First published online October 13, 2005
Stem Cells
Vol. 24 No.
2
February 2006, pp.
333
-336
doi:10.1634/stemcells.2005-0201; www.StemCells.com
© 2006 AlphaMed Press
TISSUE-SPECIFIC STEM CELLS |
Myocardial Homing of Nonmobilized Peripheral-Blood CD34+ Cells After Intracoronary Injection
Didier Blockleta,
Michel Toungouzb,
Guy Berkenboomc,
Micheline Lambermontb,
Philippe Ungerc,
Nicolas Preumontc,
Eric Stoupelc,
Dominique Egrisea,
Jean-Paul Degautec,
Michel Goldmanb,
Serge Goldmana
a Department of Nuclear Medicine and PET/Biomedical Cyclotron Unit;
b Cellular and Molecular Therapy Unit;
c Department of Cardiology; Hôpital Universitaire Erasme, Université Libre de Bruxelles, Brussels, Belgium
Key Words. Stem cells • Labeling • Homing • Positron emission tomography • Infarction • Heart • Implantation
Correspondence: Didier Blocklet, M.D., Service de Médecine Nucléaire, U.L.B. Hôpital Universitaire Erasme, 808 route de Lennik, 1070 Brussels, Belgium. Telephone: 32-2-555-33-00; Fax: 32-2-555-68-00; e-mail: dblockle{at}ulb.ac.be
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ABSTRACT
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Granulocyte– colony-stimulating factor administered for autologous hematopoietic stem cell isolation from blood may favor restenosis in patients implanted after acute myocardial infarction (AMI). We therefore tested the isolation of peripheral-blood CD34+ cells without mobilization in six patients with AMI. After large-volume cytapheresis and positive CD34+ cell selection, 3.6 to 27.6 million CD34+ cells were obtained. We performed intra-coronary implantation of these cells and recorded no restenosis or arrhythmia. We used positron emission tomography (PET) to assess myocardial-labeled CD34+ cell homing, which accounted for 5.5% of injected cells 1 hour after implantation. In conclusion, large amounts of CD34+ cells, in the range reported in previous studies, can be obtained from nonmobilized peripheral blood. PET with [18F]-fluorodeoxyglucose cell labeling is an efficient imaging method for homing assessment.
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INTRODUCTION
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Adult myocardium has limited capacity to restore contractile function after infarction. Autologous hematopoietic stem cell (HSC) implantation in damaged myocardium has been proposed to prevent remodeling processes leading to cardiac insufficiency. HSC isolation from blood usually requires pretreatment with granulocyte–colony-stimulating factor (G-CSF). Administration of such factors in patients with acute myocardial infarction (AMI) is a matter of controversy. Several animal studies indicated a potentially beneficial role of G-CSF in the prevention of cardiac remodeling after AMI [1–4]. Human clinical studies, however, have led to the conclusion that G-CSF pre-treatment is strongly associated with higher occurrence of instent restenosis and extension of the infarcted region [5, 6]. Some authors also reported that HSCs cannot differentiate into cardiomyocytes, leading to the concept that their myocardial restoration potential depends on other capacities [7–10]. In particular, CD34+ cells could release growth factors activating cardiac stem cells; they also may differentiate into endothelial cells, leading to enhanced neoangiogenesis. Whatever the mechanism involved, it certainly requires CD34+ cell homing in the injured tissue. Evaluations of cell trafficking and homing are therefore essential. They may be studied on a gamma camera after radiopharmaceutical cell labeling, but sensitivity and resolution would be much better with positron emission tomography (PET). Nevertheless, the short half-life of PET radiopharmaceuticals allows trafficking studies for only a few hours. We may therefore take advantage of the longer half-life of tracers used for gamma camera imaging to prolong cell detection for 2 to 3 days.
In this study, we tested the feasibility of peripheral-blood CD34+ cell isolation without mobilization and applied PET and gamma camera imaging to assess their homing after intracoronary implantation in patients with AMI.
