Bone marrow cell transplantation in clinical perspective

Journal of Molecular and Cellular Cardiology 38 (2005) 225–235 www.elsevier.com/locate/yjmcc

Review article

Bone marrow cell transplantation in clinical perspective Husnain Kh. Haider, Muhammad Ashraf * Department of Pathology and Laboratory Medicine, 231-Albert Sabinway, University of Cincinnati, Cincinnati, OH-45267-0529, USA Received 20 September 2004; received in revised form 20 November 2004; accepted 10 December 2004 Available online 27 January 2005

Abstract The deficit in left ventricular performance in an ischemically damaged heart is characterized by depletion of functioning cardiomyocytes. The problem is accentuated by the inadequate intrinsic repair mechanism of the heart. Recent progress in regenerative medicine has paved way for an outside intervention to support the limited on-going reparative process in the heart through transplantation of cells with myogenic and/or angiogenic potential. The functional plasticity of bone marrow derived stem cells has been exploited for cardiac repair. The early experiences in proof-of-concept animal studies and phase-1 clinical trials have been encouraging and suggest the safety, feasibility and potential of this approach. We critically review the literature in depth to elucidate the progress in this field together with discussion of the problems and controversies that need to be addressed in order to fully exploit the potential of bone marrow stem cell transplantation with clinical relevance. © 2005 Elsevier Ltd. All rights reserved. Keywords: Bone marrow; Cardiac; Infarction; Myocardial; Stem cells; Transplantation

1. Introduction Despite enormous progress in the modern day cardiovascular therapeutics, treatment of heart failure still remains a therapeutic challenge. Orthotropic heart transplant is considered as the gold standard to treat the patients who remain refractory to maximum medical therapy. However, the approach is limited by problems such as donor organ rejection, untoward effects of immunosuppression and more so because of the disparity between the number of the donors and recipients. Risk factor reduction, pharmacological treatment using b-blockers and angiotensin converting enzyme inhibitors, and conventional surgical interventions for revascularization together with implantation of left ventricular assist devices have improved the quality of life for heart failure patients [1]. Most of these therapeutic options fail to address the root cause of the problem, which is characterized by compromised blood circulation to the myocardium due to coronary artery occlusion together with multifactorial irreversible cardiomyocyte dysfunction and loss [2,3]. The problem is accentuated by the inadequate intrinsic repair mechanism of the heart, which needs an outside intervention to

prevent the heart from entering into compensatory vicious cycle of left ventricular remodeling. The existence of resident cardiac muscle stem cells is a good omen but their number is too little to significantly contribute in the repair mechanism [4]. Reports have been published showing the ability of cardiomyocytes for their renewal [5,6]. Of the various cellular and molecular approaches adopted, therapeutic angiogenesis and adult stem cell based therapy have given encouraging results [7–12]. The safety and effectiveness of both these approaches have been well documented in proof-of concept animal studies as well as in the human trials [13–16]. A step further is to combine both these approaches to achieve concurrent angiogenesis and myogenesis for cardiac repair [17–19]. Myocardial regeneration based on stem cell transplantation is tantalizing and exciting [20–23]. The underlying principle of this approach is to transplant donor cells with myogenic or/and angiogenic potential to supplement for the cardiomyocyte loss and improve regional blood flow [24–27]. Donor cells from various origins and with differing potentials have been used [28–34]. 2. Bone marrow derived stem cells

* Corresponding author. E-mail address: [email protected] (M. Ashraf). 0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.12.005

Bone marrow has a complex environment, housing a vast repertoire of both committed as well as non-committed cells.

