Human CD34+ Cells in Experimental Myocardial Infarction
Long-Term Survival, Sustained Functional Improvement, and Mechanism of Action
Rationale: Human CD34+ cells have been used in clinical trials for treatment of myocardial infarction (MI). However, it is unknown how long the CD34+ cells persist in hearts, whether the improvement in cardiac function is sustained, or what are the underlying mechanisms.
Objective: We sought to track the fate of injected human CD34+ cells in the hearts of severe combined immune deficiency (SCID) mice after experimental MI and to determine the mechanisms of action.
Methods and Results: We used multimodality molecular imaging to track the fate of injected human CD34+ cells in the hearts of SCID mice after experimental MI, and used selective antibody blocking to determine the mechanisms of action. Bioluminescence imaging showed that injected CD34+ cells survived in the hearts for longer than 12 months. The PET signal from the injected cells was detected in the wall of the left ventricle. Cardiac MRI showed that left ventricular ejection fraction was significantly improved in the treated mice compared to the control mice for up to 52 weeks (P<0.05). Furthermore, treatment with anti-α4β1 showed that generation of human-derived cardiomyocytes was inhibited, whereas anti–vascular endothelial growth factor (VEGF) treatment blocked the production of human-derived endothelial cells. However, the improvement in cardiac function was abolished only in the anti-VEGF, but not anti-α4β1, treated group.
Conclusions: Angiogenesis and/or paracrine effect, but not myogenesis, is responsible for functional improvement following CD34+ cells therapy.
Adult progenitor cells have been used to treat patients with acute myocardial infarction (MI) or chronic ischemic cardiomyopathy with variable success.1–8 Nonlabeled autologous progenitor cells were used in these clinical trials. Hence, it was hard to track the fate of the transplanted cells in the treated hearts even postmortem.3 In animal studies, it is easier to track the transplanted human progenitor cells via the xenogeneic differences between the transplanted cells and the surrounding cardiac tissues. We have injected human CD34+ cells into the hearts of SCID mice after experimental MI and used HLA as a marker to identify the transplanted cells.9–11 Others have used GFP, sex mismatch, or allelic differences to track the transplanted cells.12 Such methodology is not dynamic and requires euthanasia of the animals to gain a single data point. To perform noninvasive monitoring of the transplanted cells, some laboratories have labeled the transplanted cells with iron oxide magnetic particles or superparamagnetic nanoparticles and followed them with MRI.13,14 Activated macrophages, however, can engulf iron oxide–labeled stem cells, making it difficult to interpret the data.15 Thus, it is important to develop more reliable way to track transplanted cells in animal studies.
The advancement of molecular imaging techniques provides tremendous potential to identify noninvasively the location, magnitude, and duration of cellular survival and fate of the transplanted cells. In principle, this approach requires the introduction of a reporter gene into the transplanted cells. A convenient reporter is firefly-Luciferase (f-Luc), an enzyme that cleaves d-luciferin and generate photon, which can be monitored by a bioluminescence detector.16 This method has been highly successful in small animal models because of the relatively short distance between the photon emitting cells and the detectors. However, this technique will not likely work in larger animals or humans because of tissue attenuation, scattering, and of the difficulty in localizing these signals inside larger bodies.
To track transplanted cells reliably in larger animals or in humans, we and others have developed PET reporter gene human herpes simplex virus type 1-thymidine kinase (HSV1-tk).17 HSV1-tk is essentially nontoxic in humans, and is currently being used in clinical gene therapy protocols as a “susceptibility” gene for treatment of cancer (in combination with ganciclovir). Importantly, the acquired sensitivity of HSV1-tk–transduced cells to ganciclovir provides an additional margin of safety because the genetically modified cells can be easily eliminated by treatment with ganciclovir. In this study, we constructed a triple fusion (TF) reporter vector encoding for enhanced-GFP (e-GFP, for cell selection), f-Luc (for bioluminescence imaging [BLI]), and HSV1-tk (for PET imaging). Using this reporter along with MRI and CT, we sought to determine the localization and persistence of the labeled CD34+ cells transplanted into living animals after experimental MI. Furthermore, we investigated whether these transplanted cells contributed to improvement in cardiac function, and what are the underlying mechanisms.
