no damage. These cells have better resistance to hypoxic conditions than many other cell types, and can be from autologous origin, but 2 to 3 weeks are necessary to establish and expand myoblasts from skeletal muscle biopsies [36]. These cells are capable to contract; that is the reason why they were expected to attach to the beating cardiomyocytes and contribute to the effective beating by integrating in the working syncytium muscle. Nevertheless, there is no electro-mechanical coupling between the implanted cells and the native cardiomyocytes. This absence of coupling turns the implanted cells into a pro-arrhythmic substrate [44]. The cause for this uncoupling is the lack of the gap junctional protein connexin 43. Therefore, the implantation of a pacemaker or a defibrillator to avoid malignant arrhythmias and sudden death would be necessary when implanting these cells, to obtain a synchronous beating of the heart and the grafted cells [26, 59]. Despite the lack of electro-mechanical coupling of the myoblasts with the host cardiac cells, improvements in the ventricular performance have been observed in animal models, even with a reduced number of grafted cells, suggesting a cytokine-mediated effect [46].

The encouraging preliminary results and its autologous origin made this cell type the first to reach clinical trials. Initial clinical trials carried out with these cells showed symptomatic improvements in the patients, but some of them experienced arrhythmias, making necessary the use of implantable defibrillators [36]. For instance, in the phase II randomized placebo controlled trial MAGIC [60], skeletal myoblasts and a cardioverter defibrillator were implanted during a coronary artery by-pass graft surgery.

#### *Umbilical Cord Blood Cells (UCBC)*

UCBC can be easily obtained from the umbilical cord and do not present ethical concerns [42]. These cells have certain plasticity and reduced risk of rejection because they show low immunogenicity [25]. Their injection in animal models has been found to improve their left ventricular function [61].

#### *Amniotic Fluid Stem Cells (AFSC)*

Amniotic fluid is extracted for prenatal diagnosis and AFSC are isolated from it. They have many characteristics of ESC and seem to be in an intermediate stage between embryonic and adult stem cells in terms of versatility. Interestingly, these cells do not present ethical concerns and do not present risk of tumorogenicity [62].

Human AFSC have been successfully differentiated into endothelial or cardiac lineages *in vitro*. When these cells were implanted in an immunosupressed rat model, they contributed to attenuate its left ventricular remodeling, to preserve the thickness of the ventricle and to improve cardiac function [63].