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These cells have a well defined proliferative hierarchy. The bone marrow derived stem cells are multipoten and have the capacity to differentiate into distinctive and diverse progenies of differentiated cells including bone, hematopoietic cells, myocardium and endothelial cells [35–37]. Plasticity of bone marrow derived stem cells is under intense investigation [38,39]. Pre-clinical studies in both small and large animal models have confirmed that transplantation of autologous bone marrow cells or their purified subpopulations, restored regional blood flow by angiogenesis, reduced infarction size and fibrosis, improved regional wall thickness and enhanced the contractile function of the left ventricle wall through myogenesis [40–43]. More recently, BMSC have been genetically modulated to over express angiogenic cytokines to achieve angiogenesis in order to treat myocardial ischemia [44]. The adult bone marrow contains hematopoietic (1–2%) and stromal (<0.05%) stem/progenitor cells. Hematopoietic stem cells (HSC) and progenitor cells are capable of reconstitution of the entire hematopoietic system and have been shown to have the ability to trans-differentiate into cardiomyocytes [45]. On the other hand, bone marrow stromal stem cells include adult mesenchymal stem cells (MSC) and multipotent adult progenitor cells, which are capable of multilineage differentiation [46]. MSC remain undifferentiated with stable phenotype and may be reprogrammed to transdifferentiate into cardiomyocytes [47–49]. In vitro treatment of BMSC with 5-azacytidine, a DNA demethylating agent has been shown to induce their cardiomyogenic differentiation through an as yet undefined mechanism [27,50]. Our group has recently shown in vitro that cell to cell contact between cardiomyocytes and bone marrow cells may be necessary for cardiomyocyte differentiation of the bone marrow cells [51]. Various studies have shown milieu dependent transdifferentiation into heart muscle cells [52–54]. Besides, bone marrow also contains endothelial progenitor cells, which have the capacity to initiate neovascularization to alleviate myocardial ischemia [55–57]. The mononuclear fraction of bone marrow consist of various subpopulations which have the potential to participate in the repair and regeneration of diseased myocardium in vivo through transdifferentiation into cardiomyocytes and initiate neovascularization by expressing various angiogenic cytokines [58–61]. Hence, the heterogeneous mononuclear fraction of the bone marrow is the most promising stem cell population for cellular cardiomyoplasty. There is still no universal marker to identify bone marrow derived stem cells. However, a variety of surface proteins have been identified for selection and enrichment of subpopulations of bone marrow cells transplantation studies in animal models [41,62–65]. The CD34 is commonly used to reconstitute hematopoietic cells. A recent study has suggested that the bone marrow stem cell plasticity resides in CD34+ population. However, HSC are not limited to the CD34+ cell population, and the role of CD34– HSC in hematopoietic reconstitution and regenerative biology is not clear [62]. BM derived CD34+ endothelial progenitor cells have been isolated from

peripheral blood and the BM in humans. Lin– c-kit– cells, known to be devoid of stem cells, failed to regenerate cardiac cells upon transplantation, whereas Lin– c-kit+ cells could generate cardiomyocytes, smooth muscle cells, and endothelial cells. Similarly, Sca-1+ cells have been shown to possess the ability to differentiate into beating cardiomyocytes [66]. The results of a phase-1 human study using AC133+ cells are in agreement with experimental studies showing the high angiogenic potential of the non-hematopoietic (CD34–) subpopulation of the BM, which is a constituent of AC133+ cells. Thus, AC133, CD34, Lin– and c-kit could be important markers that may identify more useful subpopulations of BM stem cells in terms of cardiac regeneration.

3. Transdifferentiation or fusion: a debatable issue Bone marrow mononuclear cells taken from myocardial infarction patients have been found to show tropism for damaged cardiac tissue in vitro [67]. Besides that significant debate over the presence of stem cells from extracardiac origin in the cardiac tissue [68,69], the underlying mechanism as to how the donor bone marrow stem cells contribute towards improved cardiac function remains a highly debatable issue [70,71]. The possible mechanisms include transdifferentiation of donor cells, fusion of the donor cells with the host cardiomyocytes to adopt their phenotype, dedifferentiation–redifferentiation of stem cells to adopt the required phenotype, neoangiogenesis and upregulation of multiple paracrine factors. Can stem cells cross lineage boundaries is an important issue [72–74]. A series of recent experiments have challenged the long standing dogma of lineage restriction and multipotentiality of bone marrow stem cells to transdifferentiate into various lineages have been substantiated in vitro [35,75– 82]. Contrary to these findings, several research groups have advocated independently the possibility of fusion between the donor BM cells with the host cells to adopt the phenotype of the host tissue [83,84]. Recent publications from Alvarez et al. [85] have shown fusion between donor bone marrow cells and Purkinje fibers, cardiomyocytes and hepatocytes. Back to back published recent reports from Balsam et al. [86]; Murry et al. [87] have clearly depicted lack of the ability of donor bone marrow cells to undergo differentiation after transplantation into cardiomyocytes. They have shown that despite their presence in the cardiac environment, there was little evidence that the transplanted bone marrow cells formed cardiomyocytes at the site of the graft. Similarly, a word of caution from Bel et al. [88] that isolated un-fractionated bone marrow cells failed to differentiate into cardiomyocytes. These results seriously challenge the findings of Orlic et al. [89] and have left the debate open whether bone marrow stem cells have the required plasticity to transdifferentiate into cardiomyocytes post transplantation.

