27

C H A P T E R 3

Cell Sources for Cardiac Tissue

Engineering

CHAPTER SUMMARY

Ideally, regeneration of the myocardial tissue after a major insult should involve adding new contracting cardiomyocytes into the infarct zone, which after integration with the host tissue would empower the heart contractility. Naturally, the best cell source for this purpose is fully differentiated and functional autologous cardiomyocytes. However, in a real world situation, the clinical applicability of this strategy is very limited since adult cardiomyocytes have lost their capability to proliferate and regenerate after damage. This chapter presents several clinically relevant cell sources that are in use or could potentially be used for cardiac tissue engineering and regeneration strategies. Cardiomyocytes derived from human embryonic stem cells, induced pluripotent stem cells or by direct reprogramming of somatic cells will be first introduced followed by the presentation of contemporary alternatives, such as autologous stem/progenitor cells purified from bone marrow, adipose tissue, or cardiac biopsies. The chapter concludes with a description of clinical studies performed with adult stem cells, their results, and the “paracrine theory” explaining the beneficial effects of stem/progenitor cell transplantation in improving cardiac function.

3.1INTRODUCTION

In pathological situations where large numbers of cardiomyocytes are lost, e.g., following severe ischemic injury after MI or chronic stress, the endogenous regeneration capacity is insufficient to form adequate cardiac contractile mass to maintain heart contractility. Cardiac cell therapy, therefore, ideally aims at actively replacing the damaged and non-functional cardiomyocytes with a new and viable transplantable tissue. The characteristics of the ideal cell type have been articulated by many experts: a cell type should be both quantitatively and temporally available, safe to administer, effective at engraftment, and (most importantly) induce cardiac repair. Some argue that a source of autologous therapy is ideal so as to avoid any possibility of rejection, although it should be acknowledged that allogeneic cell therapy is also emerging as a strong possibility. Practical considerations, including the cost of therapy, will ultimately bear importantly on the accessibility of a new therapy [1].

Theoretically, the natural electro-physiological, structural, and contractile properties of differentiated cardiomyocytes make them the ideal candidate as cell source for in vitro bioengineering of cardiac tissue. Indeed, in most studies cardiac patches were produced using embryonic, fetal, or

28 3. CELL SOURCES FOR CARDIAC TISSUE ENGINEERING

neonatal rat cardiomyocytes (see Chapter 6). However, cardiomyocytes are difficult to obtain and expand, are sensitive to ischemic insults, and are allogenic, that is, they can evoke immune response in the host tissue. The clinical need for human cardiomyocytes was at least partially fulfilled by introduction of human pluripotent stem cell-derived cardiomyocytes (either human embryonic or induced pluripotent stem cell-derived) (Fig. 3.1) [2, 3, 4, 5]. However, differentiation efficiency, immunogenicity, and various safety concerns (genome stability, mutations, and possible teratoma formation) are still major hurdles in the translation of applications of these cell types in clinics.

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Figure 3.1: Clinically relevant cell sources for myocardial tissue engineering and regeneration. Blastocyst-derived embryonic stem cells (ESC) have an established potential to differentiate into cardiomyocytes. An autologous alternative to ESC is represented by introduction of induced pluripotent stem cells (iPSC) that can be derived from somatic cells (e.g., fibroblasts). Moreover, protocols are developed for direct reprogramming of somatic cells into cardiomyocytes. The differentiation process goes through several stages with varying efficiency, some leading to formation of cardiac or cardiovascular progenitors. Additional adult and autologous sources for stem/progenitor cells are the bone marrow, adipose, or cardiac tissues. The differentiation potential of these cells is contradictory, and mostly limited. Nevertheless, these cells were proven to exert beneficial paracrine effects in infarcted heart upon transplantation. CSC/CPC, cardiac stem/progenitor cells; BMSC, bone marrow-derived stem cells; ADSC, adipose tissue-derived stem cells. See text for more details.

3.2. SOURCES FOR DE NOVO CARDIOMYOCYTES FOR CLINICAL APPLICATIONS 29

Meanwhile, other adult stem or progenitor cell types with some indications of cardiomyogenic differentiation potential and/or with established beneficial positive effects on infarct repair represent a possible alternative, and in most cases, autologous source (i.e., bone-marrow-derived stem cell subsets, adipose-tissue derived, and cardiac progenitors) of cells that can be used for cardiac tissue engineering and myocardial regeneration after MI (Fig. 3.1) [1, 6, 7, 8, 9, 10, 11].

3.2SOURCES FOR DE NOVO CARDIOMYOCYTES FOR CLINICAL APPLICATIONS

As mentioned, the vast majority of studies attempting to engineer cardiac patch in vitro, used rat neonatal, fetal, or embryonic cardiomyocytes as an ultimate cell source. Although these cells are easily accessible and represent a good model system for various in vitro studies, translation of cell transplantation, as well as graft engineering efforts into clinics, still suffers from the lack of appropriate cell source. As adult human cardiomyocytes are hard to obtain and have no ability to proliferate in culture, there is an urgent need to find an alternative suitable stem cell source, which will have a proven ability to effectively differentiate into functional cardiomyocytes. Not surprisingly, although having a promising potential for cardiogenesis, no cell type is perfect, each still having intrinsic drawbacks, as will be discussed below.

