Heart failure, as the inexorable consequence of myocardial infarction is a major cause of morbidity and mortality in the western world. Current conventional therapies of myocardial infarction aim for the rescue of sublethaly injured cardiomyocytes, but they cannot rescue already dead tissue. Regenerative medicine based on the cell replacement using stem cells and their derivatives holds a new promise: theoretically, any tissue could be repaired by transplantation of stem cells which can either integrate in the damaged tissue or secrete healing molecules which in turn improve impaired tissue function. Following rather straightforward and on cellular level rather simple mechanic role of cardiac muscle cells, cardiac stem cells represent a very interesting and highly promising approach for the improvement of the failing heart. In the past two decades various types of stem cells have been identified and extensively examined as potential donor cells for tissue/organ regeneration. Other potentially useful applications for stem cell derivatives, such as generation of in vitro human experimental models have emerged as well. In vitro models, for example, offer testing of new hypotheses in regard to pathophysiological mechanisms or testing of new molecules with healing potential in controlled in vitro environment.

Stem cell technology started its modern development in 1960s, when McCulloch and Till performed first successful transplantations of bone marrow stem cells to experimental animals. Only few years after, the first successful bone marrow transplantations to the patients have been performed. It was needed 20 years more to develop protocols for isolation of stem cells from other organs and in 1990s new era of genetics allowed us to make a specific cocktails and complex media which announced a revolution in stem cell cultivation. In parallel with cloning story and birth of the sheep Dolly, newly isolated and well described stem cell lines become a powerful tool for the first transplantations to human patients, including those ones with heart ischemia. In 2000s and especially in the last five years we entered a new era in treatment of heart diseases: clinical trials with stem cells for heart ischemia are being launched all-around the world and they are yielding promising results.

In the selected review article the authors are presenting the recent advances in basic and translational research concerning the cell therapy in cardiovascular disease (1). In the recent years the extensive basic research efforts has lead to fast translation from bench to bedside that is recognizable in the application of stem cell research in clinical cardiology. Several clinical trials which are testing transplantation of autologous bone marrow mononuclear cells and its subgroups (adult stem cells, ASCs), e.g. hematopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells in cardiac disease have reached the phase I and II and the most recent development is advancement to phase III. It is unclear what benefit carry engrafted stem cells since they can give rise to new cardiomyocytes when transplanted to animal models, but they can be detected only up to 8 weeks following the transplantation. It is therefore believed that engrafted stem cells stimulate proliferation and differentiation of resident cardiac stem cells rather than becoming the significant source of new cardiomyocytes. Another strategy has been taken in basic research where stem cells were first in vitro differentiated into cardiomyocytes and then transplanted into failing animal heart (2). This approach showed superior outcome over transplantation of undifferentiated stem cells. However, transplantation of differentiated cardiomyocytes is not a major focus of clinical trials at the moment. In addition to suspension of single cells, transplantation of preformed 3D tissue patches consisting of extracellular matrix, progenitor cardiac cells and vascular elements have also tested in animal models with variable success and arrythmogenicity as a major obstacle (3).

Most commonly stem cells are classified according to their origin as embryonic stem cells (ESCs) and ASCs. ESCs are isolated from an early blastocyst. They have ability for indefinite self-renewal or differentiation into derivatives of all three germ layers, including cardiomyocytes (4). Although human ESCs have the greatest differentiation potential, and therefore considered as gold standard in stem cell biology, their use is hampered by many issues, including ethical concerns, efficient derivation protocols, optimal culturing methods, functional competence of derived cardiomyocites, immune rejection and tumorigenicity.

