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Imaging and 1-day kinetics of intracoronary stem cell transplantation in patients with idiopathic dilated cardiomyopathy
Nuclear Medicine and Biology 43 (2016) 410–414
Contents lists available at ScienceDirect
Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio
Imaging and 1-day kinetics of intracoronary stem cell transplantation in patients with idiopathic dilated cardiomyopathy Luka Lezaic a,⁎, Aljaz Socan a, Petra Kolenc Peitl a, Gregor Poglajen b, Matjaz Sever c, Marko Cukjati d, Peter Cernelc c, Bojan Vrtovec b,e a
Department for Nuclear Medicine, UMC Ljubljana, Slovenia Advanced Heart Failure and Transplantation Center, UMC Ljubljana, Slovenia Department of Hematology, UMC Ljubljana, Slovenia d National Blood Transfusion Institute, Ljubljana, Slovenia e Stanford University School of Medicine, Stanford, CA, USA b c
a r t i c l e
i n f o
Article history: Received 24 September 2014 Received in revised form 18 November 2015 Accepted 29 December 2015 Available online xxxx Keywords: Stem cell imaging Stem cell labeling Heart failure Stem cell therapy IDCM
a b s t r a c t Background: Stem cell transplantation is an emerging method of treatment for patients with cardiovascular disease. There are few studies completed or ongoing on stem cell therapy in patients with idiopathic dilated cardiomyopathy (IDCM). Information on stem cell homing and distribution in the myocardium after transplantation might provide important insight into effectiveness of transplantation procedure. Aim: To assess early engraftment, retention and migration of intracoronarily transplanted stem cells in the myocardium of patients with advanced dilated cardiomyopathy of non-ischaemic origin using stem cell labeling with 99m Tc-exametazime (HMPAO). Materials, methods: Thirty-five patients with IDCM and advanced heart failure were included in the study. Autologous hematopoietic (CD34+) stem cells were harvested by peripheral blood apheresis after bone marrow stimulation, labeled with 99mTc-HMPAO, tested for viability and injected into coronary vessel supplying areas of myocardium selected by myocardial perfusion scintigraphy as dysfunctional yet viable. Imaging was performed 1 h and 18 h after transplantation. Results: Myocardial stem cell retention ranged from 0 to 1.44% on early and 0–0.97% on delayed imaging. Significant efflux of stem cells occurred from site of delivery in this time period (p b 0.001). Stem cell viability was not affected by labeling. Conclusion: Stem cell labeling with 99mTc-HMPAO is a feasible method for stem cell tracking after transplantation in patients with IDCM. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Stem cell transplantation is an emerging method of treatment for patients with cardiovascular disease, resulting in improvement of cardiac haemodynamics and functional capacity [1]. While clinical research is predominantly concentrated on ischaemic heart disease in its acute and chronic presentation, there are few studies completed or ongoing in advanced heart failure of non-ischaemic etiology [2–5]. Stem cell engraftment, retention, distribution and differentiation are the likely important events that determine the success of treatment [6]. Assessment of long-term fate of transplanted stem cells requires use of imaging techniques involving genetic manipulation which are unlikely to be used in human studies due to ethical restraints [7]. However, it is possible to evaluate the important early events of stem cell engraftment, ⁎ Corresponding author at: Department for Nuclear Medicine, University Medical Centre Ljubljana, Ljubljana, Zaloska 7, 1525, Ljubljana, Slovenia. Tel.: +386 1 522 8450; fax: +386 1 522 2237. E-mail address: [email protected] (L. Lezaic). http://dx.doi.org/10.1016/j.nucmedbio.2015.12.003 0969-8051/© 2016 Elsevier Inc. All rights reserved.
