TRANSLATIONAL AND CLINICAL RESEARCH Intracoronary Infusion of CD133ⴙ and CD133ⴚCD34ⴙ Selected Autologous Bone Marrow Progenitor Cells in Patients with Chronic Ischemic Cardiomyopathy: Cell Isolation, Adherence to the Infarcted Area, and Body Distribution EVGENIOS GOUSSETIS,a ATHANASSIOS MANGINAS,b MARIA KOUTELOU,c IOULIA PERISTERI,a MARIA THEODOSAKI,a NIKOLAOS KOLLAROS,c EVANGELOS LEONTIADIS,b ATHANASIOS THEODORAKOS,c GEORGE PATERAKIS,d GEORGE KARATASAKIS,b DENNIS V. COKKINOS,b STELIOS GRAPHAKOSa a
Stem Cell Transplant Unit; “Aghia Sophia” Children’s Hospital, Thivon and Levadias; bDepartment of Cardiology, Onassis Cardiac Surgery Center; cDepartment of Nuclear Medicine, Onassis Cardiac Surgery Center; dImmunology Department and National Histocompatibility Center; “G. Genimmatas” General District Hospital, Athens, Greece Key Words. Homing • Selected bone marrow-progenitors • Intracoronary infusion • Chronic cardiomyopathy
ABSTRACT Central issues in intracoronary infusion (ICI) of bone marrow (BM)-cells to damaged myocardium for improving cardiac function are the cell number that is feasible and safe to be administrated as well as the retention of cells in the target area. Our study addressed these issues in eight patients with chronic ischemic cardiomyopathy undergoing ICI of selected BM-progenitors. We could immunomagnetically isolate 0.8 ⴞ 0.32 ⴛ 107 CD133ⴙ cells and 0.75 ⴞ 0.24 ⴛ 107 CD133ⴚCD34ⴙ cells from 310 ⴞ 40 ml BM. After labeling these cells with 99mTc-hexamethylpropylenamineoxime, they
were infused into the infarct-related artery without any complication. Scintigraphic images 1 (eight patients) and 24 hours (four patients) after ICI revealed an uptake of 9.2% ⴞ 3.6 and 6.8% ⴞ 2.4 of the total infused radioactivity in the infarcted area of the heart, respectively; the remaining activity was distributed mainly to liver and spleen. We conclude that through ICI of CD133ⴙ and CD133ⴚCD34ⴙ BMprogenitors a significant number of them are preferentially attracted to and retained in the chronic ischemic myocardium. STEM CELLS 2006;24:2279 –2283
INTRODUCTION
[4 – 6] and initial clinical studies suggested that either intramyocardial [7, 8] or intracoronary [9 –13] delivery of BM-SCs, in both acute and chronic ischemic cardiomyopathy, might contribute to sustained improvement of cardiac function due to enhanced neovascularization and its effects on the protection/ proliferation of cardiomyocytes. However, the quantity of BMSCs that could be isolated and intracoronarilly delivered without coronary flow impairment and the homing of these cells to chronic infarcted myocardium remain to be defined.
Stem cell repair of cardiac and vascular tissue is a naturally occurring but inefficient regeneration process after myocardial infarction (MI) [1]. In this process two types of stem cells have been suggested to be involved. First, bone marrow-stem cells (BM-SC), which are mobilized to the damaged area in response to released cytokines from the injured myocardium, engraft in the myocardium and differentiate into endothelial cells enhancing neovascularization of the ischemic tissue [2]. Secondly, local resident cardiac SCs are activated, probably by the paracrine effects of recruited BM-SC, and divide, regenerating the damaged cardiac tissue [3]. Given the fact that this process occurs too slowly to compensate for the loss of infarcted cardiomyocytes, the most realistic approach of regenerating damaged myocardium would be to exogenously supply and engraft BM-SCs into the postinfarct scars. Indeed, recent experimental
MATERIALS
AND
METHODS
The present study addresses the feasibility of the isolation of a relative high number of BM-progenitors, 10 times more than the previously published number of intramyocardially implanted CD133⫹ cells [14], the safety of administering them intracoronarilly, and their ability to adhere to the infarcted area.