The mechanisms by which the transplanted progenitor cells benefit the cardiac function remain obscure. We have previously shown that adult human peripheral blood CD34+ cells injected into SCID mice transform into cardiomyocytes, endothelial cells, and smooth muscle cells in hearts injured by experimental MI.9 We have also demonstrated that most human-derived cardiomyocytes result from fusion of human CD34+ cells with mouse cardiomyocytes, whereas endothelial cells are directly differentiated from human CD34+ cells.11 Furthermore, fusion of CD34+ cells with cardiomyocytes is mediated by the interaction of VCAM-1 and α4β1, after which the fused cells re-enter the cell cycle and become new cardiomyocytes.10 The biological relevance of this finding is that myogenesis can be blocked by antibodies against VCAM-1 or α4β1 but not by antibody against vascular endothelial growth factor (VEGF). On the other hand, angiogenesis can be blocked by anti-VEGF, but not by anti-α4β1 antibodies. Thus, myogenesis can be distinguished from angiogenesis on the basis of selective antibody blockade and the relative contribution of myogenesis and angiogenesis to cardiac repair can be determined in the SCID mouse MI model.
All animal studies were approved by the Institutional Animal Care and Use Committees of the University of Texas-MD Anderson Cancer Center. Female SCID mice (C3H, The Jackson Laboratory, Bar Harbor, Me), weighing 16 to 20 g were used.
Construction of Lentivector
We excised a TF reporter gene encoding HSV1-tk, e-GFP, and f-Luc (termed as TGL)18,19 from pBluescipt II SK+ using SalI and BamHI and ligated into pENTRIA (Invitrogen, Carlsbad, Calif) generating pENTR1A-nesTGL. This vector was then recombined with a self-inactivating lentiviral destination vector encoding the human ubiquitin promoter according to the specifications of the manufacturer (Invitrogen). Lentiviral packing, concentration, and titer were performed as previously described.20,21
Isolation and Transduction of CD34+ Cells
Induction of MI and Transplantation of Human CD34+ Cells Into the Mice
After anesthesia (3% isoflurane and oxygen) and mechanical ventilation, the left anterior descending artery was ligated. 10 minutes later, we injected 1×106 CD34+ cells in 25 μL of saline directly into the periinfarcted areas. Control mice were injected with saline.
In Vitro and In Vivo BLI of f-Luc Gene Expression in CD34+ Cells
The cells were serially diluted and seeded on a 24-well plate; d-luciferin (Caliper LS, Alameda, Calif) was added at 1 μg/mL to the media and luminescent signal was measured using IVIS 200 (Caliper LS). In vivo BLI was determined 10 minutes after IP injection of d-luciferin (150 mg/kg). We manually defined regions of interest to measure signal intensities, expressed as photons/sec·cm−2×4π (photon flux).
Small Animal MRI
A 7.0 T Biospec small animal scanner (Bruker Biospin Inc, Billerica, Mass) was used. Imaging gradients with 60 mm inner diameter were used with a 35 mm inner diameter linear birdcage-style volume resonator. T1-weighted anatomic reference images were acquired using a 3D fast low-angle shot gradient echo sequence. A retrospectively gated fast low-angle shot pulse sequence was used to acquire cardiac cine images exhibiting excellent contrast between bright blood and adjacent myocardium. To measure volumetric left ventricular ejection fraction (LVEF), at least 6 short axis images were scanned at 1 mm interval from the apex to the base of the heart. End-diastolic (ED) and end-systolic (ES) left ventricular volumes were obtained by the biplane area length method, and percent LVEF was calculated with the equation (ED−ES)/ED]/100.21
A micro-CT (RS-9 tabletop CT scanner, General Electric Medical Systems, London, Ontario) was used. We took 360° at 1° increments, with each projection consisting of a 500-ms x-ray exposure. We then used a cone-beam back-projection reconstruction method, after normalizing and correcting the raw projection images for bad detector pixels. The CT value grayscale from the initial reconstructed raw data were then calibrated into Hounsfield units by sampling air, water, and bone material standards positioned within the field of view of the image.