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4. Clinical studies

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ischemic heart disease. The prospective study was designed to assess the safety and feasibility of non-surgical and less invasive catheter based cell implantation technique as a sole therapy. The patients were having stable angina and their heart function was assessed to have LVEF > 30%. Five of the eight patients were previously treated by percutaneous trasluminal coronary angioplasty (PTCA) (two with laser myocardial revascularization (LMR) and two with CABG with LMR). All patients underwent non-fluoroscopic left ventricular electromechanical mapping with NOGA system. An average of 107 cells per ml was injected at 11 scattered sites targeted in the infarcted region. The whole cell transplantation procedure was uneventful and the patients recovered well. Three months follow up revealed no arrhythmic changes on Holter monitoring. There was a significant reduction in the number of anginal episodes with concomitant reduction in nitroglycerine consumption. End point measurements using MRI showed improved left ventricle muscle wall thickness (11.6%) and wall motion (5.5%). There was an overall reduction in the hypoperfused left ventricle (from 8.8% to 5%). LVEF, however, showed no significant improvement as compared to the baseline measurements (57.6 ± 10.8). Whereas the study revealed overall safety of the catheter based delivery of bone marrow cells, the effectiveness remains questionable. NOGA mapping helped in targeted delivery of bone marrow cells to

Encouraged by the outcome of the first-in-man autologous skeletal myoblasts transplantation, researchers took a different approach [90]. Transplantation of autologous bone marrow stem cells was carried out to exploit the plasticity of bone marrow stem cells. Interestingly, most of the clinical studies reported to date have been carried out in Europe and Asia. Overall, the results are encouraging, however, the trials reported until now are observational to assess the safety and feasibility of the approach. As the studies were designed to assess the safety and feasibility of the approach, inclusion of a control group was not considered mandatory during the design of most of the documented studies. However, some of the studies have included a control group of patients to validate the results (Table 1). A summary of the reports published in refereed journals have been discussed.

5. Cell transplantation as sole therapy Tse et al. [91] at Queens Hospital, Hong Kong implanted autologous bone marrow derived unselected mononuclear cells (3.2 ± 2.4% CD34+; 7.6 ± 3.5% CD3; 43.7 ± 15.9% CD116CD15 granulocytes) in eight patients with severe

Table 1 Clinical studies using bone marrow cell transplantation with control group of patients Study reference

Patients (control)

Delivery Adjunct mode procedure

07

Patients Cell type (with cells) 14 BMMNC

Perin et al. [93]

EC

ST

Strauer et al. [108]

10

10

BMMNC

IC

PCI

Assmus et al. [110]

09

20

BMMNC/CPC

IC

PCI

Chen et al. [109]

35

34

BMMNC

IC

PCI

Wollert et al. [114]

30

30

CD34+

IC

PCI

Follow Result summary up (months) 2 and 4 Improved LVEF and reduction in LVEDV, Significant mechanical improvement in the injected segments as assessed by 2D Echo, dipyridamole SPECT perfusion scan and NOGA EMM 3 Significant reduction in infarct size Improved infarct wall motion, improved stroke volume index, LVEF and regional perfusion by DS Echo, radionuclide ventriculography 4 Improved regional wall motion in the infarct area, enhanced LVEF, profoundly reduced LVEFV, enhanced viability in the infarct zone by DS Echo, quantitative PET scan Improved wall velocity in the infracted Segments, reduction in the perfusion defects, improved LVEF, significantly reduced LVESV by Echo, NOGA EMM, PET scanning 06 Improved LVEF, 24 h Holter monitoring, functional MRI

Complications

One death each in the control and cell injected groups

None

None

BMMNC: bone marrow mononuclear cells; DS-Echo: dobutamine stress echocardiography; Echo: echocardiography; EMM: electro mechanical mapping; IC: intracoronary; LVESV: left ventricular end systolic volume; MRI: magnetic resonance imaging; PCI: percutaneous coronary intervention; PET: positron emission tomography.

