Autologous Peripheral Blood Stem Cell Transplantation for Myocardial Regeneration: A Novel Strategy for Cell Collection and Surgical Injection

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1 Autologous Peripheral Blood Stem Cell Transplantation for Myocardial Regeneration: A Novel Strategy for Cell Collection and Surgical Injection Giulio Pompilio, MD PhD, Aldo Cannata, MD, Fedro Peccatori, MD, Francesco Bertolini, MD PhD, Angelo Nascimbene, MD, Maurizio C. Capogrossi, MD, and Paolo Biglioli, MD Departments of Cardiovascular Surgery and Cardiovascular Gene and Cell Therapy Clinical Program, Centro Cardiologico Monzino IRCCS, Milan, Division of Haematology-Oncology, European Institute of Oncology IRCCS, Milan, and Laboratory of Vascular Pathology, Istituto Dermopatico dell Immacolata IRCCS, Rome, Italy Purpose. Bone-marrow and peripheral blood-derived stem cells can be used as stimulators of myogenesis and angiogenesis. We describe an original technique for collection and surgical intramyocardial injection of peripheral blood-derived stem cells. Description. Stem cells are mobilized from the bone marrow by means of subcutaneous administration of Lenogastrim (Granocyte 34 [Aventis Pharma, Milan, Italy]) for 4 days. Then the day before the operation the peripheral blood-derived stem cells are collected by means of apheresis and processed in order to obtain the CD 133 cells. Cells are injected into the myocardium in a beating heart in order to induce angiogenesis locally or myogenesis, or both. When necessary, off-pump coronary artery bypass grafting is previously accomplished. Evaluation. Thus far we have investigated 4 patients (3 patients who have received off-pump peripheral blood stem cell injection and coronary bypass grafting through median sternotomies, and 1 patient who underwent cell transplant alone through a minimally-invasive transdiaphragmatic approach). No complications were noted at a mean of 4 months after surgery. Conclusions. This novel method of peripheral bone marrow stem cell collection and intramyocardial injection seems to be safe, feasible, and reproducible. However, there is need of further evidence to definitely assess safety issues and clinical results. (Ann Thorac Surg 2004;78: ) 2004 by The Society of Thoracic Surgeons A significant body of in vitro and pre-clinical data suggest that bone marrow-derived and peripheral blood-derived stem cells (PBSC) can be used as stimulators of myogenesis [1, 2] and angiogenesis [3, 4]. Recently, research teams have used bone marrow-derived stem cells with different collection, purification, and expansion strategies to evaluate the feasibility of this strategy. Cells have been delivered to the heart for the purpose of (1) repairing damaged myocardium in patients with a recent myocardial infarction and candidates to coronary artery bypass grafting [5, 6], and (2) inducing angiogenesis in patients with refractory chronic angina [7] in which Accepted for publication Sept 8, Address reprint requests to Dr Pompilio, Department of Cardiovascular Surgery, Centro Cardiologico Monzino IRCCS, Via Parea 4, Milan 20138, Italy; giulio.pompilio@ccfm.it. conventional revascularization (surgical or percutaneous) was largely incomplete or not applicable. Technology Herein we report an original technique for intramyocardial injection of PBSC-collected by apheresis and positively selected with anti- antibody. Technique Autologous Bone Marrow Stem Cells Mobilization and Collection After study enrollment, 10 g/kg/die of the hematopoietic growth factor Lenograstim (Granocyte 34, Aventis Pharma, Milan, Italy) were administered subcutaneously to the patient for 4 consecutive days to mobilize stem 2004 by The Society of Thoracic Surgeons /04/$30.00 Published by Elsevier Inc doi: /j.athoracsur

