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作者：Evgenios Goussetisa, Athanassios Manginasb, Maria Koutelouc, Ioulia Peristeria, Maria Theodosakia, Nikolaos Kollarosc, Evangelos Leontiadisb, Athanasios Theodorakosc, George Paterakisd, George Karatasakisb, Dennis V. Cokkinosb, Stelios Graphakosa作者单位：aStem Cell Transplant Unit; Aghia Sophia Children
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3 e. y0 L4 z9 O3 t( `6 [2 z4 { 【摘要】* g5 D" N$ d6 P* u p& z+ k' L
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 ¡À 0.32 x 107 CD133 cells and 0.75 ¡À 0.24 x 107 CD133¨CCD34 cells from 310 ¡À 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% ¡À 3.6 and 6.8% ¡À 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 CD133 and CD133¨CCD34 BM-progenitors a significant number of them are preferentially attracted to and retained in the chronic ischemic myocardium.
# K0 R1 _6 W( A9 i# ~ 【关键词】 Homing Selected bone marrow-progenitors Intracoronary infusion Chronic cardiomyopathy) n! ~1 _ Y* S( D8 M
INTRODUCTION S9 o) I' q, q' i
2 X |2 Y {9 LStem cell repair of cardiac and vascular tissue is a naturally occurring but inefficient regeneration process after myocardial infarction (MI) 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 BM-SCs 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.6 Y! U3 i. B+ E9 }) w: J. H
: b6 N: g) T0 B) }& v8 wMATERIALS AND METHODS1 H# _& F) \6 J1 B) ]6 i8 g
2 \% u, i0 Q$ [: U* |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 , the safety of administering them intracoronarilly, and their ability to adhere to the infarcted area.! }& d7 d; `, E" b6 c9 }
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Patient Population, Baseline Studies, and Clinical Follow-Up3 G7 E% L5 e! C) p# u2 X0 _
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All eight patients aged 50.5 ¡À 8.2 years were men and they had had transmural MI 45 ¡À 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 3 s# b) Q* s8 ^$ \) @6 w
3 @1 ~0 b& Y: a) l* B2 `% JTable 1. Demographics of the patients3 x5 z, f2 M2 i- f0 C9 @
# H1 G4 b. U& a) l& ~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.6 X8 `8 {/ U" D0 D
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BM-Harvesting and Cell Selection
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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 Gladbach, Germany, http://www.miltenyibiotec.com) for 30 minutes at 4¡ãC. 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¨CCD34 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¨CCD34 cells was estimated by FACS using the Coulter Epics XL-MCL device (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com).% H& D. u7 x- b% M8 z7 |
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Cell Labeling and IC-Administration
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Thirty minutes before ICI, CD133 , and CD133¨CCD34 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 vessel-wall, 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.% @( _+ G8 T8 l% a2 I }
/ p; ~& n- f: x% l4 [8 z sRESULTS
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Despite the large BM-volume aspirated under local anesthesia (310 ¡À 40 ml), all patients tolerated BM harvesting well without complications. We isolated immunomagnetically 0.8 x 107 ¡À 0.32 x 107 CD133 and 0.75 x 107 ¡À 0.24 x 107 CD133¨CCD34 cells, with a purity of 92 ¡À 4.5% and 74.2 ¡À 6%, respectively (Fig. 1). The total number of infused cells was 1.6 ¡À 0.5 x 107 progenitor cells. Incorporation of 99mTc-HMPAO in percentage of added activity yielded a mean labeling efficiency of 28.5 ¡À 4.6% after 30 minutes incubation. 99mTc-labeled cells demonstrated viability >95% as assessed by the trypan blue exclusion test, whereas a reduction of 42 ¡À 12.5% and 32.4 ¡À 10.5%, in CD133 cell expansion and colony-forming unit assay, respectively, was detected in comparison with unlabeled cells.
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Figure 1. Representative fluorescence-activated cell sorting analysis post-MACS purification of bone marrow (BM)-cells. Mononuclear BM-cells 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¨CCD34 progenitor cells (B). Both eluted cells were double-stained with both phycoerythrin-anti-CD133 and fluorescein isothiocyanate-anti-CD34 antibodies. The percentage of CD133 CD34 , CD133 CD34¨C, and CD133¨CCD34 is indicated. Abbreviations OR-FL, orange fluorescence; GR-FL, green fluorescence.
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3 a8 r$ z# S* B/ hThe 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%¡À3.6 and 6.8%¡À2.4 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 . 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.
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8 ]9 v) I8 e6 r( l/ b. T; SFigure 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).* ]& i; O) @. v8 ~! }1 p% W
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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 ¡À 7.18% at baseline to 28 ¡À 7.26% at 10 ¡À 3 months follow-up, p = .044.6 g$ L7 \: C' i7 U& S5 P
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DISCUSSION
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$ o8 D9 Z+ M& P/ s/ l6 jAfter the first open-labeled pilot trials reporting intramyocardial delivery of autologous ex vivo expanded myoblasts .