retention and distribution in the heart after transplantation using established methods of cell labeling [7,8]. 99mTc-hexamethyl-propyleneamine-oxyme (HMPAO) is a well established cell label, used primarily for localization of infectious/inflammatory processes. Half-life of the radionuclide allows for tracking of labeled cells within 24 h of labeling. To date, there are no published studies using labeled stem cell imaging for assessment of cardiac stem cell transplantation in patients with nonischaemic cardiomyopathy. The aim of this study was to assess early engraftment, retention and migration of intracoronarily transplanted stem cells in the myocardium of patients with advanced dilated cardiomyopathy of non-ischaemic origin. 2. Material, Methods 2.1. Patients We included 35 patients with (Table 1) depressed left ventricular ejection fraction on echocardiography (b 30%; standard Simpson technique)
L. Lezaic et al. / Nuclear Medicine and Biology 43 (2016) 410–414
and who had been in NYHA class III or IV for at least 6 months on optimal medical therapy. Dilated cardiomyopathy was defined based on the absence of any stenotic lesions on coronary angiography, no primary valve disease on echocardiography, and no history of hypertension or alcohol abuse, according to the European Society of Cardiology position statement. 2.2. Myocardial perfusion scintigraphy Myocardial perfusion scintigraphy in resting state was performed using viability protocol. Prior to radiopharmaceutical injection, 2 puffs (0.4 μg) of nitroglycerin were applied sublingually and blood pressure and heart rate was monitored. After 10 min, 600 MBq of 99mTcsestaMIBI was injected intravenously. Dual-detector single-photon emission computed tomography (SPECT) was performed 1 h pi., using ECG-gating, 64 × 64 matrix, 36 projections, 25 s per frame (GE Millenium dual-head gamma camera, GE Corporation, USA). Myocardial perfusion was quantified using 20segment model, normalized to maximum uptake in the myocardium. 2.3. Stem cell mobilization and collection Patients received bone marrow stimulation using G-CSF after testing for appropriate bone marrow response (single test dose of G-CSF, transient increase in absolute neutrophil count N = 50%). Peripheral blood stem cells were mobilized by daily subcutaneous injections of G-CSF (5 μg/kg bid). On the fifth day a full blood count and peripheral blood CD34 + cell count were performed. Peripheral blood stem cells were then collected on the same day with the Amicus cell separator (Baxter Healthcare, IL, USA). The magnetic cell separator Isolex 300i (Nexell Therapeutics Inc., Irvine, CA, USA) was used for the immunomagnetic positive selection of CD34 + cells. In the closed system the collected cells were washed to remove the platelets, sensitized with mouse monoclonal anti-CD34 antibodies and then incubated with immunomagnetic beads coated with polyclonal sheep anti-mouse antibodies (Dynabeads-Dynal AS, Oslo, Norway). The bead/CD34+ cell rosettes were separated in the magnetic field from other cells and CD34 + cells were released from the Dynabeads using an octapeptide with an affinity for anti-CD34 antibodies. The remaining CD34+ cells were free of surface contaminants and the CD34 cell surface antigen was left intact. Final infusate was concentrated to a volume of 100 ml. 2.4. Stem cell labeling, viability and labeling stability assessment 2.4.1. Stem cell labeling Predefined volume (20%) of collected CD34+ cells was radiolabeled with 600 MBq of 99mTc-exametazime (HMPAO) and incubated for
411
20 min at room temperature. Unbound radioactivity was removed by washing the cells with cell free media. Labeled stem cells were resuspended with the remaining infusate. 2.4.2. Stem cell viability, labeling efficiency and stability assessment Viability of labeled stem cells was assessed by Trypan Blue exclusion assay 1 h after labeling at the time of intracoronary injection. A small proportion of unlabeled stem cells was used for viability assessment at identical time-points for comparison. 2.5. Stem cell transplantation/injection Using the standard right femoral approach a microcatheter (Progreat Microcatheter System, Terumo, Leuven, Belgium) was positioned in a mid portion of the target coronary artery, and repeated injections of stem cell solution were performed. Before the procedure patient was fully heparinized. To avoid trauma of the target vessel we performed no balloon inflations at any time during the procedure. Target vessel was determined by myocardial perfusion scintigraphy as one supplying areas of viable yet dysfunctional myocardium (segments of tracer accumulation of N = 50% of maximal activity in the myocardium and regional dysfunction). In patients with diffusely inhomogenous tracer distribution and regional dysfunction LAD was selected for stem cell injection as a vessel supplying the largest proportion of myocardium. 2.6. Stem cell transplantation imaging and quantification 1 h after transplantation, stem cell imaging was undertaken to assess myocardial engraftment and distribution. Planar anterior/posterior and LAO/RPO projections of the thorax and upper abdomen (128 × 128 matrix, 15 min per projection) and tomographic imaging of cardiac region (64 × 64 matrix, 36 projections, 25 s per frame) were performed on a dual – head gamma camera (GE Millenium, GE Corporation, USA). After 18 h, planar imaging was repeated to detect potential stem cell migration. Quantification of stem cell retention was performed from anterior and posterior planar images (at 1 h and 18 h post injection) using mirrored regions of interest (ROI) placed over areas of stem cell accumulation after background correction (small periventricular ROI). Whole – organ ROIs were used for quantification of activity in the liver and spleen. Round ROI, normalized at 100 pixels was used for quantification of activity in the lung parencyhma and rectangular ROI, normalized at 200 pixels and placed at the mediastinum (i.e., representing activity in the sternum and vertebral column) was used for quantification of
Fig. 1. Anterior planar image of intracoronary stem cell transplantation at 1 h (panel A) and 18 h (panel B) post injection. Target vessel for stem cell delivery was left anterior descending artery. Transplanted stem cells are distributed in the anterior myocardial wall. At delayed imaging, stem cell distribution does not appear to change.
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Fig. 2. Tomographic reconstruction of intracoronary stem cell transplantation imaging (same patient as Fig. 1). Polar image (panel A) shows distribution of stem cells in the anterior segments of the myocardium. Panel B combines tomographic images in standard axes (SA – short axis, HLA – horizontal long axis, VLA – vertical long axis) of myocardial perfusion (color images, top row in each axis) and stem cell transplantation (monochrome images, bottom row in each axis), showing corresponding areas of myocardium.
activity in the bone marrow. Conjugate-view method of quantification was used for correction of attenuation.