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MATERIALS AND METHODS
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Patients
The selection criteria were a first acute anterior myocardial infarction with severe hypokinesis or akinesis of at least two adjacent segments on the baseline left ventricular echocardiogram and the absence of reperfusion within 6 hours after the onset of symptoms. After approval by the local ethics committee, six patients admitted in our institution gave their written informed consent and were included in the study. All patients were treated by aspirin, clopidogrel, statins, beta-blockers, and angiotensin-converting enzyme inhibitors. At the time of CD34+ cell implantation, patients were 7 to 21 days after a documented AMI and 4 to 21 days after culprit coronary artery stenting. At 2 to 3 days before implantation, we assessed regional myocardial viability with PET after the injection of 370 MBq of [18F]-fluorodeoxyglucose (FDG) in a hyperinsulinemic state. External marks were used to improve coregistration with the homing studies. Dobutamine stress echocardiography for assessment of viable myocardium was carried out before stem cell implantation. Regional left ventricular wall motion analysis was performed according to the American Society of Echocardiography recommendations [11], dividing the left ventricle into 17 segments to produce a wall motion score index.
CD34+ Cell Enrichment
Autologous peripheral blood HSCs were obtained after large-volume cytapheresis (15 to 20 L) performed on a Cobe Spectra cell separator. Positive selection of CD34+ cells was performed on a Baxter Isolex 300i medical device. Selected cells were maintained at 4°C for approximately 20 hours and then labeled.
Cell Labeling and Implantation
Two groups of 2 to 4 million CD34+ cells were labeled with 74 MBq of FDG and 7.4 MBq of 111In-oxine, respectively, at the concentration of 2 x 106 cells/ml. Cells were subsequently washed in phosphate-buffered saline (PBS) and resuspended in 1 ml PBS containing 5% human albumin. During coronary angiography, labeled and unlabeled stem cells were injected through the central lumen of an over-the-wire balloon catheter (oversized by 0.5 mm) into the culprit coronary artery as described by Assmus et al. [12]. The balloon was inflated inside the stent with low pressure to completely block the blood flow during 3 minutes, and the CD34+ cell suspension (2 ml) was infused distally to the occluding balloon through the central catheter port. After a washout with saline, the balloon was deflated for 3 minutes. The maneuver was repeated two to three times, and labeled stem cells were always used in the first infusion. This procedure allows for high-pressure infusions under stop-flow conditions to facilitate the infiltration of the cells into the infarcted zone and to prevent backflow. Enoxiparin 0.5 mg/kg was used during the implantation procedure.
Homing Studies
One hour after implantation, 20-minute 3D PET images were obtained over the chest. For 111In imaging, 20-minute planar images were acquired on a gamma camera 19 hours and 43 hours after implantation. At 19 hours, tomographic acquisition was also performed.
Follow-Up
Four months after implantation, patients were reevaluated by FDG-PET, echocardiography, and coronary angiography.
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RESULTS
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CD34+ Cell Enrichment
A mean of 32.4 x 109 (range, 22 to 40 x 109) mononuclear cells containing less than 0.1% CD34+ cells were collected after treating a mean of 3.5 x total blood volume (range, 2.9 to 5.3). After CD34+ selection, a mean of 14.9 x 106 (range, 3.6 to 27.6 x 106) cells containing a mean of 30% (range, 14%–35%) CD34+ cells were obtained for implantation. Major contaminants were CD19+ B-lymphocytes (mean, 42%; range, 29%–58%); monocytes were less than 1% of all cells.
Implantation and Homing Studies
Mean (± standard deviation) labeling efficiency of implanted cells with 111In-oxine and FDG was 65% ± 8% and 6% ± 1%, respectively.
The stem cell implantation was well tolerated, without any complications, especially any early restenosis or arrhythmia (Table 1
). One hour after implantation, 5.5% ± 2.3% of the administered label remained in the myocardium. The major part of the radioactive label was present in the liver (48% ± 35%), spleen (29% ± 19%), and bone marrow.
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Table 1. Clinical characteristics, CD34+ cell preparation, PET imaging, and echocardiography findings in six patients with intra-coronary CD34+ cell implantation for acute myocardial infarction
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In four patients, detection of myocardial radioactivity was restricted to the vascular territory of the revascularized vessel, more precisely within the borders of the infarcted zone (Fig. 1). In one patient presenting with a large infarction and a no-reflow pattern, injected cells remained in a linear strip at the surface of the myocardium, suggesting a trapping in the left anterior descending artery. Apart from this observation, neither PET nor 111In imaging demonstrated cell homing in the patients’ vasculature, including the aorta.
