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a Center for Molecular Medicine;
b Division of Hematology, and
c Division of Cardiology, Department of Internal Medicine;
d Division of Cardiovascular Surgery, Department of Surgery; and
e Department of Anatomy, Jichi Medical School, Minamikawachi, Tochigi;
f Tsukuba Primate Center, National Institute of Infectious Diseases, Tsukuba, Ibaraki;
g Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan
Key Words. Nonhuman primate • Acute myocardial infarction • Stem cell transplantation • Genetic marking • Lentivirus vector • Plasticity • Neoangiogenesis
Correspondence: Yutaka Hanazono, M.D., Ph.D., Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan. Telephone: 81-285-58-7450; Fax: 81-285-44-5205; e-mail: hanazono{at}jichi.ac.jp
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo tracking and plastic properties of hematopoietic stem or progenitor cells have not been examined in primate cardiac ischemia. We have transplanted genetically marked autologous CD34+ cells to the ischemic myocardium in a nonhuman primate (cynomolgus macaque) model and tracked the in vivo fate of the cells. We have used CD34+ cells because the cells are widely used as a fraction of hematopoietic stem cells in clinical and nonhuman primate studies [12]. In addition, CD34+ cells contain vascular endothelial progenitor cells [7]. Thus, the present study can address the question of whether transplanted CD34+ cells really give rise to endothelial cells and cardiomyocytes in ischemic myocardium in primates.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of CD34+ Cells
Cynomolgus bone marrow (50 ml) was aspirated from the iliac crest under an isoflurane-induced general anesthesia. From the bone marrow, a nucleated cell fraction was obtained after red blood cell lysis with addition of ACK buffer (Biosource, Camarillo, CA). CD34+ cells were isolated using magnetic beads conjugated with anti-human CD34 (clone 561; Dynal, Lake Success, NY), which cross-reacts with cynomolgus CD34 [13]. The purity of CD34+ cells at harvest ranged from 90% to 95%, as assessed with another anti-human CD34 (clone 563; PharMingen, San Diego) that cross-reacts with cynomolgus CD34 [13]. The purity remained at the same levels after the 1-day transduction culture, which is discussed next.
Lentiviral Transduction
A simian immunodeficiency virus (SIV)-based lentivirus vector carrying enhanced jellyfish green fluorescent protein (GFP) (Clontech, Palo Alto, CA) was used for transduction. The vector was prepared as previously reported [14, 15]. All recombinant DNA experiments were approved by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
CD34+ cells (1 x 106) were seeded in six-well plates in 2 ml of StemSpan serum-free expansion medium (Stem Cell Technologies, Vancouver) supplemented with recombinant human thrombopoietin (100 ng/ml; Kirin, Tokyo), recombinant human stem cell factor (100 ng/ml; Biosource, Camarillo, CA), recombinant human Flt-3 ligand (100 ng/ml; Research Diagnostics, Flanders, NJ), and antibiotics (100 U/ml of penicillin and 0.1 µg/ml of streptomycin; Meiji, Tokyo). The cells were transduced twice each for 12 hours (total, 24 hours) with the SIV vector at 50 transducing units per target cell. After transduction, cells were cryopreserved with 10% dimethylsulfoxide (Wako, Osaka, Japan) and 1% Dextran 40 (Yoshitomi, Osaka, Japan) in a controlled-rate programmable freezer (Kryo 10; Planer Biomed, Middle-sex, UK) until transplantation. The viability of cells after thawing was 53.0 ± 6.5%, as assessed by trypan blue staining. An aliquot of transduced cells was assessed for GFP expression at 48 hours after transduction by flow cytometry using a FACScan (Becton Dickinson, Franklin Lakes, NJ) with excitation at 488 nm and fluorescence detection at 530 ± 30 nm.