2.7. Statistical methods Variables are shown as range (mean; median, interquartile range). Stem cell quantification (1 h vs. 18 h post injection) and viability (labeled vs. non-labeled) was compared using Wilcoxon Signed Rank test. Pearson product–moment correlation coefficient (Pearson's r) was used for assessment of correlation between administered activity and myocardial stem cell retention. R software package v3.0.2 (R Foundation for Statistical Computing, Vienna, Austria) was used for data analysis.
2.8. Ethical approval The study protocol was approved by the National Medical Ethics Committee and all participants gave written informed consent upon entering. 3. Results 3.1. Stem cell labeling and viability Stem cell number in the infusate was 23.0–284.2 (111.9; 102.7, 63.9)× 10 6. Activity of stem cell infusate was 51–343 (148,7; 130, 87.5) MBq. Stem cell viability after labeling was 61.9–98.4 (89.2; 91.9, 8.15)%. Viability of unlabeled stem cells at the same time point was
Fig. 3. Quantification of labeled stem cell retention in the myocardium at early and delayed imaging. *: significant efflux of transplanted stem cells from the myocardium on delayed imaging (p b 0.001).
L. Lezaic et al. / Nuclear Medicine and Biology 43 (2016) 410–414 Table 1 Patient characteristics. Gender
2 Female, 33 male
Age LVEF Pro-BNP Creatinine Therapy RAAS antagonists Aldosterone antagonists Beta-blockers Diuretics Digoxin
51.2 ± 8.4 years 24.8 ± 7.6% 2276.0 ± 2821.3 pg/mL 111.5 ± 85.5 umol/L 34 (94) 22 (61) 29 (80) 33(92) 9 (25)
Values are shown as average ± SD or as patient number (percentage of patients), as appropriate. LVEF: left ventricular ejection fraction; pro-BNP: pro-beta natriuretic peptide; RAAS: renin – angiotensin – aldosterone system.
89.0–97.1 (92.5; 91.5, 7.0)%. Difference between labeled and nonlabeled stem cells was not statistically significant (p = 0.10). 3.2. Stem cell transplantation, distribution, engraftment and kinetics No cases of significant troponin leak, acute cardiac dysfunction or distal coronary artery occlusion occurred; no reactions to murine antibodies were observed. Target vessel for stem cell deployment was LAD in 22, LCX in 3 and RCA in 10 patients. At imaging 1 h after intracoronary administration, the majority of labeled injected stem cells accumulated in the liver, spleen and bone marrow. Accumulation of stem cells in the myocardium ranged from 0 to 1.45% of injected activity in the field of view (Fig. 1, panel A and Fig. 2); myocardial stem cell retention did not correlate with administered activity (Pearson r = 0.009, p = 0.95). In 12 patients, only very poor accumulation in the myocardium could be noted on planar images, with no visualization on SPECT imaging or quantification possible. The distribution of labeled stem cells in the myocardium corresponded to the area supplied by the vessel used for administration. There was no apparent accumulation in the areas of myocardium supplied by other main vessels. At imaging 18 h post injection, the distribution of labeled stem cells appeared unchanged (Fig. 1, panel B), but the accumulation of labeled stem cells in the myocardium decreased (range 0 to 0.97% of injected activity in the field of view). The difference in myocardial accumulation at the two time points was statistically significant (p b 0.001, Fig. 3). Accumulation of stem cells in the liver, spleen and lungs also significantly decreased at delayed imaging (p b 0.001), while there was nonsignificant increase in stem cell accumulation in the bone marrow (p = 0.213); (Table 2). 4. Discussion This is the first study to evaluate the early engraftment, distribution and kinetics of cardiac hematopoietic stem cell transplantation in patients with dilated cardiomyopathy. To the best knowledge of the Table 2 Quantification of labeled stem cell retention at early and delayed imaging.
Heart Liver Spleen Bone marrow Lung
Time p.i.
Organ retention (% activity)
p (1 h vs. 18 h)
1h 18 h 1h 18 h 1h 18 h 1h 18 h 1h 18 h
0–1.44 (0.35) 0–0.97 (0.55) 21.22–50.72 (32.37) 19.16–42.81 (29.62) 9.86–34.17 (22.6) 11.23–29.61 (20.85) 0.19–1.5 (0.92) 0.23–1.42 (0.97) 0.15–2.24 (0.83) 0.16–0.99 (0.64)
b0.001
Values (except p) represent range (median).
