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ORIGINAL ARTICLE |
Frontier Lifeline Pvt. Ltd, Chennai, India
Soma Guhathakurta, PhD Tel: +91 44 42017575 Fax: +91 44 26565150 Email: husun1971{at}gmail.com, Frontier Lifeline Pvt. Ltd, R 30 C Ambattur Industrial Estate Road, Mogappair, Chennai 600 101, India.
ABSTRACT
Growing myocardial cells from human stem cells and stem cell transplantation to repair injured myocardium are new frontiers in cardiovascular research. The 1st stage of this study was conducted to determine whether transplantation of autologous bone marrow stem cells into infarcted myocardium of sheep could differentiate into beating cardiomyocytes. The 2nd stage was to demonstrate transdifferentiation of human bone marrow mesenchymal stem cells to precursor cardiomyocytes in vitro, using a novel conditioning medium. In the 3rd stage, a clinical trial of stem cell implantation in patients with severe myocardial dysfunction involved injection of peripheral blood-derived endothelial precursor cells in 11 patients and autologous bone marrow mononuclear cells in 29. A marginal improvement in myocardial function was noted at 3 months (mean increase in ejection fraction, 6% ± 1%), although it plateaued at 6 months. The trial proved to be safe because there was no procedure-related mortality. There is growing optimism that stem cell therapy may delay heart transplantation.
Key Words: Adult Stem Cells • Cardiomyoplasty • Cell Proliferation • Heart Failure • Mesenchymal Stem Cells
INTRODUCTION
Over the last 20 years, the incidence of heart failure has markedly increased, and its prevalence in patients over 65 years of age is now 10%.1 Standard surgical procedures, such as coronary artery bypass grafting, mitral valvuloplasty and/or valve replacement, have been used to treat patients with advanced left ventricular (LV) dysfunction, with good initial results. These procedures prevent further myocardial deterioration but do not act on underlying diseases such as myocardial fibrosis. At the opposite end of the spectrum, cardiac transplantation provides radical therapy, but the donor organ shortage and strict eligibility criteria mandate alternative treatments when a substantial portion of the myocardium has been destroyed. Cell transplantation has emerged as a possible means of increasing the number of contractile elements in damaged myocardium.2 The twin capabilities of self-renewal and multi-lineage differentiation make stem cells unique. In 1995, the term "cellular cardiomyoplasty" was coined at the annual meeting of the Society of Thoracic Surgeons. Cell therapy has taken a great leap in the past decade with a rush of clinical and laboratory trials based on restoring cardiomyocytes in failing myocardium. Despite rapid progress in this field, many questions remain unanswered. To determine the role of cellular cardiomyoplasty in the failing heart, we performed a series of ovine experiments followed by a clinical trial, taking our work from bench to bedside.
PATIENTS AND METHODS
This study was conducted in 3 stages. The 1st stage was conducted in the animal laboratory to find out whether bone marrow stem cells injected into infarcted myocardium could differentiate into beating cardiomyocytes. The 2nd stage was based on the concept that stem cells could differentiate into precursor cardiomyocytes instead of mature cardiomyocytes. Cell proliferation is expected in this precursor state, but not in adult cardiomyocytes. In the 3rd stage, a clinical study involved injection of endothelial precursor cells (EPC) or autologous bone marrow mononuclear cells into patients with endstage ischemic or dilated cardiomyopathy.
ANIMAL STUDY
After approval by the Committee for the Purpose of Control and Supervision of Experiments on Animals, 9 Madras Red sheep were divided into a study group of 6 and a control group of 3. All 9 sheep underwent preoperative 2-dimensional echocardiography and a 199m-sestamibi cardiac perfusion scan. A left antero-lateral thoracotomy was performed, and a diagonal branch of the left coronary artery near the apex was ligated to produce infarction. During this procedure, the 5th rib was excised from the 6 study sheep for extraction of mesenchymal bone marrow stem cells (BMSC). One sheep in each group died after coronary ligation. The BMSC were expanded for 3 weeks, and 105 to 106 cells were injected by an epicardial route into the periphery of the infarcted myocardium via a redo left anterolateral thoracotomy. The BMSC from sheep no. 1 were transfected with cytomegalovirus vector tagged with green fluorescent protein; the other 4 sheep received untagged BMSC. The 2 control sheep were not injected. All sheep underwent 2-dimensional echocardiography and 199m-sestamibi perfusion scanning at 3 weeks post-ligation and 3 months post-injection, followed by explantation of the hearts for histological evaluation. All operations were performed under general anesthesia.