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the ischemic myocardium. The authors claim to have achieved angiogenesis to reduce the overall hypoperfused area of the left ventricle; there was no mention as to how the wall motion and wall thickness showed significant improvement. Moreover, the study lacked in a control group to make a comparison of the results. Perin et al. at Texas Heart Institute in collaboration with Dohman et al. at Hospital Procardiaco, Rio de Janeiro reported 21 patients for a non-randomized phase-1 study. The first 14 patients were included as cell treatment group whereas the last seven patients admitted into the study were taken as control group [92,93]. The no-option patients included in the study were having severe LV dysfunction and end stage ischemic disease (43% control and 7% treatment group had PTCA and 86% control and 64% had CABG done previously). After a complete baseline clinical and laboratory evaluation and heart function assessment by exercise stress (treadmill), 24 h Holter monitoring, 2D Doppler echocardiography and SPECT perfusion imaging, NOGA electromechanical mapping was performed for location of viable and ischemic myocardium. BMMNC were purified from autologous bone marrow aspirate (50 ml) by Ficoll gradient. The purified cell population comprised a mean population of 2.44 ± 1.33% CD45CD34+ cells. During cell transplantation, each patient received 15 ± 2 cell injections of two million freshly isolated cells/0.2 ml each injection in the hibernating myocardium. One patient died in control group and experiment group each at 2 and 14 weeks after the commencement of the study respectively. Non-invasive 2 months follow up revealed improvement in NYHA class (from 2.21 ± 0.89 to 1.14 ± 0.26) in the cell treatment group as compared to control group (from 2.271 ± 0.75 to 2.71 ± 0.76). The echocardiographic data was interpreted by two observers blinded to the treatment protocol. Whereas the hemodynamic parameters ESV, EDV, and LVEF showed trend of deterioration in the control patients, there was significant improvement in ESV (from 146.75 ± 53.46 to 123.21 ± 47.88) EDV (from 211.35 ± 76.89 to 189.14 ± 67.54) and LVEF (from 30 ± 5.56 to 35.5 ± 7.85). Similarly, during nuclear perfusion imaging in the treatment group, the total reversible defect showed 73% reduction (from 15.15 ± 14.9% to 4.53 ± 10.61%), however, the rest defect remained almost unaltered. A 4 months invasive follow up which was restricted to the treatment group patients only, left ventricle angiography revealed that the cardiac function improvement was sustained over the period of time. EMM based segmental analysis showed a significant improvement in the contractile function of the segments where cells were transplanted. One year follow up study results highlight the safety and effectiveness of endocardial route of administration for bone marrow cell transplantation and documents the improvement of cardiac function and blood flow in the ischemic regions transplanted with BMMNC [94]. Fuchs et al. [95] at Washington Hospital Center reported percutaneous transendocardial injection of un-fractionated autologous bone marrow cells in a group of 10 patients with advanced coronary artery disease. They had previously shown

the feasibility of the approach in pigs [96]. The patients were in Canadian Cardiovascular Society (CSS) angina class III–IV and having at least one epicardial conduit >70% and one 70% diameter stenosis. These patients were diagnosed with severe symptomatic myocardial ischemia, which was not amenable to conventional revascularization procedures. All the patients included in the study had a history of CABG and 90% had percutaneous coronary intervention (PCI). Bone marrow aspirate was purified and contained 32.6 × 106 nucleated cells per ml (CD34+ 2.6 ± 1.6% of which 47.9 ± 15.1% were CD34+ CD45+ and 85 ± 14% double positive also expressed CD117). These cells were assessed for expression of VEGF and MCP1 in vitro. The patients underwent transendocardial injection of the freshly isolated cells using left ventricular electromechanical guidance. A total of 12 injections (0.2 ml each injection) were made in the ischemic myocardium, which was previously identified by SPECT perfusion imaging and NOGA EMM. The whole procedure was uneventful without complications. Three months follow up showed improved angina symptoms in eight patients (from CSS class 3.1 ± 0.3 to 2.0 ± 0.94). Myocardial perfusion studies showed improved perfusion in the stress score whereas the rest score remained unchanged. LVEF, however, showed no significant improvement (from 47 ± 10% to 52 ± 6%). Treadmill exercise duration increased (from 391 ± 155 to 485 ± 198) in nine of the patients included in the study. Beran et al. [97] have documented a case report in which a 57-year-old-male patient was treated with autologous BMMNC. The patient was administered thrombolytic therapy within 2 h of acute anterolateral myocardial infarction. Echocardiographic assessment revealed apical and mid anterolateral hypo- to akinesia and confirmed by Tc99m-MIBI rest scans. Six weeks later, 2 × 109 autologous BMMNC suspended in 5.2 ml volume were injected under NOGA guidance system at 12 different sites in the infarct border zone. Follow up at 6 months using NOGA suggested improved viability in the anterolateral regions. Tc99m–MIBI rest scans showed reduced resting defect size from 34% to 25%. LVEF obtained from SPECT showed improvement from 33% to 41%. The study vividly showed improved viability of the myocardium after BMMNC. Fernandez-Aviles et al. [98] have reported results in five patients with anterior acute myocardial infarction and isolated stenosis of the left anterior descending artery. The patients underwent successful repair by primary or facilitated angioplasty. At 10–15 days after infarction episode, bone marrow derived cells were delivered as intracoronary infusion without any procedural complications and arrhythmias. The follow up protocol at 6 months after cell transplantation included low-dose dobutamine echocardiography, MRI and ECG Holter monitoring. The results revealed that LVEDV remained unchanged (from 159 ± 25 to 157 ± 16 ml), LVESV decreased (from 77 ± 22 to 65 ± 16 ml), and the LVEF increased insignificantly (from 53 ± 7% to 58 ± 8%). The results highlight the safety of intracoronary delivery of BM cells.