b0.001
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authors, it is also the largest study to date using stem cell labeling in human subjects. Few human studies have evaluated the efficiency of intracoronary stem cell transplantation in terms of early stem cell retention and distribution. All involved patients with ischaemic heart disease in either acute (usually defined as up to 1 month after acute coronary event) or chronic phase (usually defined as at least 6 months to 1 year after an acute coronary event or heart failure of ischaemic origin). In a study using 99mTc-HMPAO as stem cell label, early myocardial retention comparable to our results was reported in patients with chronic ischaemic cardiomyopathy, ranging from 0 to 3.0% [9]; interestingly, no stem cell retention was demonstrated on delayed imaging. Similar myocardial stem cell retention rates were found using 111In (averaging to 2.5%) [10] and 18F-FDG (ranging from 0.2 to 3.3%) [11] as cell label. Higher myocardial retention rates were reported in patients transplanted in the acute phase of a coronary event, averaging from 1.5 to 6.9% [10,11,12]. Although direct comparison of results is difficult due to different stem cell subtypes and labels used in imaging studies, it appears that stem cell retention in the myocardium after intracoronary delivery is rather low, particularly in chronic cardiomyopathy. Plausible explanation for low retention rates in chronic heart failure in comparison to an acute ischaemic event is low myocardial homing signal for delivered stem cells [13], which may be even lower in non-ischaemic heart failure [14]. As early engraftment is apparently the determining factor for late functional improvement [15], methods that allow a more efficient delivery and retention of transplanted stem cells will be required. Direct intramuscular injection was already shown to be more efficient than intracoronary route in animal models and in our preliminary report in a human study [16,17]. Furthermore, significant efflux of stem cells occurs from the site of delivery, underlining the importance of early cardiac retention. The distribution of stem cells in the myocardium did not appear to change and no additional uptake was seen on delayed images as a result of potential migration and (collateral) recirculation. Similar organ distribution kinetics were also reported by other authors [9,10,11] with liver, spleen and bone marrow accumulating the vast majority of delivered stem cells and lung tissue demonstrating early uptake and fast clearance of stem cells.
4.1. Limitations Imaging approach in our study is limited by 6 h half-life of 99mTcHMPAO label. However, as the goal of the study was assessment of early stem cell engraftment, we found the 24-h imaging window to be sufficient. Moreover, in contrast to 111In label, whose half-life would allow imaging up to 7 days post transplantation, 99mTc appears to have the least effect on proliferation and migration capacity of labeled cells [18], while 111In was shown to affect stem cell viability [19]. We found no effect on stem cell viability after labeling and demonstrated good labeling stability, corroborating previous findings [10]. In addition, we found no correlation between administered activity and myocardial stem cell retention, suggesting no nonspecific binding of radiopharmaceutical in the myocardium that would account for myocardial uptake. It must be noted that subject numbers in human stem cell cardiac transplantation imaging studies, including present one, are small. Nevertheless, an important insight is provided into a promising field of cardiac therapy.
b0.001 0.213 b0.001
5. Conclusion Stem cell labeling with 99mTc-HMPAO is a feasible method for assessment of intracoronary hematopoietic stem cell transplantation in patients with idiopathic dilated cardiomyopathy.
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References [1] Strauer BE, Steinhoff G. 10 years of intracoronary and intramyocardial bone marrow stem cell therapy of the heart: from the methodological origin to clinical practice. J Am Coll Cardiol 2011;58(11):1095–104. [2] Seth S, Bhargava B, Narang R, Ray R, Mohanty S, Gulati G, et al. AIIMS stem cell study group. The ABCD (autologous bone marrow cells in dilated cardiomyopathy) trial a long-term follow-up study. J Am Coll Cardiol 2010;55(15):1643–4. [3] Schannwell CM, Köstering M, Zeus T, Brehm M, Erdmann G, Fleissner T, et al. [Humane autologe intrakoronare stammzelltransplantation zur myokardregeneration bei dilatativer cardiomyopathie (NYHA stadium II-IV)]. The Düsseldorf autologous bone marrow cells in dilated cardiomyopathy trial. ABCD trial. J Kardiologie 2008; 15:23–30. [4] Fischer-Rasokat U, Assmus B, Seeger FH, Honold J, Leistner D, Fichtlscherer S, et al. A pilot trial to assess potential effects of selective intracoronary bone marrow-derived progenitor cell infusion in patients with nonischemic dilated cardiomyopathy: final 1-year results of the transplantation of progenitor cells and functional regeneration enhancement pilot trial in patients with nonischemic dilated cardiomyopathy. Circ Heart Fail 2009;2(5):417–23. [5] Vrtovec B, Poglajen G, Sever M, Lezaic L, Domanovic D, Cernelc P, et al. Effects of intracoronary stem cell transplantation in patients with dilated cardiomyopathy. J Card Fail 2011;17(4):272–81. [6] Chavakis E, Urbich C, Dimmeler S. Homing and engraftment of progenitor cells: a prerequisite for cell therapy. J Mol Cell Cardiol 2008;45(4):514–22. [7] Wu JC, Abraham MR, Kraitchman DL. Current perspectives on imaging cardiac stem cell therapy. J Nucl Med 2010;51(Suppl. 1):128S–36S. [8] Fu Y, Kraitchman DL. Stem cell labeling for noninvasive delivery and tracking in cardiovascular regenerative therapy. Expert Rev Cardiovasc Ther 2010;8(8): 1149–60. [9] Penicka M, Lang O, Widimsky P, Kobylka P, Kozak T, Vanek T, et al. One-day kinetics of myocardial engraftment after intracoronary injection of bone marrow mononuclear cells in patients with acute and chronic myocardial infarction. Heart 2007; 93(7):837–41.
