Lakshmi Pillai (left) and Deepa Bhartiya. 
Currently, the only cure for end-stage congestive heart failure is heart transplantation – severely restricted by the non-availability of donor organs and need for life-long suppression of the immune response. The loss of cardiomyocytes is an interesting target for regenerative medicine by cell-based therapies or by mobilising endogenous stem cells. The introduced cells are expected to replace the dying cardiomyocytes and functionally integrate with the existing cardiomyocytes around the scar tissue to restore pumping action and vasculature of the heart. It is reasonable to hope that such cell-based therapies will also need to regenerate angioblasts to generate a network of capillaries and larger size blood vessels for supply of oxygen and nutrients to both the affected myocardium and to the newly implanted cardiomyocytes.
Cell therapy could occur by introducing (i) committed cells e.g. skeletal myofibroblasts and fetal cardiomyocytes; (ii) progenitor cells e.g. endothelial progenitor cells or resident cardiac progenitor cells; (iii) adult stem cells (such as bone marrow- derived hematopoietic cells, mesenchymal stem cells) and (iv) embryonic stem cells.
Translation from bench to bedside has already been attempted using various cell types.
Committed cells
Skeletal muscle stem cells: Myofibroblasts or 'satellite' cells are those found in skeletal muscle and readily differentiate into myocytes. They are involved in repair and growth of skeletal muscle and were the first cells used for cardiac repair. Cell therapy using autologous skeletal muscle 'satellite cells' was initiated in 2003 in cases of severe left ventricular dysfunction. These cells can be easily isolated, expanded in vitro, be available for autologous use, survive well post-transplantation and exhibit certain degree of efficacy in small animal models .
The hypothesis is that on being transplanted, they transform into cardiomyocytes. However, available literature indicates that this does not happen; they remain committed to skeletal muscle fate and do not integrate with the host myocardium, keeping the contractile activity in the scar tissue independent of neighboring cardiomyocytes 2 .
Fetal cardiomyocytes: Transplanting fetal cardiomyocytes has been shown to protect against the induction of ventricular tachycardia – a cardiac arrhythmia in which the muscles of the ventricles contract irregularly impairing the normal pumping of blood. Results from multiple laboratories suggest that fetal cardiomyocytes can couple functionally with host myocytes, stimulate formation of new blood vessels, and improve myocardial function behaving as 'biological pacemakers 3 '. However, their use for therapy is limited on ethical grounds and also because of their limited availability.
Progenitor Cells
Endothelial progenitor cells (EPCs): Adult bone marrow contains EPCs easily mobilised from the bone marrow following either an exogenous or endogenous stimuli. After mobilisation they enter the peripheral circulation, and home into sites of injury and repair. They possess the ability to differentiate into both arterial wall cells (endothelium and small muscle cells) as well as cardiomyocytes, inhibit cell death and release proangiogenic factors.
Resident cardiac progenitor cells: These cells do not terminally differentiate to express the full repertoire of cardiac muscle specific contractile protein. A small number of human cardiomyocytes retain the capacity to proliferate and regenerate in response to ischemic injury. However, whether these cells are derived from a resident pool of cardiomyocyte stem cells or from a renewable source of circulating bone marrow-derived stem cells that home to the damaged myocardium is at present not known.
Adult Stem Cells
Adult stem cells are invariably found in all the body parts including discarded organs such as milk teeth, menstrual blood, placenta and cord blood. However, they tend to diminish in number with age and have different properties depending on their source. Marrow- derived stem cells include (i) haematopoietic stem cells (HSCs) that give rise to the blood cells, (ii) mesenchymal stem cells (MSCs) found in the bone marrow stroma are multipotent stem cells and have the ability to differentiate into wide variety of cell types and (iii) endothelial stem cells. Bone marrow stem cells have shown considerable myogenic and angiogenic potential in vitro and thus rapidly moved from bench to bedside for autologous use, since they are safe.
Most studies have been carried out using autologous transplant of bone marrow mononuclear cells (a heterogeneous population of mononuclear cells with 0.1% stem cells) and delivered either through intracoronary route or direct intra-myocardial injection. Most of these trials have lacked proper control thus limiting assessment of efficacy. A systematic review and meta-analysis of the available trials5,4 indicate that BMC transplantation may improve LV ejection fraction, infarct scar size, and LV end-systolic volume. However, the precise mechanisms underlying these benefits are unknown and the specific advantages of one BMC type over another remain to be determined. Meta-analyses results estimated an increase in 3-4% in left ventricular ejection fraction after stem cell therapy in comparison with control patients. However, these beneficial effects appear to fall in the range of established therapies.