Adult stem cells (ASCs) comprise a group of several types of stem cells that reside in human tissues and organs. They are distinct in their differentiation potential, e.g. bone marrow stem cells are considered to be multipotent giving rise to several different blood cell types, while resident tissue stem cells are considered to be unipotent and being able to differentiate into only one cell type. While having questionable differentiation potential ASCs are also free of ethical issues and questions of immunogenicity and graft rejection. Nevertheless, ASCs are only group of stem cells used in clinical trials for the repair of failing heart that reached phase III clinical trial (1). The safety and feasibility of ASCs groups of stem cells in the cell therapy in myocardial infarction have been demonstrated, however the reports on the regeneration efficacy have been less consistent.

A significant advancement was made by generation of induced pluripotent stem (iPS) cells for which Yamanaka received Nobel prize in 2012 (5). iPS cells are genetically engineered cell lines generated by introducing factors of pluripotency (e.g. Oct4, NANOG, Sox2, Lin28 etc.) into already terminally differentiated cells such as fibroblasts. These cells represent a renewable source of autologous cells as can be generated from the individual patient. Thus, human iPS cell are easily available and not hampered by ethical and probably immunogenicity issues. However, differentiation potential of iPS cells is still under extensive investigation and many studies are currently addressing their genetic stability, differentiation potential, culturing methods, immunogenicity and tumorigenicity. Another problem with the use of iPSs for myocardial regeneration is the use of viral vectors for introduction of stemness factors that incorporate into DNA and alter host genome. This issue can be potentially circumvented by direct introduction of stemness proteins instead of their coding sequence with viral vectors. Our recent study demonstrated efficient generation of human iPS cells by reprogramming foreskin fibroblasts with introduction of nonepisomal plasmids which independently encode four reprogramming factors, OCT4, NANOG, SOX2 and LIN28 (6).

The School of Medicine University of Zagreb recognized a need for development of research aimed toward stem cells applications. Currently there are several groups at Zagreb School of Medicine which are investing their potential and manpower with a goal to follow a fast progress of this technology on an everyday basis. Apart from Laboratory for Stem Cells at Croatian Institute for Brain Research which is mostly focused on regenerative potential of stem cells in the ischemia of brain tissue (7), there are several more groups which use stem cells in research focused on regeneration of bone and heart.

In collaboration and support from Dr. Zeljko J. Bosnjak, Medical College of Wisconsin, in whose laboratory majority of experiments have been performed, our research efforts focused on development of experimental human cell models generated from stem cells that, unlike experimental animal models, reliably recapitulate human genotype- driven cardiomyocyte phenotype. We found that preconditioning with inhalational anesthetic isoflurane elicits comparable responses and cytoprotection in human stem cell-derived cardiomyocytes and adult human and rat cardiac cells (8). Moreover, using iPS-cardiomyocytes derived from type 2 diabetic patients, we found that inefficient preconditioning in diabetics is in part blocked by diabetic phenotype and that high ambient glucose exacerbates the negative effects (9). We also conducted extensive characterization of cardiomyogenesis in human iPS cells as a part of global efforts in introducing human iPS cells to translational research and regenerative medicine (10). We have improved methodology of “directing differentiation” of stem cells into cardiomyocytes, which is based on endoderm-induced cardiomyogenic signaling with bone morphogenetic protein-4, fibroblast growth factor and activin-A. This highly efficient methodology yields 60 and 80% of cardiomyocytes in beating cell clusters when human iPS cells and ESCs are used, respectively. Although cardiomyogenesis is less efficient in human iPS cells than in ESCs, the extensive gene expression profiling demonstrated comparable cardiomyogenesis in these lines further corroborating competence of human iPS cells for efficient cardiomyogenesis and in vitro modeling. Our current interest in stem cell research is aimed at dissecting mechanisms underlying inefficient preconditioning in diabetics using cardiomyocytes differentiated from iPS cells that are generated from diabetic patients.

Although faced with restrictions in research funding, scientists involved in stem cell research will continue to develop this methodology at Zagreb School of Medicine. Recently awarded European projects and strong collaborations with numerous groups from EU and USA are helping to keep a pace with stem cell research in the most advanced countries.