In Vitro Endothelial Differentiation
CD34+ cells were seeded on fibronectin-coated plates (Becton Dickinson) in M199 medium (Invitrogen, Carlsbad, CA) with 20% fetal calf serum and bovine pituitary extracts (Invitrogen) as previously described [7]. After 7 days in culture, cells were examined for the uptake of DiI-acetylated low-density lipoprotein (LDL) and for the expression of CD31, von Willebrand factor (vWF), vascular endothelial (VE)-cadherin, and vascular endothelial growth factor receptor (VEGFR)-2. Briefly, adherent cells were incubated with 1 µg/ml of DiI-acetylated LDL (Molecular Probes, Eugene, OR) for 4 hours at 37°C. For immunofluorescence staining, after fixation in ice-cold 4% paraformaldehyde for 10 minutes and blocking in 1% bovine serum albumin (BSA) for 15 minutes, cells were incubated with a primary antibody: mouse anti-human CD31 (VM-59; Becton Dickinson), rabbit anti-human vWF (DakoCytomation, Glostrup, Denmark), mouse anti-human VE-cadherin (55-7H1; Becton Dickinson), or rabbit anti-mouse VEGFR2 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature. Cells were then incubated with a secondary antibody, Texas red–conjugated horse anti-mouse immunoglobulin G (IgG) (Vector, Burlingame, CA) or goat anti-rabbit IgG (Vector) for 30 minutes at room temperature.
Myocardial Infarction and Transplantation
All operations on cynomolgus monkeys were performed under an isoflurane-induced general anesthesia. Thoracotomy was conducted, the pericardium was opened, and the left anterior descending coronary artery was ligated with 5-0 prolene sutures. One to 2 hours after the ligation, GFP-transduced, autologous CD34+ cells in normal saline were injected with a microsyringe through a 27-gauge needle into 10 sites (5 µl/site) in the peri-ischemic zone. In the control group, saline alone was injected in the same way. The pericardium and chest were closed. The animals then received butorphanol tartrate (0.5 mg/kg, intramuscularly) daily for 5 days to alleviate the pain associated with the operation and myocardial infarction.
Echocardiography
Echocardiographic imaging was obtained using a Sonos 5500 system (Philips Medical Systems, Andover, MA) before transplantation and at 2 weeks after transplant. The echocardiography was conducted by independent technicians irrelevant to our study group. In one animal (BM97080), it was additionally performed at 12 weeks. Short-axis two-dimensional images at the midpapillary level of the left ventricle were stored, and percent fractional shortening (%FS) was calculated to assess cardiac function.
Myocardial contrast echocardiography (MCE) was performed at day 0 (just before transplantation) and at 2 weeks after transplant to assess regional blood flow and blood flow defect size. In one animal (BM97080), chronic assessment was performed at 12 weeks after transplant. The electrocardiograph-triggered end-systolic intermittent imaging was conducted in short-axis views at incremental pulsing intervals (triggering intervals of 1, 2, 3, 4, and 8 beats) using an S12 probe. Once optimized, the settings of depth (4 cm), mechanical index (0.9), and focus (3 cm) were fixed. The contrast agent (perflutren; Yamanouchi, Tokyo) consisted of lipid-coated microbubbles of perfluorocarbon [16]. Perflutren diluted with saline (1:10) was administered intravenously at a constant rate (0.01 ml/kg per min). For the assessment of regional blood flow, MCE images were analyzed using ORIGIN 6.0J (Lightstone, Tokyo), and the blood flow was calculated as previously described [17]. Data are presented as a blood flow ratio (the periinfarct versus nonischemic control region or the infarct versus nonischemic control region). For the assessment of blood flow defect, MCE images obtained at triggering interval of four beats were analyzed using National Institutes of Health Image software (version 1.61). Data are presented as percent defect compared with the total blood flow.
Microspheres
Colored microspheres (15 µm ± 2% diameter; E-Z Trac, Los Angeles) were used to evaluate regional blood flow 2 weeks after transplant [18], with the exception of one animal (BM97080), in which evaluation was performed 12 weeks after transplant. A set of microspheres (2 x 106) was diluted in 2 ml of saline and injected into the left ventricle over 30 seconds. A reference blood sample was withdrawn at a constant rate of 5 ml/min through the femoral artery. After the collection of blood samples, monkeys were irrigated with saline for mercy killing and blood was completely washed out. The heart was excised from each monkey. Tissue samples from the infarct, peri-infarct, and nonischemic regions (one sample per region) were digested, microspheres were collected, and the blood flow was calculated according to the manufacturer’s instructions. Data are presented as blood flow ratio (the peri-infarct versus nonischemic control region or the infarct versus nonischemic control region).