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6. Cell transplantation adjunct to routine revascularization procedure 6.1. Cell transplantation together with CABG Hamano et al. [26] at Yamaguchi University reported a small study including five patients (three male and two female; average age 65 years) who were scheduled to undergo CABG. Autologous BMMNC cells were purified and injected intramyocardially. Each patient received 5 × 107–1 × 108 cells at 6–22 injection sites in the area of ischemic myocardium. All patients had an uneventful peri and postoperative course without any complications. One year follow up results revealed significant improvement in regional perfusion on Tc99m scintiscans and in overall cardiac function in three out of the five patients. Moreover, there was no calcification or teratogenic effects in the cell graft region. Galinanes et al. [99,100] from University of Leicester, UK, investigated the safety of transplanting un-manipulated autologous bone marrow into infarcted myocardium and assessed its efficacy to improve cardiac function. The procedure was carried out as an adjunct to coronary bypass surgery in 14 patients. Autologous bone marrow was obtained by sternal bone aspirate at the time of surgery, diluted in autologous serum at a ratio of 1:2, and then injected 1 cm apart into the mid-depth of the left ventricular scar. There were no deaths or peri-operative myocardial infarctions, and ventricular arrhythmias. Dobutamine stress echocardiography demonstrated overall improvement in the global and regional left ventricular function 6 weeks and 10 months after surgery. Of 34 infarcted left ventricular segments, 11 were injected with bone marrow alone, 13 were revascularized with a bypass graft alone, and 10 received bone marrow transplantation and a bypass graft in combination. Only the left ventricle segmental wall motion score of the areas injected with bone marrow and receiving a bypass graft in combination improved at low dose and at peak dobutamine stress. The regional wall motion score decreased significantly from an average of 2.41 prior to injection to 2.16 after 6 weeks and 2.09 after 10 months. The global wall motion score also decreased significantly from 1.96 before surgery to 1.64 at 6 weeks. The effects were sustained until 10 months of observation. In another interesting study by Stamm et al. [101] at Rostock, Germany, six patients suffering from transmural acute myocardial infarction was enrolled for cell transplantation as an adjunct to CABG. All patients were having distinct area of akinesis and hypokinesis in the left ventricular wall. The baseline measurements for cardiac function were obtained from coronary angiography, transthoracic echocardiography and myocardial SPECT imaging. Unlike the other studies, which used whole unselected mononuclear cell fraction of bone marrow cells, ACC133+ subpopulation of the mononuclear cells was purified and used for transplantation. The rationale to use purified ACC133+ cells was to avoid injection of large number of leukocytes and their progenitors, which have limited plasticity and the presence of which in large number may

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give rise to unwanted inflammatory response at the site of the graft. A total of 1 × 106 cells were injected at 10 sites (0.2 ml per injection site). All patients survived the procedure without any complications. The same group has added six more patients to the cohort and reported a total of 12 patients [102]. These patients have received escalating dose of ACC133+ stem cells (from 1.5 × 106 to 2.8 × 106). This number of cells is however, still far less than the number, which has been reported by other groups. An interesting observation of the study was that patients with severely deteriorated LVEF responded better to cell transplantation. LVEF in patient #1 (21%), #3 (39%), #5 (25%) and #10 (35%) showed significant improvement to 46%, 58%, 43% and 55%, respectively. To the contrary, patients with comparatively better LVEF such as patient #2 (42%), #4 (47%), #8 (43%) and #11 (47%) showed insignificant improvement in LVEF 45%, 48%, 42% and 42%, respectively, during the follow up studies. However, it is difficult to infer such relationship for sure as the patient population was very small. Moreover the study did not include a control group besides also lacking in blinded assessment of the data. Thallium-201 SPECT scan showed improved regional perfusion in the previously hypoperfused or non-perfused areas although the center of the akinetic infarct regions remained unchanged. Importantly, there were no procedure related complications up to 14 months post-operatively, including arrhythmia or neoplasia. As the study was conducted adjunct to CABG, it remained, however, difficult to ascertain and delineate the improvement in cardiac function and regional blood flow resulting from cell therapy from the beneficial effects of CABG. Prosper et al. [103] at Hospital Clinico de Salmanca, Spain reported a phase-1 study involving patients (n = 6) with coronary artery disease (LVEF < 35%) and undergoing routine coronary artery bypass grafting. The patients were grafted adult autologous stem cells (CD34+/CD45–). The patients were pre-operatively assessed by stress thallium SPECT imaging and echocardiography for mapping the ischemic regions in the left myocardium. Cell transplantation procedure was without peri-operative arrhythmias or neurological or ischemic events. Six months follow up revealed significantly improved LVEF (25–30% to 35–55%). Yoon at Yonsei University Medical School, Seoul South Korea, carried out transplantation of BMMNC in seven patients who have been assessed for heart function preoperatively by echocardiography, MIBI scans and MRI and recommended for average two coronary grafts [104]. Autologous bone marrow was aspirated from the patients and processed to purify 1.5 × 109 mononuclear cells (7.3 × 106 CD34+ cells and 2.4 × 106 AC133+ cells). The cells re-suspended in 10 ml volume were injected into the non-graftable ischemic myocardium adjunct to off pump CABG. After an uncomplicated 2–7 months course, the preliminary data for patients follow up revealed improved LVEF (43–47%) and regional myocardial perfusion.