Micro-PET R4 scanner (Concorde Microsystems, Knoxville, Tenn) was used. PET imaging was performed 2 hours after intravenous administration of the radiolabeled nucleoside analog [18F]-labeled 2′-deoxy-2′-fluoro-5-methyl-1-β-d-arabinofuranosyluracil ([18F]FEAU), which was synthesized with high specific activity as previously described.24 The data acquisition, image reconstruction, and [18F]FEAU-derived radioactivity quantification were performed as previously described.17
Coregistration of MRI, CT, and PET Images
To accurately coregister images, we used a chamber that was manufactured in-house. We registered PET to the reference anatomic CT through the coregistration chamber’s marker set, and this was further refined through a normalized mutual information cost function. We relied on an indirect MRI to CT registration process to register MRI and PET together. First, relatively high resolution MRI was registered to the reference CT via a normalized mutual information cost function.25–27 Independently, the diastole phase of a high planar- but low axial-resolution gated MRI data set was registered to the low resolution MRI of the same animal via a simpler correlation cost function.26 The geometric transformations (eg, PET to CT, CT to MRI, and MRI to gated MRI) were finally combined together to register gated MRI slices to PET images via the CT surrogate. Gated MRI slices thus provided the fine anatomy details that permitted the exact localization of the [18F]FEAU-PET uptake in relation to the myocardium and the engraftment. After data were retrieved from the scanners, all processing and visualization paradigms28,29 were achieved through an ad hoc hybrid combination of commercial platforms and in-house developments.
In Vivo Antibody Blocking of Myogenesis and Angiogenesis
We designed an experimental protocol (Figure 4A) based on our previous observation.10 Four groups of mice received anti-α4β1 or anti-VEGF antibodies or their respective isotype-matched control antibodies. The fifth group of mice received cells only, and the sixth group received neither cells nor antibodies. At the end of the study, hearts were removed and cellular subsets were analyzed with fluorescence-activated cell sorting (FACS) to ascertain the effects of antibody treatments. Antibodies were administered to the animals by IP injection at a concentration of 50 μg/mouse in 0.3 mL PBS 30 minutes before injected with CD34+ cells.
Preparation of Single-Cell Suspension From the Heart and Cell Staining for FACS
Single cell suspension was obtained by enzyme digestion. Isolated cells were washed, fixed, permeabilized, and incubated with monoclonal anticardiac troponin T (1:200, clone 1A11; Advanced Immunochemical), or with a polyclonal anti-VE cadherin (1:100, Bender MedSystems) for 30 minutes at 4°C. The secondary antibodies were conjugated with Alexa Fluor 633 (Molecular Probes), or conjugated with Alexa fluor 488, respectively. After 3 washes, all cells were incubated with antihuman HLA-ABC conjugated with PE (Cedarlane Laboratories) for 30 minutes before FACS.
5 -micrometer serial sections were collected on slides, fixed with 3.7% paraformaldehyde (pH 7.4) at 4°C for 5 minutes, rinsed in PBS 3 times, and blocked at room temperature for 30 minutes in PBS containing 5% horse serum. Slides were then incubated with primary antibodies at room temperature for 1 hour, rinsed 3 times, and incubated with the secondary antibodies at room temperature for 30 minutes. The slides were rinsed in PBS 3 times again and sealed with a mounting medium containing DAPI (Vector Laboratories). Antibodies used were: anti–HLA-ABC (Cedarlane Laboratories); anticardiac troponin T (Abcam); anti-CD31(Abcam). Secondary antibodies (Invitrogen) were Alexa fluor 488–conjugated goat anti-mouse IgG; Alexa fluor 568 rabbit anti-rat; and Alexa fluor 568 donkey anti-rabbit.
Student t test was used to compare the means of 2 groups. When 3 or more means were compared, 1-way ANOVA followed by multiple comparisons among means was used. All data collection and analyses were performed in a blinded fashion.
Efficiency of Lentiviral Transduction of CD34+ Progenitor Cells In Vitro
We used e-GFP to monitor TGL expression efficiency in transduced CD34+ cells. The average transduction efficiency was 38.2±4.3% at multiplicity of infection of 50. A linear correlation was observed between transducted cell numbers and photon flux (Online Figure I, available at http://circres.ahajournals.org).