Immunohistochemistry
Tissue samples from the infarct, peri-infarct, and nonischemic regions at 2 weeks after transplant were embedded in optimal cutting temperature compound (Sakura, Zoeterwoude, Netherlands) and frozen in liquid nitrogen. Sections were prepared (6 µm), fixed for 10 minutes at 4°C in 4% paraformaldehyde in phosphate-buffered saline (PBS), and blocked with 1% BSA in PBS. The sections were incubated at room temperature with a primary antibody, monoclonal mouse anti-human CD31 (1:200; Becton Dickinson), followed by a secondary antibody, biotin-conjugated horse anti-mouse IgG (1:500; Vector). The sections were then treated with avidin-alkaline phosphatase (ABC AP kit; Vector) for 30 minutes. The reaction was developed with a Vector Red substrate kit (SK-5100; Vector). In the case of double staining of CD31 and GFP, the above sections were further incubated with polyclonal rabbit anti-GFP (1:200; Clontech) followed by biotin-conjugated anti-rabbit IgG (1:500; Vector) and treated with avidin-peroxidase (ABC Elite kit; Vector). The reaction was developed with a Vector SG substrate kit (SK-4700; Vector). The sections were counterstained with hematoxylin, mounted in glycerol, and examined under a light microscope.
In Situ Polymerase Chain Reaction
In situ detection of transduced cell progeny was performed by amplifying proviral sequences as previously reported [19]. The following primer set for the GFP gene was used: 5'-CGT CCA GGA GCG CAC CAT CTT C-3' and 5'-GGT CTT TGC TCA GGG CGG ACT-3'. The polymerase chain reaction (PCR) mixture consisted of 420 µM dATP, 420 µM dCTP, 420 µM dGTP, 378 µM dTTP, 42 µM digoxigenin-labeled dUTP (Roche, Mannheim, Germany), 0.8 µM of each GFP primer, 4.5 mM MgCl2, 1 x PCR buffer (Mg2+ free), and 4 U of Takara Taq DNA polymerase (Takara, Kyote). Sections were prepared with a Takara slide frame (Takara) from the infarct, peri-infarct, and nonischemic regions at 2 weeks after transplant. PCR was performed using a PTC100 thermal cycler (MJ Research, Watertown, MA) with the following conditions: 94°C for 1 minute and 57°C for 1 minute with 10 cycles. The digoxigenin-incorporated DNA fragments were detected using horseradish peroxidase (HRP)-conjugated rabbit F(ab') anti-digoxigenin antibody (DakoCytomation). The sections were then stained for HRP using a Vector SG substrate kit (Vector). Finally, the sections were counterstained with a Kernechtrot solution (Muto, Tokyo) that stains nucleotides, mounted in glycerol, and examined under a light microscope.
ELISA
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) levels in tissue lysate or medium were assessed by ELISA (R&D Systems, Minneapolis) according to the manufacturer’s instructions. Tissue lysate was obtained from the peri-infarct region (three samples from each monkey) at 2 weeks after transplant. Briefly, tissue was homogenized and suspended in lysis buffer containing 10 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 150 mM NaCl, and protease inhibitor cocktail tablets (Complete Mini, Roche). The suspension was rocked at 4°C for 20 minutes and centrifuged at 16,000g and 4°C for 30 minutes. The supernatant was used for ELISA. The protein concentration of lysate was determined with DC Protein Assay (Bio-Rad, Hercules, CA).
