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Heart failure remains to be amongst one of the prominent health challenges worldwide. With increasing knowledge of the mechanisms underlying the development of heart failure, specifically targeting major pathological components is evolving; this concept looks to prevent the disease progression and involves a pharmacological basis for treatment. Reversal of heart failure phenotype is based mainly of gene therapy strategies for heart failure treatment. Studies on stem cell therapy treatment for heart failure focuses on repair by replacing have been emerging and indeed some demonstrate new advances which could potentially aid to tackle heart failure.
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Fig.1 The Development of heart failure. Adapted from Dr. Mark Leyland 'Introduction to cardiac hypertrophy and heart failure' lecture 1 In the development heart failure, conditions such as hypertension, coronary heart disease and the major contributor myocardial infarction2 can cause cardiomyocytes to die. The heart has limited regenerative capacity; diseased cardiomyocytes are removed by macrophages and replaced by scar tissue4. Remaining cardiomyocytes are required to work harder to make up for the loss. In pathophysiological conditions during decompensation, the remaining cardiomyocytes working harder undergo hypertrophy. Their increase in cell size places extra strain on the heart which spirals from remodeling to apoptosis (FIG.1).
The key question being addressed by researchers is can we reverse this effect by replacing the cardiomyocytes lost in the development of heart failure? The concepts are evolving and there has been advances particularly treating heart failure with stem cell therapy. Replacing the cardiac cells lost in heart failure has involved using cells from different sources. Skeletal myoblasts from skeletal muscle cells can fuse to form myotubes which can be injected into the heart. Skeletal muscle cells (myoblasts) and cardiac muscle cells exhibit some similarities (both express N-cadherin and connexin-43 in culture). However Reinecke et al did not support skeletal myoblasts and myotubes transdifferentiating into cardiomyocytes (lack of atrial natriuretic peptide expression, cardiac troponin I and α-myosin-heavy chain)3. The use of replacing cardiomyocytes with exactly the same cell type (cardiomyocytes) has given some positive results. There are problems such has to where to obtain the cardiomyocytes from as it is difficult to attain the cardiomyocytes from the patients own heart and to then expand tem in culture in the lab and then reinject them particularly as we already know that these cardiomyocytes display dysfunctional properties. The results are not as clear cut (due to cell numbers, immune response etc).
Stem cell therapy being studied to replace cardiomyocytes in the treatment of heart failure look more promising. These cells derived from the bone marrow and the blood are discussed as the most advanced in terms of transplantation into animal and clinical trials. The significance is that certain stem cells can develop into cell types including cardiomyocytes and also have the ability to change into other cell types too.
Fig.2 transplantation strategy used experimentally for cardiac repair. Adapted from Passier et al 27
The types of stem cells for the stem cell therapy of heart failure are derived mainly from embryonic stem cells and adult stem cells (TABLE 1). The transplantation strategy (FIG.2) for cardiac repair devised by Passier et al.27 highlights the importance of transplantation, this procedure is implemented into recent beneficial studies. Particular types of stem cells hold advantages in the treatment of heart failure compared to others.
Types of stem cells for stem cell therapy of heart failure
EMBRYONIC STEM CELLS
ADULT STEM CELLS
- Cardiac origin
c-Kit positive cells
Sca1 positive cells
Side population cells
- Non cardiac origin (Bone marrow derived)
Haematopoietic stem cells
Side population cells
Mesenchymal stem cells
Endothelial progenitor cells
Multipotent adult progenitor cells
TABLE 1: Types of cells used for stem cell therapy treatment for heart failure. Adapted from 5
The first evidence of transdifferentiation of adult stem cells derived from the bone-marrow into cardiomyocytes to restore the function of infarcted hearts was given in 2001 by Orlic et al5. Green-florescent -protein GFP tagged bone marrow stem cells (from donor mice) was injected into a mouse heart (that had been experimentally infarcted). EGFP developed into cardiomyocytes, this was confirmed by the appearance of myosin (FIG. 3A,B). The presence of transcription factor Nkx2.5 (which is present only during fetal development in cardiomyocytes) was also confirmed (FIG. 3C). Improvements in heart function were observed as lower ventricle end diastolic pressure was lower and lower ventricle function was higher.
Fig3. Bone-marrow cells regenerate infarcted myocardium. A. EGFP cells develop into cardiomyocytes. B. myosin staining confirms cardiomyocytes. C. Nkx2.5, fetal cardiomyocyte marker, seen to be present. 5
In this same study Orlic et al also reported that bone marrow derived hematopoietic stem cells once injected into infarcted mice are able to develop into cardiomyocytes, this accounted towards the rapid regeneration of the infarcted ventricle consequently showing improved cardiac function5. Despite this finding there has been recent evidence that only certain bone marrow derived stem cells differentiate into cardiomyocytes; it was concluded that although hematopoietic cells generate cardiomyocytes at a low frequency, it is achieved through cell fusion and not transdifferentiation17.