2 Ann Thorac Surg POMPILIO ET AL 2004;78: STEM CELL BASED MYOCARDIAL REGENERATION 1809 Fig 1. (Left) Electron microscopy evaluation of cells collected by apheresis from the peripheral blood of G-CSF-mobilized patients and purified by CliniMACS (Miltenyi Biotech, North Rhine-Westphalia, Germany). Cells have a small size and lymphoid morphology. (Right) Intramyocardial injection of cells on a beating heart. cells from the bone marrow to the peripheral blood. As expected, Lenograstim (Aventis Pharma) induced a significant increase in the white blood cell count, which resulted in neutrophilic leukocytosis (range, 20,000 to 50,000 white blood cells/mm 3 ). Routine blood tests and twelve-lead electrocardiograms were obtained on a daily basis during the mobilization phase. On days 4 and 5, stem cells mobilized by Lenograstim (Aventis Pharma) were collected by apheresis using the COBE Spectra Apheresis System (Gambro BCT, Inc, Stockholm, Sweden). This automated procedure implied blood collection (blood processing by centrifugation) to collect white cells in the size range of stem cells and blood return to the patient [8]. The duration of the procedure ranged from 3 to 4 hours. The product of apheresis collection was processed by a CliniMacs device (Miltenyi Biotech, Germany) to purify the cells fractions, which are highly enriched for multipotent stem cells [9] (Fig 1 left). Typically, from an apheresis collection containing 20 to of white blood cells, this procedure allowed a collection of 1 to cells/kg in a final volume of 10 to 15 ml (see Table 1). All these procedures were European community-approved and did not imply the use of reagents at possible risk for bovine spongiform encephalopathy transmission. cells were resuspended in a European community-approved medium (Miltenyi Biotech, North Rhine-Westphalia, Germany) and stored at 4 C. cell purity was always greater than 90% (range, 92% to 96%), and cell viability (evaluated by 7AAD staining and flow cytometry, was always greater than 85%) (Table 1). The operation was scheduled for the day after the apheresis to maintain cellular viability. Clinical Experience Study Design In December 2002, after approval by the local scientific and ethics committees, we performed a pilot clinical phase-1 trial to assess the feasibility and safety of PBSC transplantation for myocardial regeneration and angiogenesis. were assessed before surgery with leftheart catheterization and coronary angiography, stress and rest 2-dimensional transthoracic echocardiography, and myocardial single-photon emission computerized scintigraphy. Inclusion criteria for study enrollment were: (1) patients with evidence of a large ischemic area in the left ventricle with target coronary artery not suitable for traditional treatment; (2) patients who were candidates for coronary artery bypass grafting with a distinct large recent infarction of more than 10 days but less than 3 months before admission. Major exclusion criteria were an age greater than 75 years, need of revascularization on an urgent basis, concomitant cardiac procedures, previous or current documented malignant disease, and hematologic disorders. were followed up for 6 months after surgery. After hospital discharge, clinical data, electrocardiogram, and routine hematology were collected monthly. Stress and rest echocardiography and single-photon emission computerized scintigraphy were scheduled at 3 and 6 months after cell transplantation. Informed consent was obtained by all patients. Thus far we have investigated 4 male patients (Table 1). 1 and 4 were treated to induce angiogenesis for anterolateral and inferior ischemic areas, respectively; patients 2 and 3 were enrolled to induce cardiomyogenesis in an infarcted wall. All patients received adjunctive off-pump coronary artery bypass grafting (OPCAB) through median sternotomies, with the exception of patient 4 who was treated with PSBC implant alone through a transdiaphragmatic mini-laparotomy approach. Patient 4 underwent coronary artery bypass grafting 6 years ago, but 1 year after the operation he complained of recurrent angina. A single-photon emission computerized scintigraphy documented inducible ischemia in the inferior wall. The coronary angiography bypass grafts to the left anterior descending artery and to an obtuse margin branch were perfectly patent, but the right coronary