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RESULTS |
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). Before transplantation, CD34+ cells were genetically marked with GFP using an SIV-based lentivirus vector. The ex vivo transduction results are summarized in Table 1. The transduced cells were frozen until transplantation. An aliquot of the transduced cells was examined in vitro for the endothelial differentiation ability. After the differentiation culture, a vessel-like structure was observed (Fig. 1A). The ability of cells to take up DiI-acetylated LDL and the expression of CD31, vWF, VE-cadherin, and VEGFR-2 suggested the endothelial lineage (Fig. 1B). We and others have already confirmed the ability of hematopoietic differentiation of the cells [20, 21]. Taken together, the SIV-mediated GFP gene transfer does not spoil the differentiation abilities of CD34+ cells. In addition, on average, 41% of cells fluoresced 48 hours after transduction, and 56% of endothelial cells still fluoresced after in vitro differentiation (Table 1), showing that the GFP expression is stable during the in vitro differentiation to endothelial cells. Thus, GFP was expected to serve as a good genetic tag after transplantation.
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One of the control monkeys (CTR01061 died of heart failure 5 days after myocardial infarction, and the other control monkeys showed a decrease in %FS at 2 weeks after infarction (Fig. 2). Thus, all four control animals showed the deteriorated cardiac function. In the cell-treated group, one monkey (*, BM01051) underwent ventricular fibrillation immediately after the ligation and survived after cardiopul-monary resuscitation but eventually developed a ventricular aneurysm. Only this animal showed a decrease in %FS despite CD34+ cell treatment; the other animals receiving CD34+ cells showed an increase in %FS (Fig. 2). CD34+ cell treatment may not be able to rescue such a heavily impaired heart but otherwise had a significant effect on cardiac function. Even an old monkey (BM90047, Table 1
) showed improved %FS.
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) and colored microspheres (Fig. 3B). An excellent correlation was found between the two methods (Fig. 3C; correlation coefficient = 0.93). Two groups (CD34+ cell–treated and saline-treated) were well separated on the panel, showing an obvious positive effect of CD34+ cell injection on the blood flow in the peri-infarct zone after acute myocardial infarction. In fact, the average myocardial blood flow in the peri-infarct region in the absolute value was 0.988 ml/g per minute and 0.383 ml/g per minute for the cell-treated and saline-treated groups, respectively. Of note, the blood flow in the peri-infarct zone was ameliorated even in the animal with a ventricular aneurysm. On the other hand, the relative blood flow in the infarct to nonischemic region did not show a significant difference between the CD34+ cell–treated and saline-treated groups. The peri-infarct region was the injection site, and thus the highest degree of change would be expected there.
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In Vivo Tracking of Transplanted Cells
Two weeks after the transplantation, tissue sections were prepared from the infarct, peri-infarct, and nonischemic regions. Immunostaining of an endothelial marker CD31 demonstrated more vessels in the peri-infarct region of the CD34+ cell-treated than saline-treated myocardium (Fig. 4A). In fact, the capillary density of the peri-infarct region was significantly better preserved in the cell-treated than saline-treated group, although there was no significant difference in the capillary density of the nonischemic control regions between the two groups (Fig. 4B).
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). The result clearly indicates that at least some transplanted CD34+ cells gave rise to endothelial cells. However, we found that the transplanted cell progeny accounted for only a small fraction of endothelial cells after examining more than 100 sections of the peri-infarct region. In situ PCR for proviral GFP sequences also showed that few CD31-positive endothelial cells contained the GFP-provirus (Fig. 5B). There were no GFP-positive cardiomyocytes in more than 100 sections. Most of the transplanted cell progeny were found not incorporated in vessels (Fig. 5C). Hematoxylineosin staining did not show any noncardiac tissue regeneration in the myocardium.
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). In addition, in vivo VEGF levels in the peri-infarct tissue were significantly higher in the CD34+ cell–treated than saline-treated group (Fig. 6B, left), although in vivo levels of bFGF differed little between the two groups (Fig. 6B, right).