[10] Schächinger V, Aicher A, Döbert N, Röver R, Diener J, Fichtlscherer S, et al. Pilot trial on determinants of progenitor cell recruitment to the infarcted human myocardium. Circulation 2008;118(14):1425–32. [11] Kang WJ, Kang HJ, Kim HS, Chung JK, Lee MC, Lee DS. Tissue distribution of 18F-FDGlabeled peripheral hematopoietic stem cells after intracoronary administration in patients with myocardial infarction. J Nucl Med 2006;47(8):1295–301. [12] Musialek P, Tekieli L, Kostkiewicz M, Majka M, Szot W, Walter Z, et al. Randomized transcoronary delivery of CD34(+) cells with perfusion versus stop-flow method in patients with recent myocardial infarction: early cardiac retention of 99(m)Tc-labeled cells activity. J Nucl Cardiol 2011;18(1):104–16. [13] van Weel V, Seghers L, de Vries MR, Kuiper EJ, Schlingemann RO, Bajema IM, et al. Expression of vascular endothelial growth factor, stromal cell-derived factor-1, and CXCR4 in human limb muscle with acute and chronic ischemia. Arterioscler Thromb Vasc Biol 2007;27(6):1426–32. [14] Theiss HD, David R, Engelmann MG, Barth A, Schotten K, Naebauer M, et al. Circulation of CD34+ progenitor cell populations in patients with idiopathic dilated and ischaemic cardiomyopathy (DCM and ICM). Eur Heart J 2007;28(10):1258–64. [15] Liu J, Narsinh KH, Lan F, Wang L, Nguyen PK, Hu S, et al. Early stem cell engraftment predicts late cardiac functional recovery: preclinical insights from molecular imaging. Circ Cardiovasc Imaging 2012;5(4):481–90. [16] Li SH, Lai TY, Sun Z, Han M, Moriyama E, Wilson B, et al. Tracking cardiac engraftment and distribution of implanted bone marrow cells: comparing intra-aortic, intravenous, and intramyocardial delivery. J Thorac Cardiovasc Surg 2009;137(5): 1225–33. [17] Lezaic L, Socan A, Vrtovec B, Poglajen G, Sever M, Domanovic D, et al. Comparison of two methods of stem cell delivery into the myocardium. In: Sinusas AJ, editor. 3rd multimodality cardiovascular molecular imaging symposium—poster abstractsJ Nucl Med 2012;53 (4):664–71. [18] Botti C, Negri DR, Seregni E, Ramakrishna V, Arienti F, Maffioli L, et al. Comparison of three different methods for radiolabelling human activated T lymphocytes. Eur J Nucl Med 1997;24(5):497–504. [19] Nowak B, Weber C, Schober A, Zeiffer U, Liehn EA, von Hundelhausen P, et al. Indium-111 oxine labeling affects the cellular integrity of haematopoetic progenitor cells. Eur J Nucl Med Mol Imaging 2007;34:715–21.
Contents lists available at ScienceDirect
Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio
Imaging and 1-day kinetics of intracoronary stem cell transplantation in patients with idiopathic dilated cardiomyopathy Luka Lezaic a,⁎, Aljaz Socan a, Petra Kolenc Peitl a, Gregor Poglajen b, Matjaz Sever c, Marko Cukjati d, Peter Cernelc c, Bojan Vrtovec b,e a
Department for Nuclear Medicine, UMC Ljubljana, Slovenia Advanced Heart Failure and Transplantation Center, UMC Ljubljana, Slovenia Department of Hematology, UMC Ljubljana, Slovenia d National Blood Transfusion Institute, Ljubljana, Slovenia e Stanford University School of Medicine, Stanford, CA, USA b c
a r t i c l e
i n f o
Article history: Received 24 September 2014 Received in revised form 18 November 2015 Accepted 29 December 2015 Available online xxxx Keywords: Stem cell imaging Stem cell labeling Heart failure Stem cell therapy IDCM
a b s t r a c t Background: Stem cell transplantation is an emerging method of treatment for patients with cardiovascular disease. There are few studies completed or ongoing on stem cell therapy in patients with idiopathic dilated cardiomyopathy (IDCM). Information on stem cell homing and distribution in the myocardium after transplantation might provide important insight into effectiveness of transplantation procedure. Aim: To assess early engraftment, retention and migration of intracoronarily transplanted stem cells in the myocardium of patients with advanced dilated cardiomyopathy of non-ischaemic origin using stem cell labeling with 99m Tc-exametazime (HMPAO). Materials, methods: Thirty-five patients with IDCM and advanced heart failure were included in the study. Autologous hematopoietic (CD34+) stem cells were harvested by peripheral blood apheresis after bone marrow stimulation, labeled with 99mTc-HMPAO, tested for viability and injected into coronary vessel supplying areas of myocardium selected by myocardial perfusion scintigraphy as dysfunctional yet viable. Imaging was performed 1 h and 18 h after transplantation. Results: Myocardial stem cell retention ranged from 0 to 1.44% on early and 0–0.97% on delayed imaging. Significant efflux of stem cells occurred from site of delivery in this time period (p b 0.001). Stem cell viability was not affected by labeling. Conclusion: Stem cell labeling with 99mTc-HMPAO is a feasible method for stem cell tracking after transplantation in patients with IDCM. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Stem cell transplantation is an emerging method of treatment for patients with cardiovascular disease, resulting in improvement of cardiac haemodynamics and functional capacity [1]. While clinical research is predominantly concentrated on ischaemic heart disease in its acute and chronic presentation, there are few studies completed or ongoing in advanced heart failure of non-ischaemic etiology [2–5]. Stem cell engraftment, retention, distribution and differentiation are the likely important events that determine the success of treatment [6]. Assessment of long-term fate of transplanted stem cells requires use of imaging techniques involving genetic manipulation which are unlikely to be used in human studies due to ethical restraints [7]. However, it is possible to evaluate the important early events of stem cell engraftment, ⁎ Corresponding author at: Department for Nuclear Medicine, University Medical Centre Ljubljana, Ljubljana, Zaloska 7, 1525, Ljubljana, Slovenia. Tel.: +386 1 522 8450; fax: +386 1 522 2237. E-mail address: [email protected] (L. Lezaic). http://dx.doi.org/10.1016/j.nucmedbio.2015.12.003 0969-8051/© 2016 Elsevier Inc. All rights reserved.