Tomita et al. investigated bone-marrow derived mesenchymal stem cells (MSC)7. MSC pre-treated with 5-azacytidine were competent of cardiac engraftment and differentiation in myocardial scar tissue and improved myocardial function in a rat model. Several other studies in animal models of myocardial infarction have demonstrated improved heart function through the regeneration of cardiac tissue upon the addition of MSC8. However it was found that engraftment of mesenchymal stem cells may carry risks of unwanted cell types, for example it was observed by Breitbach et al. after injecting bone-marrow derived MSC, the osteocalcin accumulation in the injected cells revealed that these formed bone tissue in the heart (FIG.4)9.
Fig. 4 Bone formations. Cytosolic and extracellular (arrows) osteocalcin staining (Cys red) proves bone formation in cryoinfarcted heart after injecting 5 x 106 EGFP+ Bone-marrow derived cells (green). Panel A insert: Von Kossa staining indicate calcifications 9.
The most recent advances regarding MSC demonstrated in April 2009 (Arsalan Shabbir et al.10) cross talks between injected MSC and host tissues are highlighted as cardiac repair in a failing hamster heart is shown. Intramuscularly injected MSC was highlighted as the best approach for cell delivery with comparison to intracoronary infusion, intramyocardial injection or intravenous infusion. Intramuscularly injected MSC or MSC conditioned medium are both effective in that they improved ventricular function by promoting myocardial regeneration, attenuated apotosis and fibrotic remodeling, recruited bone marrow progenitor cells and induced myocardial expression of multiple growth factor genes.
K. Guan et al. further reviewed bone marrow derived endothelial progenitor cells (EPC). In animal models of ischemia, EPC delivered in the heart (systematically administration or direct intramyocardial injection) moved to the infarcted tissue diminishing its size11. In this case EPC focus more on transdifferentiation than restoring a failing heart function therefore results remain to be inconclusive. Recent advances demonstrate how EPC play a more important role when transplanting differentiated non-cardiomyocytes for example mice with mutated transforming growth factor β-receptor gene Endoglin suffer from impaired vascularisation (therefore cardiomyocytes surrounding the infarct are not protected) this results in impaired cardiac function after myocardial infarction. These mice were rescued by intravenous injection of healthy donor mononuclear cells. This demonstrates that transplantation (in patients with heart failure and vascular impairments) of healthy endothelial cells from human embryonic stem cells or endothelial progenitor cells may help retain their cardiac function.
With regard to bone marrow derived multipotent adult progenitor cells (MAPC) it was stated in 2007 11 that MAPCs have not been tested for therapeutic or differentiation potential in animal models of cardiovascular diseases. In the same year Pelacho B et al.12 found that transplantation of MAPC differentiated into derivatives of all germ layers and this improved cardiac function through the release of inflammatory and vascular growth factors. This advance demonstrates the fast moving research particular on stem cell therapy for the treatment of heart failure.
Adult stem cells of cardiac origin e.g. c-Kit positive cells, Sca-1 positive cells and side population cells show promising results as these cardiac stem cells have been shown to be present in the human heart and are capable of differentiating into cardiomyocytes, vascular smooth muscle cells and endothelial cells (both in vivo and in vitro) 13 (FIG.5) and so to repair the heart transplantation directly into a mice or other species would be required..
Fig.5 cardiac progenitor cells in the fetal and adult heart. In particular Kit+, Sca1+ and SP cells derived from the human heart. Note, these cardiac progenitor cells from both human heart and blastocyst differentiates (in vivo and in vitro) into cells that can be transplanted into mice and other species13.
With particular reference to Kit-positive cells (cardiac stem cells CSC) Antonio Beltrami et al. 14 showed that when these cells were delivered via the intravascular route to a ischemic heart not only was the infarcted myocardium regenerated but ventricular function was significantly improved; heart function was restored. In January, Shinka Miyamoto et al. published work looking into the characterisation and long term culturing of cKit+ CSC18. It was demonstrated that not only did cKit+ CSC upregulate GATA-4 expression to enhance cardiomyocytes differentiation but cKit+ CSC co-cultured with adult rat cardiomyocytes increased cardiomyocytes survival due to the stimulation of growth factors IGF-1 and VEGF. These successful findings have demonstrated not only the potential cardiac regeneration but most importantly survival of cardiomyocytes; two major factors that contribute to the treatment of heart failure.
Pfister et al 15 confirmed side population cell differentiation is limited into cells positive for Sca1 and negative for CD31 (stem cell marker). A year on after this finding Wang X et al. demonstrated the transplantation of Sca1+ CD31- in infarcted mouse heart did not only regenerate cardiomyocytes but improved cardiac function; increased left ventricular ejection, decreased left ventricular end diastole and decreased left ventricle end systole16.
Human embryonic stem (ES) cells have shown to be the most successful for cardiac regeneration as they hold the capacity of directed differentiation into cardiomyocytes and so have shown to restore cardiac function. Kehat et al. first identified the formation of cardiomyocytes from human ES; they found myofibrillar structures stained positive for anti-human cardiac myosin HC, anti-actin, anti-desmin and anti-cTnI antibody (confirming cardiomyocytes), moreover it was found that the cells expressed a number of the cardiomyocyte specific genes, GATA4 and Nkx2.5 (confirming cardiomyocytes)19. Passier et al. reviewed the potential of human-ES cell derived cardiomyocytes to function as biological pacemakers in electrophysiologically silenced or AV blocked hearts20. An advantage with this is only a few human ES-cell derived cardiomyocytes are required to exert their effect. Interestingly, recent advances involve generating human ES in much higher numbers (millions) giving the benefit of investigating their regenerative potential.