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DISCUSSION |
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As a result of this marking study, we found only a few GFP-positive cells incorporated into the vascular structure in the ischemic myocardium at 2 weeks after transplantation. GFP-positive cardiomyocytes were not detectable. The existence of GFP-positive endothelial cells can be explained by fusion events [27, 28]. However, if that is the case, GFP-positive cardiomyocytes should have also been detected, given that cardiomyocytes are even easier targets of fusion than endothelial cells [11, 29]. Whether fusion occurred or not, only a few transplanted cells gave rise to nonhematopoietic cells in our primate model.
There are several possible explanations for the very low prevalence of transplanted cell–derived endothelial cells or cardiomyocytes in the ischemic myocardium. First, 2 weeks was too short or the number of transplanted cells was too small to see the nonhematopoietic differentiation. However, the cardiac function and regional blood flow were ameliorated by this time point and with this number of transplanted cells. Thus, if transplanted cell–derived, non-hematopoietic differentiation was a reason for the improvement, transplanted cells at this number should have given rise to such cells by this time point. In fact, Orlic et al. [8] observed transplanted cell-derived endothelial cells and cardiomyocytes within 11 days after transplant in mice. In addition, we observed the endothelial differentiation from CD34+ cells within 7 days in vitro (Fig. 1). However, we cannot formally rule out a possibility that inflammatory responses after generation of infarction might have negative effects on engraftment of transplanted cells. Second, the SIV vector failed to transduce stem or progenitor cells that might be responsible for nonhematopoietic differentiation. Even if the transduction was successful, the cytokine treatment during the transduction or GFP expression in the cells spoiled the differentiation abilities. However, we have shown that the SIV vector successfully transduced cells that were capable of differentiating into GFP-expressing endothelial cells (Fig. 1). We have not examined the differentiation ability to cardiomyocytes, because the method to differentiate CD34+ cells to cardiomyocytes in vitro has not been well established. Thus, we cannot formally rule out the possibility that the ex vivo culture spoiled the ability to differentiate to cardiomyocytes or reduced the ability to differentiate to endothelial cells. Third, cells expressing xenogeneic GFP were rejected via immune responses. However, 2 weeks is too short to allow immune elimination of GFP-expressing cells in monkeys [30, 31]. Fourth, the GFP expression was shut down because of transcriptional silencing in vivo, resulting in negative immunostaining with anti-GFP. To examine this possibility, we tried to detect the provirus (vector integrated into genome) in the cardiac tissue by in situ PCR and found again that only a few CD31-positive endothelial cells contained the GFP-provirus (Fig. 5B), thus arguing against transcriptional silencing-based negative immunostaining with anti-GFP. Taken together, we concluded that most transplanted cell progeny were not incorporated into the repaired, nonhematopoietic tissues.
Our results are in agreement with recent reports that transplanted hematopoietic cells are unable to transdifferentiate into nonhematopoietic cells in ischemic myocardium in mice [9–11]. Our studies confirm and extend these findings in a couple of ways. First, we show that the cardiac function can be indeed significantly improved after injection of hematopoietic cells in a nonhuman primate model, although the above studies used murine myocardial infarction models and did not address the potential beneficial effects of hematopoietic cell injection. Second, the improvement is unlikely to be the result of generation of transplanted cell–derived endothelial cells or cardiomyocytes. Finally, we have found that cultured CD34+ cells secrete VEGF and that the CD34+ cell–treated myocardium contains a significantly higher level of VEGF than the saline-treated myocardium. This observation raises a possibility that some angiogenic cytokines secreted from transplanted cells (paracrine effects) potentiate angiogenic activity of endogenous cells. VEGF would be a candidate. Despite this, the delivery of a single agent (VEGF) failed in clinical trials for cardiac ischemia [32]. In situ multiple cytokine production and coordinated action may be essential for clinical benefits [33, 34]. It will be important to explore and identify cytokines responsible for the paracrine effect. If transplanted cells serve as cytokine factories rather than stem cells in ischemic tissues, it is not surprising that not only stem cells but other types of cells may also work [35]. The concept of stem cell therapeutics for ischemic diseases needs additional consideration.
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ACKNOWLEDGMENTS |
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