retention and distribution in the heart after transplantation using established methods of cell labeling [7,8]. 99mTc-hexamethyl-propyleneamine-oxyme (HMPAO) is a well established cell label, used primarily for localization of infectious/inflammatory processes. Half-life of the radionuclide allows for tracking of labeled cells within 24 h of labeling. To date, there are no published studies using labeled stem cell imaging for assessment of cardiac stem cell transplantation in patients with nonischaemic cardiomyopathy. The aim of this study was to assess early engraftment, retention and migration of intracoronarily transplanted stem cells in the myocardium of patients with advanced dilated cardiomyopathy of non-ischaemic origin. 2. Material, Methods 2.1. Patients We included 35 patients with (Table 1) depressed left ventricular ejection fraction on echocardiography (b 30%; standard Simpson technique)
L. Lezaic et al. / Nuclear Medicine and Biology 43 (2016) 410–414
and who had been in NYHA class III or IV for at least 6 months on optimal medical therapy. Dilated cardiomyopathy was defined based on the absence of any stenotic lesions on coronary angiography, no primary valve disease on echocardiography, and no history of hypertension or alcohol abuse, according to the European Society of Cardiology position statement. 2.2. Myocardial perfusion scintigraphy Myocardial perfusion scintigraphy in resting state was performed using viability protocol. Prior to radiopharmaceutical injection, 2 puffs (0.4 μg) of nitroglycerin were applied sublingually and blood pressure and heart rate was monitored. After 10 min, 600 MBq of 99mTcsestaMIBI was injected intravenously. Dual-detector single-photon emission computed tomography (SPECT) was performed 1 h pi., using ECG-gating, 64 × 64 matrix, 36 projections, 25 s per frame (GE Millenium dual-head gamma camera, GE Corporation, USA). Myocardial perfusion was quantified using 20segment model, normalized to maximum uptake in the myocardium. 2.3. Stem cell mobilization and collection Patients received bone marrow stimulation using G-CSF after testing for appropriate bone marrow response (single test dose of G-CSF, transient increase in absolute neutrophil count N = 50%). Peripheral blood stem cells were mobilized by daily subcutaneous injections of G-CSF (5 μg/kg bid). On the fifth day a full blood count and peripheral blood CD34 + cell count were performed. Peripheral blood stem cells were then collected on the same day with the Amicus cell separator (Baxter Healthcare, IL, USA). The magnetic cell separator Isolex 300i (Nexell Therapeutics Inc., Irvine, CA, USA) was used for the immunomagnetic positive selection of CD34 + cells. In the closed system the collected cells were washed to remove the platelets, sensitized with mouse monoclonal anti-CD34 antibodies and then incubated with immunomagnetic beads coated with polyclonal sheep anti-mouse antibodies (Dynabeads-Dynal AS, Oslo, Norway). The bead/CD34+ cell rosettes were separated in the magnetic field from other cells and CD34 + cells were released from the Dynabeads using an octapeptide with an affinity for anti-CD34 antibodies. The remaining CD34+ cells were free of surface contaminants and the CD34 cell surface antigen was left intact. Final infusate was concentrated to a volume of 100 ml. 2.4. Stem cell labeling, viability and labeling stability assessment 2.4.1. Stem cell labeling Predefined volume (20%) of collected CD34+ cells was radiolabeled with 600 MBq of 99mTc-exametazime (HMPAO) and incubated for
411
20 min at room temperature. Unbound radioactivity was removed by washing the cells with cell free media. Labeled stem cells were resuspended with the remaining infusate. 2.4.2. Stem cell viability, labeling efficiency and stability assessment Viability of labeled stem cells was assessed by Trypan Blue exclusion assay 1 h after labeling at the time of intracoronary injection. A small proportion of unlabeled stem cells was used for viability assessment at identical time-points for comparison. 