Correspondence: Evgenios Goussetis, MD, BMT-Unit, “Aghia Sophia” Children’s Hospital, Thivon and Levadias, 11527 Athens, Greece. Telephone: (30)-210-7467303; Fax: (30)-210-7778822; e-mail:
[email protected] Received November 25, 2005; accepted for publication June 15, 2006; first published online in STEM CELLS EXPRESS June 22, 2006.© AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0589
STEM CELLS 2006;24:2279 –2283 www.StemCells.com
Intracoronary Delivery of Selected BM-Progenitors
2280
8/8 8/8
Gladbach, Germany, http://www.miltenyibiotec.com) for 30 minutes at 4°C. After a single wash the cells were passed through a LC-magnetic cell sorting separation column. Unbound cells were washed out, and after removing the column from the magnetic field CD133⫹ cells were eluted. The CD133-negative cell fraction was reincubated with a monoclonal antibody against CD34 (Miltenyi Biotec), and by the same immunomagnetic procedure we isolated the CD133⫺CD34⫹ progenitor cells. The separation procedure (washing steps, incubations, and selections) was carried out under clean room A in B (clean bench in a clean room class B). The quality assurance protocol included microbial cultures, viability, recovery, and functional analysis of target cells. The purity of the isolated CD133⫹ and CD133⫺CD34⫹ cells was estimated by FACS using the Coulter Epics XL-MCL device (Beckman Coulter, Fullerton, CA, http:// www.beckmancoulter.com).
7/8
Cell Labeling and IC-Administration
Table 1. Demographics of the patients Characteristics No. of patients Age, years Transmural MI, months before ICI Previous PCI PTCA Stent implantation Previous CABG Risk factors Positive family history Smoker and ex-smoker Hyperlipoproteinemia Hypertension Medication Beta-blocker Angiotensin-converting enzyme inhibitor Statin
Patients 8 50.5 ⫾ 8.2 45 ⫾ 36 5/8 1/8 4/8 1/8 2/8 6/8 7/8 3/8
Abbreviations: CABG, coronary artery bypass grafting; ICI, intracoronary infusion; MI, myocardial infarction; PCI, percutaneous coronary intervention; PTCA, percutaneous transluminal coronary angioplasty.
Patient Population, Baseline Studies, and Clinical Follow-Up
All eight patients aged 50.5 ⫾ 8.2 years were men and they had had transmural MI 45 ⫾ 36 months before cell infusion. Six of the eight patients had been treated acutely by percutaneous coronary intervention or coronary artery bypass grafting (Table 1). The following inclusion criteria were required for patient enrollment: (a) left ventricular ejection fraction ⬍40%, (b) patent left anterior descending artery, (c) ineligibility for percutaneous or surgical revascularization, (d) no percutaneous or surgical revascularization attempt the last 6 months before enrollment, and (e) signed, informed consent. Our exclusion criteria were: (a) end-stage congestive heart failure, New York Heart Association class III/IV, (b) hepatic or renal insufficiency, (c) lack of informed consent, and (d) systemic inflammatory and hematological diseases. Patients who agreed to undergo cellular treatment underwent baseline studies, including cardiac catheterization and angiography, Tl-201 scintigram with reinjection, and echocardiogram with low-dose dobutamine infusion to assess anterior wall myocardial viability. Agreement to both tests was considered necessary to define the myocardium as being irreversibly damaged. Between 6 and 12 months post-intracoronary infusion (ICI), patients were re-evaluated by coronary angiography and echocardiography. The institutional review board of Onassis Cardiac Surgery Center approved the study protocol. We obtained informed written consent from our patients. They were informed in detail about the nature of the procedure and the radiolabeling technique.