A recent advance regarding human ES-cell derived cardiomyocytes was found by Laflamme M A et al; their work demonstrated rodents that had undergone a myocardial infarction had improved cardiac function (increase in fractional shortening, decreased end diastolic volume and increase in wall thickening) 4 weeks after transplantation of human ES derived cardiomyocytes21. It has been reported that human ES-cell has been associated with the risk of forming teratoma (tumour containing cartilage and bone), immune response poses another problem. In the same year an advance with human embryonic stem cell derived cardiomyocytes was found. Caspi O et al showed that human ES cells did not result in the generation of teratomas when ex-vivo pre-differentiated human embryonic stem cell-derived cardiomyocytes were grafted in a rat heart. These cardiomyocytes survived, proliferated and integrated with host cardiac tissue. Moreover transplantation of human embryonic stem cell-derived cardiomyocytes favoured the remodelling process and improved myocardial performance in this rat model of permanent coronary occlusion and extensive myocardial infarction22.
There are recent studies looking at the transplantation of differentiated non-cardiomyocytes derived from human ES23. In a mouse model of hindlimb ischaemia, transplantation of human ES-cell derived endothelial cells increased the number of capillaries and improved blood flow (when observed 2 weeks and 4weeks after transplantation), this shows the therapeutic potential of human Es-cell derived endotheial cells in the treatment of severe ischemic diseases. Another study, transplanted haemangioblasts derived from human ES, these differentiated into endothelial cells and haematopoietic cells. Injection of these cells reduced mortality rate after myocardial infarction and restored blood flow in mouse models of hindlimb ischemia24.
Sickle cell anemia can contribute to heart failure; it was recently discovered by Hanna J et al25 that by combining stem cell therapy with gene cell therapy. Mice were infected with OCT4-, SOX2-, KLF4- and Myc- to generate iPS cells, where necessary genetic repair by homologous recombination was carried out (FIG.6). iPS cells differentiated into hematopoietic progenitor cells which formed aggregates in culture. Most importantly these cells rescued anemic mice and these mice were cured.
In January, Sara Rankin et al. discovered a way to stimulate bone marrow
to release two other types of stem cell, between them these can repair bone, blood vessels and cartilage. Giving mice Mozobil (VEGF) boosted stem cell counts in their bloodstream more than 100-fold. This success is working towards using patients own stem cells to regenerate damaged organs. Some stem cells are able to develop into any cell type more worrying cancerous cells, this problem is overcome with this study as the stem cells used here can only grow into blood vessels, bone and cartilage, so the risk of causing cancer is eliminated26.
Fig.6. Induced pluripotent stem cells and transplantation therapy Combining stem cell therapy and gene therapy has shown to rescue anaemic mice after transplantation. Adapted from 25.
To conclude, there are various potential stem cell therapies for the treatment of heart failure; the most promising involving human ES cells. Many of the transplantations discussed derived cardiomyocytes, although studies involving transplantation of cell differentiating into non-cardiomyocytes have also been discussed. The latter focuses on heart failure indirectly, and is targeted towards the treatment of severe ischemic diseases. The unique property of human ES cells in that they are able to transdifferentiation into any cell type recent research looks to optimize differentiation depending on preferred cell type. I have appreciated the importance of transplantation strategy for cardiac repair once the particular type of stem cell derived cardiomyocytes have been isolated, moreover the method of cell delivery is also reflective on the ability of the stem cell to regenerate cardiomyocytes and restore cardiac function. This plays a significant role in animal models of stem cell therapy treatment towards heart failure which if successful can then be elaborated in human clinical trails. It has been discussed that the problems of immune response, cultured reagents (containing contaminated viruses) and cell survival in grafts are overcome by the use of adult stem cells. In the past producing a sufficient number of cells for transplantation was a problem, tough this is overcome by the ability to generate human ES cells in high numbers (millions) in culture. New hope of combining stem cell therapy with gene therapy and pharmacological treatment look promising. Several areas of stem cell therapy still need to be addressed e.g. how long will the replacing cardiomyocytes be able to restore the heart function. Also in the research discussed do certain animal models accurately reflect human heart conditions? Moreover more work needs to be carried out on the electrical properties of the cardiomyocytes derived from stem cells; are they similar to the electrical properties of resident cardiomyocytes?
Stem cell therapy is a fast moving area of research, the concept of replacing cells have not just been looked at in the heart but progression to replacing cells in the pancreas (treat patients with diabetes) and in the brain (treat patients with Parkinson's diseases) and in the spinal cord (treat patients with spinal injury) have been studied this shows how fast stem cell therapy is evolving. With regard to heart failure, much development in the past few years many are based on murine models than human models however I appreciate early stages and much development is being done to transfer many of the experiment s discussed to human clinical trails with the hope of treating the worldwide prominent disease heart failure.