2.5. Stem cell transplantation/injection Using the standard right femoral approach a microcatheter (Progreat Microcatheter System, Terumo, Leuven, Belgium) was positioned in a mid portion of the target coronary artery, and repeated injections of stem cell solution were performed. Before the procedure patient was fully heparinized. To avoid trauma of the target vessel we performed no balloon inflations at any time during the procedure. Target vessel was determined by myocardial perfusion scintigraphy as one supplying areas of viable yet dysfunctional myocardium (segments of tracer accumulation of N = 50% of maximal activity in the myocardium and regional dysfunction). In patients with diffusely inhomogenous tracer distribution and regional dysfunction LAD was selected for stem cell injection as a vessel supplying the largest proportion of myocardium. 2.6. Stem cell transplantation imaging and quantification 1 h after transplantation, stem cell imaging was undertaken to assess myocardial engraftment and distribution. Planar anterior/posterior and LAO/RPO projections of the thorax and upper abdomen (128 × 128 matrix, 15 min per projection) and tomographic imaging of cardiac region (64 × 64 matrix, 36 projections, 25 s per frame) were performed on a dual – head gamma camera (GE Millenium, GE Corporation, USA). After 18 h, planar imaging was repeated to detect potential stem cell migration. Quantification of stem cell retention was performed from anterior and posterior planar images (at 1 h and 18 h post injection) using mirrored regions of interest (ROI) placed over areas of stem cell accumulation after background correction (small periventricular ROI). Whole – organ ROIs were used for quantification of activity in the liver and spleen. Round ROI, normalized at 100 pixels was used for quantification of activity in the lung parencyhma and rectangular ROI, normalized at 200 pixels and placed at the mediastinum (i.e., representing activity in the sternum and vertebral column) was used for quantification of
Fig. 1. Anterior planar image of intracoronary stem cell transplantation at 1 h (panel A) and 18 h (panel B) post injection. Target vessel for stem cell delivery was left anterior descending artery. Transplanted stem cells are distributed in the anterior myocardial wall. At delayed imaging, stem cell distribution does not appear to change.
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Fig. 2. Tomographic reconstruction of intracoronary stem cell transplantation imaging (same patient as Fig. 1). Polar image (panel A) shows distribution of stem cells in the anterior segments of the myocardium. Panel B combines tomographic images in standard axes (SA – short axis, HLA – horizontal long axis, VLA – vertical long axis) of myocardial perfusion (color images, top row in each axis) and stem cell transplantation (monochrome images, bottom row in each axis), showing corresponding areas of myocardium.
activity in the bone marrow. Conjugate-view method of quantification was used for correction of attenuation.
2.7. Statistical methods Variables are shown as range (mean; median, interquartile range). Stem cell quantification (1 h vs. 18 h post injection) and viability (labeled vs. non-labeled) was compared using Wilcoxon Signed Rank test. Pearson product–moment correlation coefficient (Pearson's r) was used for assessment of correlation between administered activity and myocardial stem cell retention. R software package v3.0.2 (R Foundation for Statistical Computing, Vienna, Austria) was used for data analysis.
2.8. Ethical approval The study protocol was approved by the National Medical Ethics Committee and all participants gave written informed consent upon entering. 3. Results 3.1. Stem cell labeling and viability Stem cell number in the infusate was 23.0–284.2 (111.9; 102.7, 63.9)× 10 6. Activity of stem cell infusate was 51–343 (148,7; 130, 87.5) MBq. Stem cell viability after labeling was 61.9–98.4 (89.2; 91.9, 8.15)%. Viability of unlabeled stem cells at the same time point was
Fig. 3. Quantification of labeled stem cell retention in the myocardium at early and delayed imaging. *: significant efflux of transplanted stem cells from the myocardium on delayed imaging (p b 0.001).
L. Lezaic et al. / Nuclear Medicine and Biology 43 (2016) 410–414 Table 1 Patient characteristics. Gender
2 Female, 33 male
Age LVEF Pro-BNP Creatinine Therapy RAAS antagonists Aldosterone antagonists Beta-blockers Diuretics Digoxin
51.2 ± 8.4 years 24.8 ± 7.6% 2276.0 ± 2821.3 pg/mL 111.5 ± 85.5 umol/L 34 (94) 22 (61) 29 (80) 33(92) 9 (25)
Values are shown as average ± SD or as patient number (percentage of patients), as appropriate. LVEF: left ventricular ejection fraction; pro-BNP: pro-beta natriuretic peptide; RAAS: renin – angiotensin – aldosterone system.