BM-Harvesting and Cell Selection We harvested BM from the posterior iliac crest under local anesthesia and used Ficoll-density centrifugation to isolate mononuclear cells (MNC). BM-MNC were then incubated with a monoclonal antibody conjugated with magnetic beads against the human stem-cell marker CD133 (Miltenyi Biotec, Bergish
Thirty minutes before ICI, CD133⫹, and CD133⫺CD34⫹ cells were incubated under sterile conditions in a 10-ml tube with 140 MBq of Tc99m-hexamethylpropylenamineoxime (HMPAO) (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com)/ 107 cells for 30 minutes in a saline solution containing 2.5% human albumin. Labeling efficiency was estimated after removing the excess of unbound radioactivity by washing the cells with the saline-2.5% human albumin solution. Radioactivity was measured with a dose calibrator (PTW Curiementor 2). Viability of the cells was assessed by trypan blue exclusion. To further assess the influence of radiolabeling on ex vivo proliferation and differentiation of isolated SCs, liquid expansion cultures (StemSpan medium, Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell. com) of radiolabeled SCs supplemented with thrombopoetin 50 ng/ml, Flt-3 ligand 100 ng/ml, and stem cell factor 100 ng/ml (all human recombinant cytokines were provided by R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and methylcellulose semisolid cultures (Methocult; Stem Cell Technologies) were performed. Cells were infused through an over-the-wire balloon catheter at the proximal portion of the left anterior descending artery (LAD). To maximize the adherence of cells to the vesselwall, the balloon was inflated for 5 minutes during the infusion. Planar and SPECT images were obtained 1 hour after ICI in all patients and 24 hours after ICI in four patients. Uptake was defined as the percentage of myocardium-originated number of counts, compared to the total number of counts of radiolabeled cells.
RESULTS Despite the large BM-volume aspirated under local anesthesia (310 ⫾ 40 ml), all patients tolerated BM harvesting well without complications. We isolated immunomagnetically 0.8 ⫻ 107 ⫾ 0.32 ⫻ 107 CD133⫹ and 0.75 ⫻ 107 ⫾ 0.24 ⫻ 107 CD133⫺CD34⫹ cells, with a purity of 92 ⫾ 4.5% and 74.2 ⫾ 6%, respectively (Fig. 1). The total number of infused cells was 1.6 ⫾ 0.5 ⫻ 107 progenitor cells. Incorporation of 99mTcHMPAO in percentage of added activity yielded a mean labeling efficiency of 28.5 ⫾ 4.6% after 30 minutes incubation. 99m Tc-labeled cells demonstrated viability ⬎95% as assessed by the trypan blue exclusion test, whereas a reduction of 42 ⫾ 12.5% and 32.4 ⫾ 10.5%, in CD133⫹ cell expansion and colony-forming unit assay, respectively, was detected in comparison with unlabeled cells.
Goussetis, Manginas, Koutelou et al.
Figure 1. Representative fluorescence-activated cell sorting analysis post-MACS purification of bone marrow (BM)-cells. Mononuclear BMcells were incubated with a monoclonal antibody conjugated with magnetic beads against CD133 and then were passed through a separation column. Unbound cells were washed out and after removing the column from the magnetic field CD133⫹ cells were eluted (A). The CD133 negative cell fraction was reincubated with a monoclonal antibody against CD34 and by the same immunomagnetic procedure we isolated the CD133⫺CD34⫹ progenitor cells (B). Both eluted cells were doublestained with both phycoerythrin-anti-CD133 and fluorescein isothiocyanate-anti-CD34 antibodies. The percentage of CD133⫹CD34⫹, CD133⫹CD34⫺, and CD133⫺CD34⫹ is indicated. Abbreviations ORFL, orange fluorescence; GR-FL, green fluorescence.