IN-VITRO EXPERIMENTS WITH HUMAN MESENCHYMAL STEM CELLS
To aid the formation of precursor cells, a novel induction medium was devised.3 This was added to the BMSC culture medium without 5-azacytidine, and the results were assessed under inverted microscopy. Immunohistochemistry and reverse transcription poly-merase chain reactions were performed to study cardiac transcription factors so as to devise a simpler method of application of stem cell therapy in patients.
CLINICAL TRIAL
An initial study of stem cell implantation involved injection of endothelial precursor cells into 11 patients, and autologous BMSC into 29 patients, up to December 2008. All statutory permissions were obtained before stem cell therapy, including informed consent of the patient. The institutional research committee, ethical committee, and Committee for Stem Cell Research and Therapy gave approval for this clinical trial. All patients had endstage ischemic or dilated cardiomyopathy with LV ejection fractions of 20%–35%. The etiology included coronary artery disease, anomalous left coronary artery from the pulmonary artery (ALCAPA), noncompaction of the LV, and irreversible pulmonary hypertension. The stem cells were delivered by various routes: 9 patients underwent epicardial injection as a concomitant procedure, 25 had intracoronary artery injections, and 6 had intrapulmonary artery injections (Table 1
). One patient with noncompaction of the LV had a complex congenital heart defect and received BMNC epicardially during closure of a ventricular septal defect and a patent ductus arteriosus. All epicardial injections were performed around the scar with 5 mL of BMNC concentrate containing 107 to 109 cells per milliliter. The intracoronary volume had a 3-times dilution of the cells and was made up to 15 mL. Two pediatric patients with dilated cardiomyopathy were given intracoronary injections after obstructing the coronary sinus for better homing. An acellular bovine pericardial support was used as a scaffold for more effective homing of epicardial injections in 3 patients with coronary artery disease. An appropriate marker study using flow cytometry (CD34+ for EPC; CD90, CD105, and CD45 for BMNC) and an endotoxin study were also conducted. Preoperative patient evaluation included brain natriuretic peptide estimation and 2-dimensional echocardiography. These tests were repeated at 3, 6, and 12 months postoperatively.
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FIRST STAGE
The 5 experimental animals that survived coronary artery ligation showed definite improvements in perfusion, which were not seen in the 2 control sheep. Echocardiography demonstrated marginal improvements in ejection fraction at 3 months post-injection, just before explantation. Trichrome staining of explanted heart tissue from the injection site revealed subendocardial sparing of muscle tissue in the scar tissue. Distributed within the scar tissue, in both control and experimental sheep, were island of muscle tissue that could not be differentiated from the surrounding muscle tissue. Histopathology in the 4 sheep that underwent untagged stem cell injection showed a large island of newly formed muscle within the scar, which appeared different from the surrounding myocardium (Figure 1). In the animal given tagged stem cells, the muscle cells within the scar were fluorescent Figure 2A. GFP tagged & Figure 2B H&E Stain. The neo-muscular area contracted at a rate of 40 beats per minute (Figure 3).
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). Transmission electron microscopy revealed that these structures did not have gap junctions, but sarcomeric protein was observed (Figure 5). Immunocytochemistry was positive for alpha myosin heavy chain and troponin I on fluorescent microscopy, comparable to 5-azacitidine-induced mesenchymal stem cell differentiation. Reverse transcription polymerase chain reaction tests revealed expression of GATA-4 (MGC126629, location: 8p23.1p22), Nkx 2.5-E (location: 5q34), hANP (location: 1p36.2), MLC-2v (location: 7p43), and MLC-2a (location: 21p33).
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& 6B). The stem cell injection was performed along with the Takeuchi procedure to correct the ALCAPA anomaly. Other EPC-injected patients produced mixed results, from no improvement to a 7% increase in ejection fraction, with no mortality.
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Despite medical advances, heart failure management remains a challenge. New modalities of treatment have enhanced lifespan, but these patients ultimately succumb to heart failure. Heart transplantation is the final option, but donor hearts and external devices that bridge to transplantation are inaccessible to the majority of the population, for various reasons. Among the many causes of heart failure, ischemic or dilated cardiomyopathy can be addressed by stem cell therapy.4 Myogenic cell grafting is a promising treatment for cardiac failure. The efficacy of fetal cardiomyocyte or autologous skeletal myoblast transplantation in the failing hearts of experimental animals has been etablished.5,6 Menasché and colleagues7 reported that transplanted autologous skeletal myoblasts, expanded in culture, increased ejection fractions by 9% in patients with myocardial infarction; however, 25%–30% required implantation of an automatic cardiac defibrillator to prevent ventricular arrhythmias. Chiu and colleagues8 also reported favorable results of cellular cardiomyoplasty.