In Vivo BLI Revealed Long-Term Persistence of Transplanted CD34+ Cells After MI
We performed in vivo noninvasive BLI in SCID mice injected with TGL-expressing CD34+ cells to monitor cell localization and survival (measured as bioluminescent activity) over 12 months in living animals subjected to MI. We observed a bioluminescent signal only in the chest overlying the hearts (Figure 1A), suggesting that transplanted CD34+ cells did not migrate to peripheral organs in sufficient numbers to be detected. The change in photon flux over time in hearts is shown in Figure 1B. Three days after injection, maximum photon flux was observed (2.98±0.31 x105), this decreased to 7.20±0.13×104 by week 25, and maintained at that level for greater than 1 year (Figure 1B). Photon flux was 2 to 3 fold higher during the first 2 weeks after cell injection (P<0.01). This suggests that transplanted CD34+ cells had multiplied. Alternatively, the protein expression of f-Luc may have increased following transplantation. Importantly, no signal above the noise level was observed from other organs in both the experimental and the control mice, indicating that no teratoma was induced by the transplantation of the transduced CD34+ cells.
Coregistration of MRI, Micro-CT, and Micro-PET Images Confirmed the Localization of Transplanted CD34+ Cells
Through the coregistration of MRI/CT/PET images (Figure 2 and Online Video I), we can precisely localize the transplanted CD34+ cells in the periinfarction area. PET was used to localize the TGL-expressing CD34+ cells using [18F]FEAU as the substrate for HSV1-tk. Phosphorylation of [18F]FEAU by HSV1-tk retained the radio-labeled PET probes inside the transplanted CD34+ cells and can be imaged by a PET scanner. As previously reported, nonspecific [18F]FEAU uptake was observed in the gallbladder and kidneys.30 As shown in Online Video I, there was no PET signal in the cardiac region in a sham-operated mouse. However, PET signals were clearly visible in the cardiac region in a CD34+ cell transplanted mouse (Online Video II).
Improvement in Cardiac Function in Infarcted Hearts After Transplantation of CD34+ Cells
We used MRI to evaluate the therapeutic benefit of CD34+ cell transplantation into the infarcted hearts (Figure 3 and Online Video III). ES and ED volumes, measured for up to 52 weeks, were used to calculate the LVEF (Figure 3A). One day and 3 days after MI, the decreases in EF were similar in both the control and cell-treated groups (Figure 3B). These results suggest that our MI protocol was carried out consistently and achieved a similar degree of cardiac damage in both the control and the cell treated group. The EF began to diverge in a statistically significant way between the control and cell-treated group 1 week following MI and improvement in LVEF in the cell treated animals was sustained up to 52 weeks.
Angiogenesis and/or Paracrine Effect Is Responsible for Functional Improvement
To the mechanism of action, we used FACS to analyze cellular subsets in the hearts 2 months after MI. Figure 4B shows that between 0.78% and 0.88% of all cardiac cells in the anti-VEGF antibody and isotype control groups were HLA+/troponin T+ cells. However, anti-α4β1 antibody reduced the percentage of HLA+/troponin T+ cells to 0.13%. Thus, myogenesis was effectively blocked by the anti-α4β1 antibody but not by the anti-VEGF antibody. On the other hand, only the anti-VEGF antibody reduced the percentage of HLA+/VE-cadherin+ cells to 0.05% (Figure 4C). The FACS results were also supported by immunohistochemical staining of cardiac sections (Online Figures II and III).
Cardiac MRI performed at the end of the study showed that the LVEF had dropped to 28% from the baseline value of 51% in the group of mice that did not receive cells (Figure 4D). However, mice that received cells had less of a drop in LVEF (from 50% to 37%). So, cell therapy produced a 10% net improvement in LVEF. In contrast, anti-α4β1 antibody did not reduce the decline in LVEF compared with the LVEF seen in isotype-matched control mice (from 50% to 36%). Interestingly, anti-VEGF antibody completely blocked the improvement in LVEF (from 50% to 25%) seen in the CD34+ cell treatment group. To further understand the effect of our treatment protocol on angiogenesis, we ascertained the number of CD31+ vessels in the periinfarct zone. As shown in Online Figures IV and V, the number of CD31+ vessels in the anti-VEGF treated hearts was similar to the hearts that did not receive cell therapy, whereas other groups showed a significant increase in the number of CD31+ vessels. Thus, our results suggest that angiogenesis and/or paracrine effect, but not myogenesis, is responsible for improving cardiac function following cell therapy.