89.0–97.1 (92.5; 91.5, 7.0)%. Difference between labeled and nonlabeled stem cells was not statistically significant (p = 0.10). 3.2. Stem cell transplantation, distribution, engraftment and kinetics No cases of significant troponin leak, acute cardiac dysfunction or distal coronary artery occlusion occurred; no reactions to murine antibodies were observed. Target vessel for stem cell deployment was LAD in 22, LCX in 3 and RCA in 10 patients. At imaging 1 h after intracoronary administration, the majority of labeled injected stem cells accumulated in the liver, spleen and bone marrow. Accumulation of stem cells in the myocardium ranged from 0 to 1.45% of injected activity in the field of view (Fig. 1, panel A and Fig. 2); myocardial stem cell retention did not correlate with administered activity (Pearson r = 0.009, p = 0.95). In 12 patients, only very poor accumulation in the myocardium could be noted on planar images, with no visualization on SPECT imaging or quantification possible. The distribution of labeled stem cells in the myocardium corresponded to the area supplied by the vessel used for administration. There was no apparent accumulation in the areas of myocardium supplied by other main vessels. At imaging 18 h post injection, the distribution of labeled stem cells appeared unchanged (Fig. 1, panel B), but the accumulation of labeled stem cells in the myocardium decreased (range 0 to 0.97% of injected activity in the field of view). The difference in myocardial accumulation at the two time points was statistically significant (p b 0.001, Fig. 3). Accumulation of stem cells in the liver, spleen and lungs also significantly decreased at delayed imaging (p b 0.001), while there was nonsignificant increase in stem cell accumulation in the bone marrow (p = 0.213); (Table 2). 4. Discussion This is the first study to evaluate the early engraftment, distribution and kinetics of cardiac hematopoietic stem cell transplantation in patients with dilated cardiomyopathy. To the best knowledge of the Table 2 Quantification of labeled stem cell retention at early and delayed imaging.
Heart Liver Spleen Bone marrow Lung
Time p.i.
Organ retention (% activity)
p (1 h vs. 18 h)
1h 18 h 1h 18 h 1h 18 h 1h 18 h 1h 18 h
0–1.44 (0.35) 0–0.97 (0.55) 21.22–50.72 (32.37) 19.16–42.81 (29.62) 9.86–34.17 (22.6) 11.23–29.61 (20.85) 0.19–1.5 (0.92) 0.23–1.42 (0.97) 0.15–2.24 (0.83) 0.16–0.99 (0.64)
b0.001
Values (except p) represent range (median).
b0.001
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authors, it is also the largest study to date using stem cell labeling in human subjects. Few human studies have evaluated the efficiency of intracoronary stem cell transplantation in terms of early stem cell retention and distribution. All involved patients with ischaemic heart disease in either acute (usually defined as up to 1 month after acute coronary event) or chronic phase (usually defined as at least 6 months to 1 year after an acute coronary event or heart failure of ischaemic origin). In a study using 99mTc-HMPAO as stem cell label, early myocardial retention comparable to our results was reported in patients with chronic ischaemic cardiomyopathy, ranging from 0 to 3.0% [9]; interestingly, no stem cell retention was demonstrated on delayed imaging. Similar myocardial stem cell retention rates were found using 111In (averaging to 2.5%) [10] and 18F-FDG (ranging from 0.2 to 3.3%) [11] as cell label. Higher myocardial retention rates were reported in patients transplanted in the acute phase of a coronary event, averaging from 1.5 to 6.9% [10,11,12]. Although direct comparison of results is difficult due to different stem cell subtypes and labels used in imaging studies, it appears that stem cell retention in the myocardium after intracoronary delivery is rather low, particularly in chronic cardiomyopathy. Plausible explanation for low retention rates in chronic heart failure in comparison to an acute ischaemic event is low myocardial homing signal for delivered stem cells [13], which may be even lower in non-ischaemic heart failure [14]. As early engraftment is apparently the determining factor for late functional improvement [15], methods that allow a more efficient delivery and retention of transplanted stem cells will be required. Direct intramuscular injection was already shown to be more efficient than intracoronary route in animal models and in our preliminary report in a human study [16,17]. Furthermore, significant efflux of stem cells occurs from the site of delivery, underlining the importance of early cardiac retention. The distribution of stem cells in the myocardium did not appear to change and no additional uptake was seen on delayed images as a result of potential migration and (collateral) recirculation. Similar organ distribution kinetics were also reported by other authors [9,10,11] with liver, spleen and bone marrow accumulating the vast majority of delivered stem cells and lung tissue demonstrating early uptake and fast clearance of stem cells.
4.1. Limitations Imaging approach in our study is limited by 6 h half-life of 99mTcHMPAO label. However, as the goal of the study was assessment of early stem cell engraftment, we found the 24-h imaging window to be sufficient. Moreover, in contrast to 111In label, whose half-life would allow imaging up to 7 days post transplantation, 99mTc appears to have the least effect on proliferation and migration capacity of labeled cells [18], while 111In was shown to affect stem cell viability [19]. We found no effect on stem cell viability after labeling and demonstrated good labeling stability, corroborating previous findings [10]. In addition, we found no correlation between administered activity and myocardial stem cell retention, suggesting no nonspecific binding of radiopharmaceutical in the myocardium that would account for myocardial uptake. It must be noted that subject numbers in human stem cell cardiac transplantation imaging studies, including present one, are small. Nevertheless, an important insight is provided into a promising field of cardiac therapy.
b0.001 0.213 b0.001
5. Conclusion Stem cell labeling with 99mTc-HMPAO is a feasible method for assessment of intracoronary hematopoietic stem cell transplantation in patients with idiopathic dilated cardiomyopathy.
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