The use of bovine serum-cultivated skeletal myoblast implantation in clinical trials has resulted in serious ventricular arrhythmias to the extent of sudden cardiac death.9 Bovine serum subjects patients to immunogenic reactions as well as xenozoonosis, thus we chose a serum-free medium for BMSC culture. Mandatory use of an internal cardiac defibrillator should be considered with skeletal myoblasts, which is not practical in the Indian scenario for economic reasons.10 The high probability of the need for an intracardiac defibrillator hinders the use of this modality in developing countries. Skeletal myoblast transplantation has many advantages: it is autologous, resistant to ischemia, and large numbers of cells are available. This procedure has also proved useful in nonischemic heart failure. We considered fetal cardiomyocytes to be unfeasible because they are difficult to acquire, and the immunogenic and ethical issues are obvious. Embryonic stem cells emerged as promising for cardiac repair in the late 1990s. Although it had been known for some time that these cells could give rise to cardiomyocytes, several difficulties limit their application in cardiac repair, such as the technical difficulty of growing these cells and keeping them undifferentiated, the low efficiency with which cardiac differentiation occurs spontaneously, and the challenge of purifying cardiomyocytes from the many other cell types that form during spontaneous differentiation.11
When adult stem cells have been used to regenerate myocardium, different results have been obtained experimentally by various groups.4 In-vitro experiments with human BMSC in conjunction with various growth factors or induction media, such as 5-azacytidine, showed cardiomyogenic potential.12 Systemic delivery of BMSC is a noninvasive approach to myocardial repair. Krause and colleagues13 tested this strategy in a pig model of myocardial infarction; intravenous delivery of BMSC limited myocardial infarct size, indicating an attractive approach to tissue repair. However, in practice, cardiac failure is associated with multiorgan dysfunction, hence systemic delivery of stem cells to the heart is a mere probability. The novel conditioning medium may be a future option for systemic therapy if the factors can be made injectable, because it induces stem cells to form precursor cardiomyocytes. In standardizing the various procedures for stem cell culture from multiple sources, it was hypothesized that the ubiquitous autologous stem cell sources transdifferentiate into cardiomyocytes in vivo.14 Initially, EPC from peripheral blood with granulocyte colony-stimulating factor augmentation were the cells of choice following the rationale of Asahara and colleagues15 and other reports.16 It has also been hypothesized that paracrine factors are the cause of this type of cardiac regeneration along with neo-angiogenesis, which is the primary function of EPC.17
Zohlnhofer and colleagues18 noted insignificant improvements in infarct size in a randomized controlled trial of progenitor cell injection of cardiac muscle in acute myocardial infarction. The inflammatory condition may hinder survival of the injected cells. Most of our patients had chronic ischemic and/or dilated cardiomyopathy. The child with ALCAPA showed considerable improvement that was possibly due to the young myocardium. One month after surgery, the LV appeared to be thickened and hypertrophied, but regressed later. Most patients who received EPC showed a 4%–5% increase in ejection fraction at the 3-month follow-up. The artificial stimulation of bone marrow by granulocyte colony-stimulating factor was a matter of controversy in our research group, and autologous bone marrow-derived mononuclear cells for cellular cardiomyoplasty have gained wider acceptance worldwide. In our clinical trial of BMNC, 2 pediatric patients with dilated cardiomyopathy had cells delivered by catheter to the coronary artery, with coronary sinus occlusion for 10 min. This was tolerated because of the opening up of sinusoidal veins in the right side of the heart, which is common in dilated cardio-myopathy. Yoon and colleagues19 reported calcification and bone formation after BMSC injection into the myocardium. So far in our studies, no calcification has been noticed on echocardiography or radiography.
Stem cell therapy has a definite regenerative role in myocardial injury. The route of delivery and cell type have yet to be optimized. Regular use of the NOGA catheter for endocardial stem cell injection and scaffold support for better homing remain difficult in facilities with limited resources. This clinical trial proved to be safe because there was no procedure-related mortality. The plateau in the ejection fraction observed after 6 months has been noted in other studies, and it indicates that a single injection is insufficient; probably stem cell therapy has to be offered at intervals to patients to provide sustained improvemnet in cardiac function.4
ACKNOWLEDGMENTS
This study was partly supported by the Indian Council for Medical Research. We gratefully acknowledge the assistance and expertise of Dr. G Subramaniam, Director, Sree Venkateswara Institute of Medical Sciences. The surgical and cardiology teams at Frontier Lifeline Pvt. Ltd. fully supported this study by their expertise in delivery of stem cells. The scientists of the stem cell laboratory of the institution gave wholehearted support in making this research successful.
REFERENCES
Asian Cardiovasc Thorac Ann 2009;
17:581-586
© 2009 by SAGE Publications
DOI: 10.1177/0218492309349363
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