A variety of adult progenitor cells have been used as cellular therapy for acute MI.2 Kawamoto et al reported that CD34+ cells purified from peripheral blood mononuclear cells can preserve myocardial integrity and function after MI.31 Using similarly prepared human CD34+ cells in a mouse model of experimental MI, we demonstrated that transplanted CD34+ cells became cardiomyocytes, endothelial cells, and smooth muscle cells. Thus, CD34+ cells have the potential to be a clinically useful adult progenitor cells for cardiac repair. Recently, Losordo et al published the results of a phase I/IIa study using autologous CD34+ cells for intractable angina, showing clinical improvements in patients treated with CD34+ cells for up to 6 months.32 Our study demonstrates that human CD34+ cells persist in the heart for up to 12 months following injection into the periinfarct zone of SCID mice, and furthermore, the improvement in LVEF is also preserved for at least 6 months. Thus, these results suggested that transplanted CD34+ cells may similarly persist in the clinical setting, a finding which may correlate with myocardial repair.
It is difficult to track the fate of injected cells in clinical studies. Hofmann et al have labeled non purified bone marrow cells with 2-[18F]-fluoro-2-deoxy-d-glucose ([18F]FDG) and infused them into the infarct-related coronary artery. Fifty to 75 minutes after cell transfer, 1.3% to 2.6% of [18F]FDG-labeled bone marrow cells were detected in the infarcted myocardium using 3D PET imaging. Whereas after transfer of [18F]FDG-labeled CD34-enriched cells, 14% to 39% of the total activity was detected in the infarcted myocardium.33 However, this method cannot be used to track the long-term fate of injected CD34+ cells.
Efficient expression of a given reporter is critical for in vivo tracking transplanted cells using live animal imaging techniques. This is particular critical for multimodality imaging using a fusion of multiple reporter genes because the fusion tends to decrease activity of each reporter. Lentivirus can effectively introduce and integrate reporter genes into the genome of CD34+ cells, achieving long-term and stable reporter gene expression. In this study, we constructed lentivectors expressing the TGL reporter gene driven by human ubiquitin promoter. Remarkably, the transducted cells with these vectors survived in the infarcted hearts for longer than 1 year. We have also confirmed that the transplanted CD34+ cells contribute to restoring cardiac function in the long-term. The profile of LVEF recovery is comparable with results reported elsewhere.21,34 These findings validate that multimodality molecular imaging is a powerful method for tracking progenitor cells in vivo.
BLI enabled us to determine the location, magnitude, survival, and especially the long–term time-spatial kinetics of the transplanted CD34+ cells. BLI, however, is not clinically applicable because of several limitations (see introduction). To generate a clinically applicable molecular imaging protocol, we included the HSV1-tk gene in the TF reporter gene and used [18F]FEAU-PET concomitant to BLI to monitor the transplanted CD34+ cells.17,30,35 Micro-PET imaging displayed similar tempo-spatial kinetics for HSV1-tk gene expression as BLI. The coregistered MRI/CT/PET images provided us with detailed morphological picture of CD34+ cell therapy. This paradigm can be applied clinically to track the fate of progenitor cell therapy in humans.
Previous studies have shown that several factors contribute to improvement in cardiac function, including the formation of new muscle cells and blood vessels9–11,36 from the transplanted cells. In addition, the secretion of cytokines from the transplanted cells can have a paracrine effect on improving cardiac function. Here, we showed that angiogenesis and/or paracrine effect, rather than myogenesis, is responsible for the functional improvement. Following the injection of 1 million CD34+ cells to the periinfarct region, we have consistently observed that approximately 0.8% to 0.9% of total cardiac cells were HLA+/troponin T+. One would assume that these human-derived cardiomyocytes contributed to the improvement in cardiac function following cell therapy. However, when the formation of the human-derived cardiomyocytes was blocked by anti-α4β1, the gain in LVEF persisted. Thus, myogenesis appears not to play a significant role in the improvement in cardiac function following CD34+ cell therapy in our model. Our finding that anti-VEGF treatment, which abolished the formation of human-derived endothelial cells and the paracrine effect mediated by VEGF, abrogated the improvement in cardiac function further suggests that angiogenesis3 and/or paracrine effect37 play a critical role in the improvement in cardiac function in this murine model.