The cell infusion was well tolerated. There were no increases in troponin serum levels in any of the patients 1 day after ICI, indicating that the procedure did not cause additional ischemic damage to the myocardium. Scintigraphic images of the patients obtained 1 (eight patients) and 24 hours (four patients) after ICI showed 9.2%⫾3.6 and 6.8%⫾2.4 of the radioactivity to be localized in the area of the damaged myocardium, respectively (Fig. 2). No significant activity was detected in other regions of the myocardium. At both time points of imaging the remaining radioactivity accumulated in liver, spleen, and bladder. A high intestine uptake was observed 24 h after ICI. Leakage of 99mTc-HMPAO from labeled cells and excretion through renal and hepatobiliary pathways rather than homing of cells are the reasons for the high uptake of radioactivity in liver, bladder, and intestine [15]. Between 6 and 12 months post-cell infusion a follow-up angiogram was performed in six of the eight patients to detect in-stent restenosis (intimal hyperplasia causing a ⬎50% diameter stenosis at the treatment site) or progression of vascular lesions (defined as new coronary lesion with ⬎50% diameter stenosis) along the artery where the progenitor cells were administered. No such complications were noted in these patients. Two additional patients refused a repeat angiography; however their clinical status has remained stable during follow-up. All the patients we studied were in well-compensated and mildly symptomatic (functional class I) congestive heart failure despite the low ventricular ejection fraction. As a consequence we did not observe any significant change in their symptomatology. An improvement in global ejection fraction at rest was documented; from 25.12 ⫾ 7.18% at baseline to 28 ⫾ 7.26% at 10 ⫾ 3 months follow-up, p ⫽ .044. www.StemCells.com
2281
Figure 2. Adhesion to the myocardial ischemic area and biodistribution of 99mTc-hexamethylpropylenamineoxime (HMPAO)-labeled bone marrow (BM) progenitors. Black, no uptake; blue-red-yellow-white, increasing uptake. Anterior view of chest and upper abdomen of a patient 1 hour (A), 24 hours (B), and whole-body scans 1 (C) and 24 hours (D) after infusion of labeled BM-progenitors into the left anterior descending coronary artery. At both time points of imaging, significant uptake was obtained in anterioapical wall (ischemic area) of the heart, liver, and spleen. A high bladder and intestine uptake, which was observed 24 hours after intracoronary infusion, is attributed to the excretion of 99mTc-HMPAO through renal and hepatobiliary pathways. Abbreviation: h, hour(s).
DISCUSSION After the first open-labeled pilot trials reporting intramyocardial delivery of autologous ex vivo expanded myoblasts [16] and unselected BM-cells [7, 8] for myocardial regeneration in patients after myocardial infarction, one recent randomized-controlled study [17] has shown that ICI of unselected or selected CD34⫹ BM cells during the early postinfarction period improves left ventricular function, including a significant reduction in infarct size. However, selection of CD34⫹ cells may lead to depletion of CD133⫹CD34⫺ cells; the latter cells have shown potential to differentiate into mesenchymal, endothelial and hematopoietic cells and have been associated with myocardial regeneration [18]. These findings prompted Stamm et al. to implant surgically up to 1.5 ⫻ 106 BM-CD133⫹ cells into the borders of the infarction in six patients during coronary artery bypass grafting [14]. We assumed, based on published engraftment studies in animals [19 –21], that to achieve successful engraftment of such a number of progenitor cells via the intracoronary route, 10 times more cells should be infused. We decided, therefore, to select all known progenitor cells with hematopoietic and angiogenic potential by isolating first the CD133⫹ cell fraction and subsequently the CD133⫺CD34⫹ cells. In fact, we could isolate and infuse as many as 1.6 ⫾ 0.5 ⫻ 107 BM-progenitor cells without any complication. In line with our results, Bartunek et al. demonstrated that selection and ICI of 1.26 ⫾ 0.22 ⫻ 107 CD133⫹ BM-progenitors in 19 patients with recent myocardial infarction was feasible [22]. The homing of BM-progenitor cells to the damaged myocardium, which results in cell engraftment, may play a key role
Intracoronary Delivery of Selected BM-Progenitors
2282