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7. Transplantation adjunct to percutaneous coronary intervention Intracoronary delivery approach for gene delivery and cell therapy is gaining popularity as it allows less invasive and targeted delivery of the agent at the site of interest [103,105]. Stem cells placed in the coronary vessel lumen are expected to extravasate and engraft in the region of the infarcted myocardium [106,107]. Strauer et al. [108] at Heinrich-Heine University, Dusseldorf exploited the advantages of intracoronary route for BMMNC transplantation. They have reported a phase-1 safety study in 20 patients of acute myocardial infarction. All the patients underwent conventional revascularization by PTCA and stent placement. Ten of these patients were given additional treatment of autologous BMMNC transplantation. The data from the other BMMNC non-treated patients was used to validate the results. A total of 1.5–4 × 106 cells in 2–3 ml heparanized saline were infused under high pressure in the infarct related artery around the infarct border zone during the routine procedure. The composition of the BMMNC preparation was 0.65 ± 0.4% AC133+ and 2.1 ± 0.28% CD34+ cells with potential to form mesenchymal cells in tissue culture. A 3 months follow up of all the patients revealed significant difference in their left ventricle dynamics of BMMNC treated and non-treated patients. Perfusion defect in BMMNC treated patients significantly decreased by 26% (P = 0.016) together with improvement in the percentage of hypokinectic and akinectic segments of left ventricle (from 30 ± 13 to 12 ± 7% P = 0.005). LVEF also showed improvement, albeit insignificantly. Although the therapeutic benefits of BMMNC transplantation are vivid from the results, and the safety of intra coronary route for BMMNC transplantation is obvious from the lack of arrhythmia and related problems, the study lacks a blinded control group. Moreover, like in many other studies, it is hard to assess the real benefits of BMMNC transplantation in the presence of PTCA. Chen et al. [109] at Nanjing Hospital, China have recently documented a thus far largest phase-1 study undergoing bone marrow cell transplantation for cardiac repair. Out of 78 patients who underwent PCI within 12 h after the onset of myocardial infarction episode, 69 patients were included in the study after they passed the established inclusion and exclusion protocol. The patients were randomized to receive bone marrow cell transplantation (n = 34) and only PCI (n = 35) as control group. Sixty milliliters of autologous bone marrow was aspirated from each patient and purified. After culture for 10 days in vitro, the cells were harvested, washed and re-suspended in 6 ml heparinized saline solution at a concentration of 8–10 × 109 cell per ml. The cells were transfused into the infarct related target coronary artery through an inflated over-the-balloon catheter with high pressure as described previously [108]. The control group patients received 6 ml normal saline solution without cells using the same method. All of the 69 patients underwent 18Ffluorodeoxyglucose PET scanning and echocardiography on

the day of cell transplantation, and 3 and 6 months after cell transplantation. EMM was also performed for 15 patients (cell transplantation group) and eight patients in the control group 1 day before and 3 months after cell transplantation. Three and 6 months follow up results were encouraging and showed significant differences between the two groups of patients. There were no deaths and the follow up until 6 months did not register any untoward effects of the treatment. The percentage of hypokinectic, akinectic and dyskinectic areas in the left myocardium significantly decreased (from 32 ± 5% to 13 ± 11%) whereas wall motion velocity (from 2.17 ± 1.3 to 4.2± 2.5 cm/s) and LVEF (from 49 ± 9% to 67 ± 11%) registered significant increase in the cell transplanted patients. Perfusion defects detected by PET also decreased significantly to 134 ± 66 cm2 as compared to the baseline 185 ± 87 cm2. Similar trend was also observed for hemodynamic parameters including LVEDV and LVESV. Echocardiography showed no arrhythmias at 3 months follow up. The most encouraging finding of the study was that the indicators of improvement in cardiac performance were maintained unruffled until 6 months of observation. Assmus et al. [110] have documented preliminary results of TOPCARE-AMI (transfer of progenitor cells and regeneration enhancement in acute myocardial infarction) a randomized study (n = 20; male n = 17 and female n = 3) in which they have carried out a comparison between BMMNC (nine patients) and circulating progenitor cells (CPC) in the blood (11 patients) for cardiac repair. An internal reference group of 11 patients was included as a control for comparison of results. All patients included in the study were acutely reperfused by coronary stent replacements and were maintained on optimum medical therapy. The study was originally designed to include 20 patients in each group. The control patients were matched for LVEF, infarct localization and infarct size with those included in the study for cell therapy and they underwent the routine stent implantation therapy. BMMNC (CD34+/CD45+) were purified from autologous bone marrow aspirates of each patient undergoing BMMNC transplantation and CPC were purified and expanded ex vivo for 3 days for each patient undergoing CPC transplantation from 250 ml venous blood. Cell transplantation was carried out 4.3 ± 1.5 days after acute myocardial infarction. A total of 10 ml cell suspension was delivered in the infarct related coronary artery using balloon catheter in the form of three infusions of 3.3 ml at an interval of 3 min each followed by coronary angiography to ensure unimpeded flow of blood. Except for one patient who suffered anterior wall infarction 3 days after cell transplantation, the whole exercise was uneventful without any untoward incidents including arrhythmia or death. A 4 months follow up revealed improvement in global cardiac function, however, it did not differ between patients receiving BMMNC or CPC. In patients receiving CPC, LVEF improved from 51.3 ± 11% to 59.5 ± 9% together with regional wall motion from –1.5 ± 0.3 to –0.6 ± 0.6 and LVESV from 56.9 ± 17.6 to 48.9 ± 14.2 ml. Corresponding values for BMMNC transplanted patients were from 51.9 ±