It was shown that anti-α4β1 antibody mobilizes cardiac progenitor cells from the bone marrow to enhance cardiac repair after MI.38 Thus, anti- α4β1 antibody treatment could be a double-edged sword that, on the one hand, blocks the formation of human-derived cardiomyocytes but, on the other hand, recruits mouse progenitor cells from the bone marrow to enhance cardiac repair. We think this is unlikely based on the data shown in Figure 4E. In this experiment, animals with experimentally induced MI did not receive cell therapy. Instead, they were treated with either anti- α4β1 or anti-VEGF antibody over a 2-month period and neither anti- α4β1 nor anti-VEGF antibody altered the drop in LVEF following MI. Thus, it is unlikely that in our study the anti- α4β1 antibody enhanced the LVEF that compensated for the drop in LVEF attributable to blocked myogenesis.
Recently, Wu and colleagues reported tracking of the fate of transplanted adipose tissue- and bone marrow–derived mesenchymal stem cells using BLI.39 However, the mesenchymal stem cells survived for less than 5 weeks after transplantation and did not contribute to functional recovery. In contrast, our present study showed that CD34+ cells persisted for up to 1 year and contributed to functional recovery. Furthermore, our study also provides insights into the mechanisms of repair. Although antibody treatment may exert other effects, our FACS analysis and immunohistochemical studies suggest that our antibody treatment protocol can distinguish angiogenesis/paracrine effect from myogenesis, leading to mechanistic insights.
In conclusion, we coregistered MRI, micro-PET, micro-CT, and BLI images to successfully obtain anatomic and functional information on CD34+ cells transplanted in mice with experimental MI. We demonstrated that BLI is capable of long-term tracking of transplanted CD34+ cells and that we can use micro-PET, micro-CT, and MRI images to accurately localize transplanted CD34+ cells. Furthermore, we demonstrated that the long-term survival of the transplanted CD34+ cells contributed to improvement in cardiac function. Using selective antibody blocking, we also demonstrated that angiogenesis and/or paracrine effect accounts for the functional improvement.
We thank Drs Ryuichi Nishii and Leo G. Flores for excellent technical assistance.
Sources of Funding
This work has been supported in part by NIH grant R01 HL086983 (to J.G.G., E.T.H.Y.). J.W. is the recipient of a Research Fellowship from the Canadian Institutes of Health Research. E.T.H.Y. is the McNair Scholar of the Texas Heart Institute.
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Novelty and Significance
What Is Known?
Adult stem cells have been used to treat patients suffering from myocardial infarction or heart failure.
CD34+ cells, one type of adult stem cells, can generate new myocardium and blood vessels.
What New Information Does This Article Contribute?
Human CD34+ cells survived for up to 1 year in the mouse heart following experimental infarction.
Human CD34+ cells improved cardiac function after transplantation.
Human CD34+ cells did this by forming new blood vessels or producing beneficial chemicals, but not by forming new myocardium.
Adult stem cells have been used to treat patients experiencing from myocardial infarction or heart failure. However, it is not known how long these cells survive after transplantation and how these cells improve cardiac function. To address these questions, we transplanted human CD34+ cells, a type of adult stem cells that can be purified from blood, into the heart of immune-deficient mice that had sustained experimental myocardial infarction. These CD34+ cells were labeled with reporters that allowed us to see them from outside the body using cardiac imaging machines that detect light signals or positrons. We show that the injected cells survived for up to 1 year in the hearts of mice and contributed to improvement in cardiac function. We also show that the transplanted cells effected this improvement by forming new blood vessels or producing beneficial chemicals but not by forming new myocardium. This study provides new insights into the mechanisms whereby one type of adult stem cells helps to repair the heart. We expect that our results will be helpful in improving the design of clinical trials to determine the best way to provide cell therapy to patients with heart disease.
↵*Both authors contributed equally to this work.
This work was presented in part at the American Heart Association Scientific Sessions, Orlando, Fla, November 3-7, 2007, and published in abstract form (Circulation. 2007;116[suppl II]:II-22).
Original received January 15, 2010; resubmission received April 11, 2010; revised resubmission received April 23, 2010; accepted April 26, 2010.