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The homing of BM-progenitor cells to the damaged myocardium, which results in cell engraftment, may play a key role 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 , whereas we found that 99mTc-HMPAO-induced damage of the in vitro proliferation-differentiation of BM-progenitors was 30%¨C40% 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., d1 _3 R3 \1 h0 o; U& `
' T0 t' C4 A$ `7 lCompared 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 . 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.# l i6 g. n2 \' [
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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 x 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¨C39% of radioactivity was detected in the infarcted myocardium 50¨C75 minutes after infusing CD34 BM cells in three patients during the early postinfarction period . 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.2 z. o4 O( j! W3 A. b) l
1 L) t6 e- Q1 U% O( |0 DOur 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.8 l$ s$ H3 g" t \7 ?
) P4 D9 o5 e Z) vDISCLOSURES
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The authors indicate no potential conflicts of interest./ Q$ U2 Q0 E9 g# r0 Q
2 O1 U9 E. s8 H5 S; A; M" LACKNOWLEDGMENTS G$ j0 k" G2 M3 g+ X+ g
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We thank G. Kontostolis and A. Kouzoumi for their assistance in performing scintigraphic imaging.
1 q; L3 F/ }; U( E 【参考文献】 Q; {. G" [& M2 B: |( F0 Y+ W4 R
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0 Y) j0 c# A1 r
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¨C1402.
. G( u6 u' S' E: v, [1 R* F6 j$ h
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¨C436.
5 I: A5 y3 @5 t9 B0 \1 ]3 g* r9 x S- ?# b
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¨C8697.
T# O% Q* k3 L( Z' z7 Q
B$ A j, p4 N" XOrlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701¨C705.
- w# f' U5 [$ J9 `- p8 O+ J7 H, d' } P$ \! k8 S
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¨C463.
6 J. r8 O( p* J- ^0 K
8 e4 E+ p+ M3 h# K6 y% r! CYoshioka 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¨C364.6 C5 b% D# v, c
) R. ]) {6 C8 I5 M0 x9 G& ?' [7 JTse HF, Kwong YL, Chan JK et al. Angiogenesis in ischemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet 2003;361:47¨C49.
* L. G6 J9 {! Q% F9 e. H v$ M* d& u- k
Perin EC, Dohmann HF, Borojevic R et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003;107:2294¨C2302.
/ B$ I; D' w) H- l' I/ {/ ?
& b/ G" E8 ~& z' H/ q% Y2 Z8 VStrauer 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¨C1918., a3 l, ^" ~0 G$ p
; T, A/ ?3 {, R4 N" B, D, F5 q
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¨C3017.
3 d8 |+ z* N3 i; T% ^/ _. ^
1 m0 O- u% @7 h6 OBritten 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¨C2218.$ Z/ h* ~5 T) B, U8 W9 @
9 Y, r$ A: l6 M7 q }0 y+ ~# c
Fern¨¢ndez-Avil¨¦s F, San Rom¨¢n JA, Garcia-Frade J et al. Experimental and clinical regenerative capability of bone marrow cells after myocardial infarction. Circ Res 2004;95:742¨C748.* U: y" k5 Y- Z* V1 _6 R
5 n/ y6 V7 p d/ L0 j; ~ p
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¨C1658.
+ G* G/ |4 ]/ {5 V2 K: n9 o& W$ H' S
7 }: y. V, [" xStamm C, Westphal B, Kleine HD et al. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet 2003;361:45¨C46.
) d2 k" Z8 |3 c4 k/ S( Q9 Y0 c' ^6 m9 k) b& D$ U9 j* w
Peters AM. The utility of 99mTc-HMPAO-leukocytes for imaging infection. Semin Nucl Med 1994;24:110¨C127.
8 f: w2 D. y4 l+ G5 p5 P# o. A& O) b C3 A/ o
Menasche P, Hagege AA, Scorsin M et al. Myoblast transplantation for heart failure. Lancet 2001;357:279¨C280.
6 \* g% x/ p& J7 b
8 [4 e7 ~8 P A. DWollert 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¨C148.
+ ]" v# U( d) X
6 @: X) P L9 b3 {1 p! HGallacher 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¨C2820.* s- {' j( o% J- w* E/ I4 Z$ p+ |
) K4 `' ]; `4 b/ z) ]! IBrenner 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¨C518.
D2 { \, O7 ], @- o+ G' Y9 J8 @% B. Y" u' g: B
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¨C868.
3 h6 ^$ u1 r' @! a6 D* V0 W' S
! l9 @# j- |# e4 f5 R$ ]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¨C2139.
" E7 _9 g. o& n$ }. R3 F9 n1 f* O
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¨C83.+ n& }1 k& S- w3 c% T1 D
& [3 J4 Z1 ~( W
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¨C703.
* f: \+ h/ ^ Q& w0 W
9 U0 a, g/ J0 {, Y6 j; jArbab AS, Yocum GT, Kalish H et al. Efficient magnetic cell labelling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood 2004;104:1217¨C1223.
/ H# A& L1 {0 u" E. ]: H9 o
/ A4 A) O! ]8 z% t2 c( iKraitchman DL, Heldman AW, Atalar E et al. In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. Circulation 2003;107:2290¨C2293.& p0 W6 t+ U, ^3 I
; W1 e. W+ I0 C# K/ }4 {
Hofmann M, Wollert KC, Meyer GP et al. Monitoring of bone marrow cell homing into the infarcted human myocardium. Circulation 2005;111:2198¨C2202.& l! b# d. h9 Z. _* O
8 a p& V) t$ k8 d$ l1 f$ S# N
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¨C336. |
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发表于 2015-6-1 08:54