3.2.1HUMAN EMBRYONIC STEM CELLS

Embryonic stem cells (ESCs) have emerged as one of the most promising sources of cardiac cells for transplantation purposes. Human ESCs (hESCs) first isolated in 1998 by James Thomson and co-workers from the inner cell mass of preimplantation embryos [12], are pluripotent cells capable of differentiating into virtually every cell type including cells of the heart [13, 14]. In the decade following the isolation of hESCs, protocols to differentiate these cells into cardiomyocytes have been refined [15]. Several groups have successfully isolated cardiomyocytes or cardiac progenitor cells from differentiating ESCs grown either in three-dimensional clumps termed embryoid bodies or 2D cultures treated with various extracellular proteins and/or growth factors and cytokines (i.e., BMP-4, activin A) that increase the yield of cardiac cells. Importantly, the ESC-derived cardiomyocytes not only share molecular markers with primary cardiomyocytes, but ultrastructural (electron microscopy), electrophysiological (action potential measurements), and mechanical (determination of contractility) studies of the ESC progeny indicate that they also exhibit all hallmarks of cardiomyocytes. Of potential importance, ESC-derived cardiomyocytes have been shown to exhibit a phenotype reminiscent of fetal, rather than adult cardiomyocytes. Given the observed differences between fetal and adult cardiomyocytes, ESC-derived cardiomyocytes with features of adult cells would probably be preferable for clinical transplantation purposes. Nonetheless, ESC-derived cardiomyocytes have already been used in transplantation experiments in rodent models of cardiac diseases [14]. These results show that hESC-derived cardiomyocytes can couple electromechanically with cardiomyocytes of the host, and therapeutic effects in the MI model have been reported

30 3. CELL SOURCES FOR CARDIAC TISSUE ENGINEERING

four weeks after transplantation [3]. However, in a study with a longer follow up, no effect on cardiac function could be documented twelve weeks after transplantation [16]. Thus, the long-term effects of ESC-derived cardiomyocytes to injured myocardium need to be evaluated further.

Two of the obstacles that stand in the way of the therapeutic use of ESC are immunological rejection (due to non-autologous nature) and the propensity of undifferentiated ESCs to form teratomas when injected in vivo [10]. As knowledge of pathways for ESC differentiation increases, cell differentiation will become more controllable,and this,together with effective selection protocols, could limit teratoma formation.

3.2.2INDUCED PLURIPOTENT STEM CELLS

Recently, a novel way of generating stem cells from differentiated cells has been described. This technique, pioneered by Shinya Yamanaka and colleagues, relies on the reprogramming of fully differentiated somatic cells to ESC-like cells, known as induced pluripotent stem cells (iPSCs) [5, 17]. This conversion was achieved by lentiviral-based transduction of four transcriptional factors, Oct3/4, Sox2, Klf4, and c-Myc. IPSCs exhibit the two key features of ESCs, in that they can be expanded over many passages in vitro and give rise to cells of all three germ layers, both under appropriate in vivo and in vitro differentiation conditions. Originally established for mouse embryonic fibroblasts with a genetic selection strategy for identifying reprogrammed cells, the basic iPSC derivation protocol has been refined by many groups, permitting reprogramming of human cells, reprogramming without genetic selection and chemicals enhancing reprogramming efficiency [13, 15]. The ability of both mouse and human iPSC to differentiate into functional cardiomyocytes has recently been demonstrated [4, 18, 19, 20, 21]. IPSCs were shown to differentiate efficiently into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate the developmental ontogeny of cardiogenesis. The study of iPSC from selected cohorts of patients was found to be a very efficient way to uncover molecular mechanisms of disease. This was shown, for instance, by iPSC generation from patients with long QT syndrome, with subsequent differentiation of iPSC to cardiomyocytes, which recapitulated the electrophysiological features of the disorder [22, 23].

Similar to ESC-derived cardiomyocytes, iPSC-derived cells are largely immature and most analogous to fetal stages of development, exhibiting automaticity (spontaneous contraction), fetaltype ion channel expression, fetal-type gene expression patterns, and fetal-type physical phenotypes [15]. Three major subtypes of ESC or iPSC-derived cardiomyocytes can be derived that have atrial-, ventricular-, or nodal-like phenotypes as determined by electrophysiological analysis of action potentials, and the specific required type can be potentially enriched [15].

Being derived from adult cells, iPSCs bypass the ethical issues regarding the use of embryonic human tissue to cure disease, and immunocompatibility is not an issue because the starting material, i.e., skin fibroblasts, can be obtained from the patient. However, there are currently caveats with the iPSC reprogramming procedure that need to be addressed before this elegant technology can be put to clinical use. One important aspect is that the original protocol for reprogramming of human cells to iPSCs relies on the use of viruses integrating into the genome of cells undergoing