3 1810 POMPILIO ET AL Ann Thorac Surg STEM CELL BASED MYOCARDIAL REGENERATION 2004;78: Table 1. Patient Data Age Infarct Localization Ischemia Localization Bypass Graft To Injection Site Purity Viability Cells Injected Count Basal Count Peak Count at Discharge 1 59 Lateral D, LPD Lateral 85% 99% ,400 22,900 8, Inferior LAD, OM Inferior 92% 93% ,500 45,600 9, Anterior, septum LAD, LPD Anterior 98% 99% ,900 54,800 9, Inferior None Inferior 79% 99% ,700 41,500 6,300 D diagonal branch; LAD left anterior descending coronary artery; LPD left posterior descending coronary artery; OM obtuse marginal branch; peripheral white blood cells per mm 3. artery was occluded and not amenable to any form of conventional revascularization. Surgical Procedures THE STERNOTOMY APPROACH. The sternotomy approach was used in patients 1, 2, and 3. After a midline sternotomy, the harvest of the coronary grafts was performed. The pericardium was opened, and the myocardial regions target of PBSC injection were recognized and inspected. After pre-load and after-load optimization, deep pericardial traction sutures ( Lima stitches ) were placed into the oblique sinus in order to obtain optimal exposure of the coronary vessels and myocardium. Off-pump coronary bypass grafting was accomplished by means of a cardiac wall stabilizer and endoluminar shunts. Then the solution containing the autologous PBSC was injected into the target myocardial areas of a beating heart by means of gentle hand injection through a 22-gauge butterfly needle. The plastic cover around the needle was left in place and shortened by approximately 3 mm to control injection of PBSC into the myocardium at a constant depth, avoiding insufficient or excessive penetration (Fig 1 right). The needle was stabilized during injection by grasping the winglets of the butterfly needle with a pair of forceps. Fifteen to 20 injections of 0.5 to 1.0 ml of the solution containing BMSC were performed in the target myocardial regions. In an attempt to induce myocardial repair, injections were accomplished along the border of the myocardial scar, directly visualized on the beating heart. Conversely, when cell therapy was conducted to create therapeutic angiogenesis, cells were delivered into the chronically ischemic ungraftable myocardium, identified preoperatively by means of stress scintigraphy and 2-dimensional echocardiography. THE TRANSDIAPHRAGMATIC MINI-LAPAROTOMY APPROACH. This approach was used in patient 4. Approximately a 10-cm median incision was performed using the xiphoid process to open the abdomen. An Olivier (Aesculap, Tuttlingen, Germany) retractor was positioned at the left and right costal arches to better expose the abdominal side of the diaphragm. Then a transverse incision of approximately 5 cm was made on the central tendon of the diaphragm to expose the inferior wall of the left ventricle. Injections were accomplished as previously described. Patient Outcomes The patient outcomes are detailed in Table 2. Both the procedures and postoperative course were uneventful, and no complications related to PBSC collection and administration were noted. In particular, mobilization and apheresis were very well tolerated. The pharmacologically induced leukocytosis was not associated with relevant clinical symptoms, electrocardiographic changes, or myocardial enzymes release. The apheresis did not cause any clinical or hemodynamic compromise. In any case we did not observe malignant ventricular arrhythmias in the postoperative period. Patient 2 had atrial fibrillation on postoperative day 3, which was pharmacologically treated. No pericardial effusion was observed. At hospital discharge, all patients had peripheral white blood cell counts return to baseline values. were recovering well at a mean of 4 months from the intervention. 1 and 2 completed the 6-month follow-up. Single-photon emission computerized scintigraphy and echocardiographic reinvestigation in patient 1 showed significant improvement in perfusion of the lateral wall, and in patient 2 it showed restoration of Table 2. Patient Outcome Postoperative Complications Postoperative LOS (Days) Viability Improvement Perfusion Improvement Follow-Up (Months) 1 AF 7 Lateral wall Inferior wall * * 2 * waiting myocardial perfusion reinvestigation scheduled at 3 months after cell transplantation. AF atrial fibrillation; LOS length of stay.