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Figure 1. First row: successive transverse chest PET slices of FDG uptake testing the regional myocardial viability evaluation (gray scale). FDG uptake is markedly reduced in the antero-apical region of the left ventricle. Second row: superimposed on the PET-FDG slices presented in the first row, FDG-labeled CD34+ cell homing is detected 1 hour after intracoronary injection in the hypometabolic infarcted area, except in the central core of the myocardial lesion (orange-to-yellow scale). Cells detected in the postero-median aspect of the chest correspond to CD34+ cell homing in thoracic vertebral bone marrow. Abbreviations: FDG, fluorodeoxyglucose; PET, positron emission tomography.
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One patient exhibited cell homing in the middle part of the anteroseptal region; this patient had small septal arteries emerging at the distal extremity of the stent. At 19 hours after implantation, 111In radioactivity was detected in the liver (71% ± 10%), spleen (29% ± 10%), and bone marrow. Only one of the six patients had detectable, although faint, activity in the myocardium. At 43 hours, the activity’s distribution was similar.
Patient Follow-Up
No restenosis or arrhythmia occurred in any patient. Echocardiography demonstrated enhancement in wall motion score index (p < .05) in all patients (Table 1
), but left ventricular ejection fraction did not increase significantly (from 56% ± 7% to 60% ± 7%). FDG-PET follow-up studies demonstrated no alteration of the viable myocardium. A restricted and nonquantifiable extension of FDG uptake was detected in four patients.
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DISCUSSION
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We have shown that large amounts of CD34+ cells can be obtained from nonmobilized peripheral blood by large-volume cytapheresis followed by positive selection of CD34+ cells. The amounts of CD34+ cells available for therapeutic administration were in the range of those reported in previous studies [12, 13]. The procedure does not require mobilization by G-CSF and generates few monocytes (less than 1% of the selected cells). Although G-CSF permits the collection of much larger amounts of cells, this benefit is balanced by the risk of enhancing the restenosis process and the infarction extension, as reported in recent clinical studies [5, 6]. Our study therefore opens the way to clinical studies testing CD34+ cell administration without the confounding effects of G-CSF. This study was not designed to assess the clinical efficacy of the cell-therapy protocol proposed. This would require a study on a larger number of patients and with a longer follow-up period. Such a study would also help confirm the inocuity of the procedure in terms of restenosis and arrhythmia occurrence.
Double radioactive labeling allowed for the assessment of cell trafficking and homing of injected cells. The sensitive and high-resolution PET study demonstrated deposition and early homing of CD34+ cells in the myocardium surrounding the necrotic area. One patient exhibited cell homing in the middle part of the antero-septal region; as this patient had small septal arteries emerging just after the distal extremity of the stent, we postulate that labeled cell infusion preferentially occurred in these arteries rather than in the left anterior descending artery. As 4%–7% of the labeled cells were detected in the myocardium, we can reasonably conclude that CD34+ cells constitute a major part of them because other labeled cells were essentially B-lymphocytes, a cell type that is not prone to adhere to, and migrate through, diseased coronary endothelium (monocytes were < 1% of all cells) [14, 15]. Cell trapping most probably occurred in the left anterior descending artery in one patient. Despite the report of bone marrow–derived progenitor cells homing in the aorta of atherosclerotic animals, we observed no other vascular labeling [16]. Apart from its component reflecting myocardial homing, the observed radioactivity distribution is in accordance with the normal homing of CD34+ cells after intravascular injection. The imaging follow-up performed at 43 hours with the 111In gamma camera tracer suggested that the activity sequestrated outside the myocardium did not substantially recirculate to secondarily reach the lesioned heart. Homing assessment is of major importance for comparison of cell-based therapeutic strategies. Satisfactory cell deposition and homing in the target area are indeed therapeutic prerequisites. They may greatly vary depending on the methods of cell collection, preparation, and implantation [17, 18]. The short half-life of the PET label that we used limited the duration of sensitive homing assessment. Still, the method revealed unexpected sites of deposition in two patients, indicating that individual factors also greatly influence cell distribution after intracoronary administration. This observation further strengthens the need for powerful cell imaging tools, such as PET, to correctly interpret cell-based trials that emerge in the various domains of regenerative medicine.
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CONCLUSION
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Large amounts of CD34+ cells, in the range reported in previous studies, can be obtained from nonmobilized peripheral blood. PET with FDG cell labeling is an efficient imaging method for homing assessment.
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ACKNOWLEDGMENTS
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This study was supported by the FNRS and the Fondation pour le Chirurgie Cardiaque (Belgium).
DISCLOSURES
The authors indicate no potential conflicts of interest.
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Received May 4, 2005;
accepted for publication October 3, 2005.
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Abstract
Introduction