in the success of cell therapy. After acute ischemic myocardial injury, serum stromal-cell derived factor-1 levels rise significantly, and this chemokine appears to be one key homing signal that regulates homing of stem and progenitor cells to the ischemic myocardium [23]. In patients with chronic ischemic cardiomyopathy, however, local homing signals may not be as intense as in the early postinfarction phase, and therefore the coronary route might not be optimal for cell engraftment. To address this issue we tracked the distribution pattern of ICinfused BM-progenitors using 99mTc-HMPAO for cell labeling, which has been widely accepted for safe radiolabeling of leukocytes to localize areas of inflammation [15]. The 6-hour half-life of 99mTc allowed us to monitor cell distribution for about 24 hours, which we considered a clear advantage over 18 F-fluorodeoxyglycose (FDG) radiopharmaceuticals with the short physical half-life of 110 minutes. We preferred to use 99m Tc-HMPAO rather than 111In-oxine, because the latter radionucleotide causes complete impairment of the in vitro CD34⫹ cell proliferation and differentiation [19], whereas we found that 99mTc-HMPAO-induced damage of the in vitro proliferation-differentiation of BM-progenitors was 30%– 40% less than the potential of the unlabeled cells. In the hematopoietic stem cell transplantation setting similar in vitro damage during cell manipulation does not necessarily correlate with the ultimate efficacy of cell therapy. In addition in our study the large number of progenitor cells with intact viability used might balance any potential induced harm. Based on the above, we consider the toxicity of our radiolabeling method acceptable. Compared to nuclear medicine techniques, magnetic resonance imaging (MRI)-visible contrast agents (ferumoxides-protamine sulfate complexes), recently approved by the Federal Drug Administration, offer specific advantages. They do not alter cell metabolism, function, proliferation, viability, or differentiation capacity and are not associated with short- or long-term cell toxicity [24]. In addition they enable serial tracking and quantification of small numbers of stem cells engrafted in myocardium [25]. However, there has been concern that cells might not retain the iron particles and that cell death might provide false-positive findings due to
macrophage uptake of the particles. Further clinical studies might be necessary to clarify these issues. For future perspective, MRI techniques may represent the most promising approach for noninvasive detection of labeled stem cells. Our data revealed that the level of BM-progenitors homing to the irreversibly ischemic myocardial area was about 10% of the infused activity, which corresponded to 1.5 ⫻ 106 selected BM progenitors. Whether these cells retained in the chronically damaged myocardium result in cell engraftment and finally improve cardiac function remains to be investigated. Recently, Hofmann and colleagues showed that after ICI of 18F-FDG-labeled-CD34⫹ cells, 14 –39% of radioactivity was detected in the infarcted myocardium 50 –75 minutes after infusing CD34⫹ BM cells in three patients during the early postinfarction period [26]. While writing our results Blocklet et al. using a double radioactive labeling with 18 F-FDG and 111In-oxine, demonstrated that after intracoronary implantation of nonmobilized peripheral-blood CD34⫹-enriched cells, 5.5% of these cells were detected in the infarcted myocardium in six patients with recent myocardial infarct [27]. This relatively low percentage of cells adhering in the infracted myocardium may be due to the fact that nonmobilized peripheral blood progenitor cells display less homing capacity than BM progenitors because they do not express adhesion receptors that are necessary for the homing process. The rather poor purity on CD34⫹ cells (30%) of infused cells might be an additional reason for the low-level of engrafted cells in this study. Our study provides clear evidence that adhesion and retention of BM progenitors via ICI to infarcted myocardium is feasible and safe not only in the period of acute ischemic injury, when homing signals from injured tissue are intense, but also in the late phase of chronic ischemic cardiomyopathy.
REFERENCES
7
Tse HF, Kwong YL, Chan JK et al. Angiogenesis in ischemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet 2003;361:47– 49.
8
Kocher AA, Schuster MD, Szabolcs MJ et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevent cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7:430 – 436.
Perin EC, Dohmann HF, Borojevic R et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003;107:2294 –2302.
9
Strauer BE, Brehm M, Zeus T et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002;106:1913–1918.
3
Urbanek K, Torella D, Sheikh F et al. Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure. Proc Natl Acad Sci U S A 2005;102:8692– 8697.