A recent murine comparative analysis of the efficacy of different cell candidates like bone marrow mononuclear cells, mesenchymal stem cells, skeletal myoblasts and fibroblasts for the treatment of heart disease, evaluated their therapeutic efficacy6. Live cell imaging studies revealed acute donor cell death of mesenchymal stem cells, skeletal myoblasts and fibroblasts within three weeks after transplantation. By contrast, cardiac signals were still present after six weeks in the mononuclear cells group. This is the first report of a comparative study demonstrating that only the mononuclear cells might have the clinical potential to survive and go on to translate into a therapeutic option for preservation of cardiac function.
The advantage of using MSCs for cell therapy is that they can be easily isolated from various sources and expanded in culture for autologous use, without any associated ethical issues. Another advantage is that they are immune-privileged cells and thus may also have allogeneic potential. Preclinical studies have shown that they may facilitate both myocardial repairs as well as induce neo-angiogenesis. However, whether they undergo transformation or it is just a mechanism to protect the cells from damage by which they exert an effect, remains to be elucidated.
Embryonic Stem Cells
Human ES (hES) cell lines have immortal and pluripotent embryonic stem cells with the ability to form any of the 210-odd cell types of the human body. They are derived from surplus human embryos from in vitro fertilization clinics. This year we celebrate the 10th anniversary of the discovery of hES cells separately by Thomson7 and Gearhart8. Indian laboratories have also derived hES cell lines, that will be used to initiate differentiation studies towards translational research10,11
Since skeletal myoblasts and bone marrow cells fail to transdifferentiate into cardiomyocytes, do not integrate with host cardiomyocytes or help generate active force in the infracted area, hES cells appear to be the only ideal source of stem cells for cardiac regeneration. Fetal cardiac cells and resident cardiac stem cells exhibit good potential but are scarce in numbers. Thus hES cells derived cardiomyocytes engraft and restore contractile function in mouse models unlike bone marrow derived cells.
Various studies have documented that hES cells derived cardiomyocytes show cardiomyocytes specific molecules, structure, electrophysiology and contractile properties. They electro- mechanically integrate with the resident cardiomyocytes in the scar tissue, augment pump function and act as a biological pacemaker. Interestingly, transplanted cardiomyocytes proliferate in the scar region up to seven fold and there may be no need to inject large number of cells. Thus hES cells derived cardiomyocytes hold substantial promise as potential therapeutic agents for cardiac repair12.
Emerging data from several laboratories have shown that hES cells can be differentiated into cells with several characteristics of cardiomyocytes. In the last five years, preclinical studies,13,14,15,16,17 have been undertaken in lower animal models to assess the effect of human ES cell transplantation towards myocardial regeneration after acute myocardial injury . The incidence of tumor formation in these reports was insignificant and employment of appropriate enrichment strategies during translation may help avoid the risk in future.
Most preclinical evaluation of hES cells derived cardiomyocytes-like cells has been done in rodents having almost double the heart beat of humans. There's great optimism for hES cell therapy in non-human primate models, which will be a more physiologic approach, keeping in mind that sustained pacing at a high rate is a standard method to induce heart failure in large animals (guinea pigs, swine). Moreover, the cardiac function post-therapy, has been measured at four weeks interval. Thus there's a greater need to study long-term effects in non-human primates.
Looking ahead
A lot of hurdles need to be crossed before the clinical potential of stem cells is realised in regenerative medicine. In addition, tissue engineering for cardiac repair as also use of plant products for regeneration are exciting and fast developing fields.
First up, the route of cell delivery needs to be optimised including injecting the heart. Almost 90% of cells delivered to the heart are lost at the injection site and extensive cell death is observed in the transplanted cells within the first week. Various approaches18,19 such as heat shocking the cells before transplantation or over-expressing antiapoptotic proteins has been attempted to overcome this hurdle.
Immunological compatibility and risk of tumor formation is another issue. Understanding the in vivo stem cell behavior after transplantation requires novel imaging techniques to longitudinally monitor stem cell localization, proliferation, and viability.
Only a deep understanding of the benefits versus the risks and the mechanisms involved in cell mediated cardiac repair, will allow us to design clinically valuable tools and fulfil the potential of this exciting 21st century approach to treating cardiovascular disease.
It is evident that despite great potential, embryonic stem cells have lagged behind due to ethical reasons whereas other safe and easily-accessible sources of stem cells have been used in several clinical trials with modest results.
The ultimate cell type or therapy for cardiac repair is not known at this time point. The bottom line is that all areas of stem cell research should remain open and be encouraged by regulatory bodies and governance. Research involving embryonic stem cells has been internationally guided by religious ideologies and not by science, but the improving governance and the emerging data in the field of embryonic stem cells will help cover the lag that this research has faced.
The authors are from the Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai, India.