4 Ann Thorac Surg POMPILIO ET AL 2004;78: STEM CELL BASED MYOCARDIAL REGENERATION 1811 Fig Tallium single-photon emission computerized scintigraphy myocardial perfusion scans. Scans done before and after coronary artery bypass grafting and stem cell injection in patient 2. (A, B) The images on the left show the short axis, (C, D) whereas those on the right show the long-axis of the heart. (A, C) Initially, the inferior wall at rest had a lack of myocardial perfusion. (B, D) Six months after cell transplantation, the same inferior wall revealed a significant improvement in perfusion. viability of the inferior wall, where a lack of viability was preoperatively detected (Fig 2). Comment Previous experimental works [1 3] and preliminary clinical studies [4 7, 10, 11] have reported the beneficial effects of stem cells delivery to regenerate infarcted myocardium and to induce therapeutic angiogenesis in chronically ischemic myocardium. Moreover, there are evidences that stem cells are spontaneously mobilized from bone marrow after an acute myocardial infarction [12] and may spontaneously contribute to new cardiac myocyte formation [13]. However, further studies are needed in order to assess best strategies for stem cell therapy in humans affected by myocardial ischemia. We believe that this is the first report documenting a combined surgical strategy of the administration of peripheral blood-derived stem cells by intramyocardial injection on a beating-heart and cell collection by means of apheresis combined with positive stem cell selection. Previous clinical studies have proposed needle aspiration for cell collection [5 7, 10] and cardioplegic arrest for surgical intramyocardial injections [10]. Off-pump techniques allow safe and reproducible exposure of all myocardial walls. These techniques can be usefully adopted for intramyocardial PBSC injection and may offer some advantages. The first advantage is to avoid the effects of exposure of PBSC to hyperkalemic solutions, such as cardioplegia, which is unknown. The second advantage is myocardial motion, which can easily be evaluated while inspecting the beating heart under physiologic hemodynamic load to better recognize areas to be injected (ie, scar tissue, hypokinetic areas). In order to prevent cell injury during injection, we have adopted a 22-gauge needle, because the use of finer needles (ie, 24-gauge and 26-gauge) has been reported to cause damage to blood cells [14]. No significant bleeding from the sites of injection was observed. The adoption of apheresis combined with positive selection for stem cell collection offers some advantages: (1) It avoids the drawbacks related to the direct aspiration of bone marrow in patients affected by severe coronary artery disease; (2) general anesthesia is not required; (3) only a minimal blood volume (approximately 200 ml vs 1L to 2 L) is subtracted to the patient, and (4) the number of well characterized stem cells obtained is significantly higher with respect to needle aspiration [5, 10]. Moreover, cell culture is not needed, and cells with a very high degree of purity and viability can be immediately injected after collection. No absolute contraindications are reported for Lenograstim (Aventis Pharma) administration in patients with coronary artery disease. Hematopoietic growth factors have been demonstrated to improve collateral flow in patients with coronary artery disease. On the contrary, the use of cytokine in a setting of myocardial ischemia per se, has the potential to promote collateral growth [15]. The neutrophilic leukocytosis induced by Lenograstim (Aventis Pharma) was well tolerated by patients, and a quick postoperative drop in white cell count was observed in all patients within 1 week after surgery. Potential limitations of this technique are (1) the time span necessary for peripheral blood mobilization and collection before intramyocardial cell injection (5 days vs 1 day with needle aspiration) [5, 6, 10], and (2) the higher costs compared with needle aspiration, due to Lenogastrim (Aventis Pharma) administration and the apheresis process. The primary issue of this pilot study was to establish the feasibility and reliability of our methods. It is advisable to have a larger number of patients to definitively assess safety, although our preliminary results are en-