10 Assmus B, Schachinger V, Teupe C et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:3009 –3017.
4
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
11 Britten MB, Abolmaali ND, Assmus B et al. Infarct remodelling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI). Circulation 2003;108:2212–2218.
5
Agbulut O, Vandervelde S, Al Attar N et al. Comparison of human skeletal myoblasts and bone marrow-derived CD133⫹ progenitors for the repair of infracted myocardium. J Am Coll Cardiol 2004;44:458 – 463.
12 Ferna´ndez-Avile´s F, San Roma´n JA, Garcia-Frade J et al. Experimental and clinical regenerative capability of bone marrow cells after myocardial infarction. Circ Res 2004;95:742–748.
6
Yoshioka T, Ageyama N, Shibata H et al. Repair of infracted myocardium mediated by transplanted bone marrow-derived CD34⫹ stem cells in a nonhuman primate model. STEM CELLS 2005;23:355–364.
1
2
Jackson KA, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395–1402.
ACKNOWLEDGMENTS We thank G. Kontostolis and A. Kouzoumi for their assistance in performing scintigraphic imaging.
DISCLOSURES The authors indicate no potential conflicts of interest.
13 Strauer BE, Brehm M, Zeus T et al. Regeneration of human infarcted heart muscle by intracoronary autologous bone marrow cell transplantation in chronic coronary artery disease. J Am Coll Cardiol 2005;46: 1651–1658.
Goussetis, Manginas, Koutelou et al.
2283
14 Stamm C, Westphal B, Kleine HD et al. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet 2003;361: 45– 46.
21 Aicher A, Brenner W, Zuhayra M et al. Assessment of the tissue distribution of transplanted human endothelial progenitor cells by radioactive labeling. Circulation 2003;107:2134 –2139.
15 Peters AM. The utility of 99mTc-HMPAO-leukocytes for imaging infection. Semin Nucl Med 1994;24:110 –127.
22 Bartunek J, Vanderheyden M, Vandekerckhove B et al. Intracoronary injection of CD133-positive enriched bone marrow progenitor cells promotes cardiac recovery after recent myocardial infarction. Circulation 2005;112(9 Suppl):78 – 83.
16 Menasche P, Hagege AA, Scorsin M et al. Myoblast transplantation for heart failure. Lancet 2001;357:279 –280. 17 Wollert KC, Meyer GP, Lotz J et al. Intracoronary autologous bone marrow cell transfer after myocardial infarction: the BOOST randomised-controlled clinical trial. Lancet 2004;364:141–148. 18 Gallacher L, Murdoch B, Wu DM et al. Isolation and characterization of human CD34(-)Lin(-) and CD34(⫹)Lin(-) hemopoietic stem cells using cell surface markers AC133 and CD7. Blood 2000;95: 2813–2820. 19 Brenner W, Aicher A, Eckey T et al. 111In-labeled CD34⫹ hematopoietic progenitor cells in a rat myocardial infarction model. J Nucl Med 2004;45:512–518. 20 Barbash IM, Chouraqui P, Baron J et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium. Feasibility, cell migration, and body distribution. Circulation 2003;108: 863–868.
www.StemCells.com
23 Askari AT, Unzek S, Popovic ZB et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischemic cardiomyopathy. Lancet 2003;362:697–703. 24 Arbab AS, Yocum GT, Kalish H et al. Efficient magnetic cell labelling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood 2004;104:1217–1223. 25 Kraitchman DL, Heldman AW, Atalar E et al. In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. Circulation 2003;107:2290 –2293. 26 Hofmann M, Wollert KC, Meyer GP et al. Monitoring of bone marrow cell homing into the infarcted human myocardium. Circulation 2005; 111:2198 –2202. 27 Blocklet D, Toungouz M, Berkenboom G et al. Myocardial homing of non mobilized peripheral-blood CD34⫹ cells after intra-coronary injection. STEM CELLS 2006;24:333–336.