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9% to 60.7 ± 9% for LVEF, –1.6 ± 0.2 to –0.4 ± 0.8 for regional wall motion and from 55.2 ± 24 to 34.9 ± 13 for LVESV. In contrast, the control group patients without cell therapy revealed no significant changes in the respective parameters of observation. Coronary artery reserve measurements and myocardial viability using 18F-fluorodeoxy glucose PET scanning also registered significant improvement. The study results on hand highlight the safety and efficacy of the procedure like many of the previously reported studies. Moreover, it also showed that transplantation of CPC, the collection and availability of which is relatively easy and less invasive as compared to BMMNC, is as effective as BMMNC in improving contractile function of the heart and coronary artery flow reserve. The same research group has extended their previous report to include 28 patients [111] out of which the data for 14 has been previously reported [110]. They have used serial contrast enhanced MRI together with in vitro assessment of migratory capacity of transplanted progenitor cells, they have attempted to ascertain a causal relationship between the adult progenitor cell transplantation and improvement in cardiac function. LVEF has been found to improve from 44 ± 10% to 49 ± 10% (P = 0.003). Similarly, end systolic volume decreased from 69 ± 2 to 60 ± 28 ml whereas end diastolic volume remained unchanged. An important feature of the study is that this is the first attempt in clinical set up to have a mechanistic insight into the basis of myocardial function improvement when bone marrow or adult progenitor cells are transplanted. The results of the study highlight the significance of migratory capacity of the transplanted cells to home on to the injured myocardium rather than the total number of the cells or the type of the subpopulation of the transplanted bone marrow cells. Despite interesting data, the study fails to show the cellular mechanism associated with the improved LV contractile function. Siminiak et al. [112] in Poland, who have previously published skeletal myoblast transplantation for cardiac repair in a small phase-1 study have also reported two cases of bone marrow cell transplantation in myocardial infarction patients (patient #1: a 41-year-old-female and patient #2: a 64-yearold-male) [113]. Both patients had undergone routine percutaneous coronary artery recanalization within hours after admittance to the emergency room. Bone marrow was aspirated from sternal puncture and purified for CD34+ population of cells. The cells suspended in normal saline were transplanted in patient #1 (4.5 × 106 cells) on day 6 and patient #2 (6 × 106 cells) on day 3 by intracoronary infusion following the procedure as described by Strauer et al. [108]. The whole process of cell transplantation was uneventful and without any complications. The main aim of the study was to study the safety and feasibility of the procedure to justify a future clinical trial. BOOST (Bone marrOw transfer to enhance ST-elevation infarct regeneration) a recent randomized controlled clinical trial in Germany has been reported by Wollert et al. [114]. The study included a population of 60 patients (males n = 42;

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females n = 18) randomized to receive optimum medical treatment and PCI together with bone marrow cell transplantation (n = 30) or without cell transplantation control group (n = 30). After randomization, all patients underwent cardiac MRI. LVEF was 51.3 ± 9.3% in control subjects and 50.0 ± 10.0% in the bone marrow cell group (P = 0.59) at baseline, determined 3.5 ± 1.5 days after PCI. Autologous bone marrow was purified for CD34+ cells and re-suspended in heparanized saline solution. Within 6–8 h after purification and assessment for viability (99% by Trypan blue method) and identification by FACS, 24.6 × 108 cells were transplanted by intracoronary infusion using balloon catheter in the infarct related artery. The whole cell transplantation process consisted of four to five occlusions of 2.5–4 min each (interrupted by 3 min reperfusion). Pro-arrythmogenicity after cell transplantation was excluded by 24 h Holter monitoring. Sixmonth follow up showed that mean global LVEF had increased by 7% in the stem cell transfer group and 0.7% in the medical therapy group (P = 0.0026). Transfer of bone marrow cells improved left ventricular systolic function mostly in myocardial segments adjacent to the infarcted area. The results were analyzed by two investigators blinded to the treatment assignment. Study limitations include lack of shamoperated control subjects and inability to determine mechanisms of action of bone marrow cells.