5 1812 POMPILIO ET AL Ann Thorac Surg STEM CELL BASED MYOCARDIAL REGENERATION 2004;78: couraging in terms of patient compliance and recovery. No complications were observed related to cell mobilization, collection, and injection. Patient outcome was also uneventful. In addition, improvement in perfusion of an ischemic ungrafted area and recovery of viability of a recent infarction were observed in the 2 patients who were re-studied 6 months after cell transplant. Obviously this is not evidence for efficacy. Only phase II doubleblinded, controlled, prospective trials can indicate whether or not autologous stem cell-based therapies will assume a place in the therapeutic armamentarium of cardiac surgeons [16]. Disclosures and Freedom of Investigation Lenogastrim and the CliniMacs kit were provided free of charge by Aventis Pharma and Miltenyi Biotech, respectively. The authors of this article have no financial relationship with Aventis Pharma or Miltenyi Biotech. The authors also planned and introduced the clinical practice, proposed protocol and technology in a free and independent manner. References 1. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410: Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA 2001;98: Tomita S, Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100(Suppl II):II Smitko PE, Fedak PWM, Weisel RD, Stewart DJ, Kutryk MJB, Verma S. Endothelial progenitors cells: new hope for a broken heart. Circulation 2003;107: Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002;106: Assmus B, Schächinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002; 106:r Hamano K, Nishida M, Hirata K, et al. Local implantation of autologous bone marrow cells for therapeutic angiogenesis in patients with ischemic heart disease: clinical trial and preliminary results. Jpn Circ J 2001;65(9): Bensinger WI, Martin PJ, Storer B, et al. Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 2001;344: Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 2002;109: Stamm C, Westphal B, Kleine HD, et al. Autologous bonemarrow stem-cell transplantation for myocardial regeneration. Lancet 2003;361: Perin EC, Dohmann HFR, Borojevic R, et al. Transendocardial, autologous bone marrow cell trasnplantation for severe, chronic ischemic heart failure. Circulation 2003;107: Shintani S, Murohara T, Ikeda H, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation 2001;103: Deb A, Wang S, Skelding KA, et al. Bone marrow-derived cardiomyocytes are present in adult human heart: a study of gender-mismatched bone marrow transplantation patients. Circulation 2003;107: Kennedy C, Angermuller S, King R, et al. A comparison of hemolysis rates using intravenous catheters versus venipuncture tubes for obtaining blood samples. J Emerg Nurs 1996;22: Seiler C, Pohl T, Wustmann K, et al. Promotion of collateral growth by granulocyte-macrophage colony-stimulating factor in patients with coronary artery disease: a randomized, double-blind, placebo-controlled study. Circulation 2001; 104: Patterson C. The Ponzo effect: endothelial progenitors cells appear on the horizon. Circulation 2003;107: Disclaimer The Society of Thoracic Surgeons, the Southern Thoracic Surgical Association, and The Annals of Thoracic Surgery neither endorse nor discourage use of the new technology described in this article. INVITED COMMENTARY Pompilio and colleagues report on the mobilization, harvesting, and purification of AC133 cells followed by myocardial implantation at the time of coronary artery bypass grafting (CABG). Cell therapy offers the promise to regenerate the heart after a myocardial infarction. Skeletal myoblasts were the first cell type to be transplanted at the time of CABG into regions that could not be revascularized to assess safety and possible efficacy [1]. This phase I trial was well controlled. Recent studies demonstrated that bone marrow cells [2, 3] contributed to cardiac restoration. The studies stimulated clinical trials that evaluated the use of bone marrow mononuclear and endothelial progenitor cell preparations to repair the injured myocardium [4 6]. Cardiac stem cells [7, 8] may also contribute to cardiac regeneration and may be tested in clinical trials. Restraint, however, should be employed and clinical trials should be well designed and adequately controlled to determine the safety and evaluate efficacy of cell transplantation. We recommend the following guidelines: Cell therapy for ischemic cardiomyopathy should be reserved for patients who have at least one ischemic region that cannot be revascularized by conventional means. The viability of the region should be determined preoperatively, and patients who will likely improve with revascularization alone should be excluded. Fully informed consent must be obtained. The conditions of cell procurement and the culture of freshly isolated or cultured cells should be extensively studied before clinical application. Phase I trials should 2004 by The Society of Thoracic Surgeons /04/$30.00 Published by Elsevier Inc doi: /j.athoracsur