**3. Cell sheets**

*Cardiac Stem Cells (CSC)*

282 Regenerative Medicine and Tissue Engineering

*Skeletal Myoblasts (SM)*

cytokine-mediated effect [46].

*Umbilical Cord Blood Cells (UCBC)*

*Amniotic Fluid Stem Cells (AFSC)*

ventricular function [61].

during a coronary artery by-pass graft surgery.

and do not present risk of tumorogenicity [62].

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‐

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

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

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

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

sphere-derived cells are being evaluated in the CADUCEUS clinical trial [58].

The use of cell sheets is based on the fact that when cells are cultured in normal flasks and enzymatically digested to detach them, the adhesive proteins and membrane receptors are disrupted leaving the cell damaged [64]. The alternative is to grow cell sheets and then detach them from the culture surface in a way that keeps the electromechanical connections between the cells and benefits from the fact that cells are kept together by their own deposited ECM, as figure 1 b displays. In that way, cells maintain the adhesion and membrane proteins, as well as the natural pro-survival and maturation environmental cues that the ECM provides [65]. Altogether, this is expected to help them to survive when implanted onto the infarcted myocardium.

Cells can be cultured, for instance, on temperature-responsive poly(N-isopropylacrylamide) (PnIPAAm)-coated plates. PnIPAAm is a hydrophobic polymer at 37ºC, and cells can attach to its surface. When the temperature is lowered, PnIPAAm suffers a transition to a hydrophilic state and this change causes the attachments of the cell monolayer to the surface to disrupt, and the entire cells sheet detaches from the surface [65]. Other materials, such as a thermoresponsive methylcellulose hydrogel, have been used to successfully obtain cell sheets fragments of human amniotic fluid stem cells (hAFSCs) [66]. Results obtained with these cell sheet fragments were superior to those with dissociated cells in terms of heart function, cell retention, proliferation and vascular density. Moreover, cardiomyocyte sheets were found to functionally integrate with the host tissue in a rat myocardial infarct model [67]. New techni‐ ques based on patterning with a gelatin stamp the thermo-responsive substrates allow obtaining complex tissue structures with cells having a determined orientation [68].

The muscle mass loss following an infarction is significant, up to 50 g [69], so the amount of cells needed to overcome this loss is obviously not covered with a single sheet of cells. On the other hand, when several layers of cell sheets are superimposed, they are easier to handle. Some groups have tried to obtain thicker grafts by overlapping several monolayer cardio‐ myocyte sheets, which adhere one to another forming gap junctions and intercellular adhe‐ sions within minutes [70]. But this approach poses a problem: as cell sheets lack of vascularization, the maximum thickness that can be achieved by overlapping them is limited to the depth at which diffusion of oxygen and nutrients can take place (a maximum of three cardiomyocyte sheets can be piled up). To try to overcome this problem, three-layer thick cardiomyocyte sheets were implanted in rats at 1-, 2- and 3-day intervals [71]; in the time between transplantations it was assumed that there is enough time for the cell sheet to be vascularized. With this approach constructs of 1 mm were obtained successfully. But anyway, this option is very invasive, so its application in patients might be limited.

A different approach based on the same idea of providing cell-cell connections and ECM to the implanted cells to improve their retention and survival is to implant them as spherical cellbodies. Human amniotic fluid stem cells (hAFSC) cultured in a methylcellulose hydrogel to form cell aggregates were implanted in immunosuppressed rats as cell-bodies, and cell retention and engraftment were enhanced as compared with disaggregated cells. This enhancement led to functional improvement and limited the progression of heart failure [72].