8. Some unresolved critical issues and future directions The beneficial effects of bone marrow derived stem cell transplantation for cardiac repair have been validated in controlled phase-1 clinical studies. Putting together the data published, these studies strongly suggest the safety and feasibility of the approach. None of the studies has reported the problem of arrhythmia, peri-operative ischemic episodes or post operative complications during the follow up ranging from 2 months until 1 year post cell transplantation [93]. Only two deaths unrelated to cell transplantation procedure have been reported until now during these studies. Invariably, all the studies have shown restoration of LV wall thickness, improvement in mechanical contractile function in the area of cell engraftment, enhanced regional blood flow, improvement in hemodynamic parameters and an overall improvement in the global cardiac function. Notwithstanding these encouraging results, the demographic and clinical characteristics of the patients included in these studies have been quite variable which makes it difficult to conclusively show the effectiveness of the procedure. Similarly, the sample size in most studies is too small to be conclusive in results. Due to the adjunct procedural approach, the beneficial effects of cell therapy are indistinguishable. Most of the studies lack blinded evaluation of the results together with absence of a true placebo control arm of the experiment. There is dearth of information which may explain the mechanistic basis of the salutary effects of cell therapy on cardiac function. Some of the studies relate improvement in

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cardiac function to the restoration of LV wall thickness, which leads to improved regional contractility of LV wall [27,53]. This has been hypothesized to result from myogenic transdifferentiation of the transplanted bone marrow cells. Others have attributed this to neoangiogenesis which leads to improved regional blood flow in the ischemic area [27,58,76,91,115]. However, it is difficult to accomplish a direct evidence to support these notions in the clinical settings. Targetted delivery of the donor cells to the area of interest presents the most ideal scenario to achieve required benefits of cell transplantation. Systemic administration may not be feasible as it may spread the cell to undesired tissues and organs [116,117]. Intramyocardial route has been widely adopted for cell transplantation in both pre-clinical and clinical studies [96]. However, this entails a surgical approach and is associated with high risks and limited application only for patients who require surgical intervention. Intracoronary administration has theoretical merits of being more practical, and may result in global dissemination of the cells in the myocardium [106,107]. However, a recent study has shown the development of microinfarction when MSC were delivered by intracoronary artery route in a canine model of myocardial ischemia and warrants a more cautious approach [118,119]. Transendocardial delivery using electromechanical mapping NOGA system is gaining popularity in the clinical studies as it helps to identify and discriminate viable myocardium from the non viable and raises the possibility of more selective and site specific injection of the cells. As an alternative to these delivery approaches, bone marrow stem cell mobilization using an arbitrarily selected cytokine or a combination of cytokines is also gaining popularity [120–122]. Cytokine-mobilized cells have been shown to regenerate myocardium with the formation of myocytes resembling fetal cardiomyocytes, capillaries and arterioles [123–125]. In a recently reported study involving six patients, mobilized peripheral stem cells have been purified by aphaeresis and injected intramyocardially adjunct to CABG with promising results [126,127]. However, cytokine therapy has its associated side effects for which patients should be carefully monitored during therapy [128]. Site specific targeting of BMSC for cardiac repair has also been achieved using bispecific antibodies [129]. Another important aspect, which needs consideration is the use of whole bone marrow in comparison with the use of a specific purified subpopulation of bone marrow and the number of the cells to be injected. The results of the clinical studies until now have shown that the use of a specific lineage of bone marrow cells has no particular advantage over use of whole bone marrow. Most studies have relied on the use of BMMNC, which contain both hematopoietic as well as nonhematopoietic cell populations. The results from TOPCAREAMI study, which provided mechanistic insights of bone marrow stem cell transplantation, together with report from Heeschen et al. [130] that BMMNC from patients of chronic ischemic cardiomyopathy has compromised migratory ability and neovascularization potential, the use of BMMNC from alternative healthy donors may provide better prognosis [110].

There is a dire need for communication between the various research groups involved in the clinical studies. The target patient population who would draw clinical benefit needs to be defined and the optimal time of injection after the onset of infarction episode has to be determined. The future human studies should be randomized to have placebo control group of patients along with the bone marrow stem cell treatment group, double blinded to both patients and the observer and with uniform methods for clinical end-point measurement. A step further to this will be genetic modulation of the donor cells before engraftment to combine cell and gene therapy to achieve best of the two approaches at cellular and molecular level [131].

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