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Eur J Cardiothorac Surg 2004;26:137-143
© 2004 Elsevier Science NL

Magnetic resonance mapping of transplanted endothelial progenitor cells for therapeutic neovascularization in ischemic heart disease

Alberto Webera, Ivan Pedrosab, Atsuhiko Kawamotoa, Nathan Himesb, Jeeva Munasingheb, Takayuki Asaharaa, Neil M. Rofskyb, Douglas W. Losordoa*

a Divisions of Cardiovascular Research and Cardiovascular Medicine, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135, USA
b Department of Radiology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA

Received 8 January 2004; received in revised form 22 February 2004; accepted 2 March 2004.

* Corresponding author. Address: Division of Cardiovascular Research, St. Elizabeth's Medical Center, 736 Cambridge Street, Boston, MA 02135, USA. Tel.: +1-617-789-3474; fax: +1-617-779-6362
e-mail: douglas.losordo{at}tufts.edu


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
Objective: Intramyocardial transplantation of endothelial progenitor cells (EPCs) has been previously correlated with significant augmentation of vascularity and improvement of left ventricular function following myocardial ischemia. However, precise intramyocardial localization of the transplanted cells and the extent of in situ cell migration are unknown. We present a novel technique using magnetic resonance imaging (MRI) to localize transplanted EPCs in ischemic hearts. Methods: CD34-positive cells were isolated from human peripheral blood by magnetic bead selection: CD34-positive cells adhere to CD34-negative antibody coated magnetic beads, while CD34-negative cells do not. All cells were labeled with fluorescent DiI-dye for histological localization. CD34-positive cells or CD34-negative cells (105, 1x106 and 2x106 cells) were transplanted into non-ischemic (n=6) or ischemic myocardium (n=2) of Sprague–Dawley rats. Rats were sacrificed 24 h after cell transplantation. The resected hearts were imaged ex vivo using 3 and 8.5 T magnets. Morphological correlation between the MRI findings and fluorescent microscopy for identification of retained CD34-positive cells was evaluated. Results: CD34-positive cells were identified as areas of low signal intensity on T2*-weighted images within the myocardium. These areas increased in size with the gradual increase in the echo time due to susceptibility effect. The extent of the low signal intensity at a given echo time was proportional to cell dosage. No areas of low signal were identified in the CD34-negative cell transplanted hearts. Histological localization of DiI-labeled CD34-positive cells documented a direct anatomic correlation with the localization of transplanted cells on the MRI images. Conclusions: Magnetically labeled EPCs transplanted for therapeutic neovascularization in myocardial ischemia can be visualized with ex vivo MRI at high-field strengths.

Key Words: Angiogenesis • Ischemic heart disease • Stem cell tracking • Magnetic resonance imaging


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
Whereas most tissues in adult organs are composed of differentiated cells, stem or progenitor cells are maintained in a quiescent status locally or in the systemic circulation and are activated during physiological and pathological tissue regeneration. Haematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs) are derived from a common precursor, the hemangioblast (Fig. 1) . HSCs and EPCs share certain antigenic determinants, including Flk-1, Tie 2, cKit, Sca-1, CD133 and CD34. These markers are subsequently lost as HSCs differentiate [1]. EPCs were first isolated, by Asahara et al., as CD34-positive mononuclear cells (MNCs) from adult peripheral blood by means of magnetic beads coated with antibody to CD34, as EPCs-enriched fraction [2].



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  		Fig. 1. Differentiation profile of endothelial lineage cells. Embryonic endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) share among others CD34, lost by the second as they differentiate.

 
Circulating EPCs have been shown to home to sites of neovascularization where they differentiate into endothelial cells (ECs) in situ, consistent with ‘vasculogenesis’ [3–5]. Intravenous transplantation of cultured human EPCs applied in a model of myocardial ischemia in the nude rat demonstrated that transplanted human EPCs can incorporate into rat myocardial neovascularization, preserving left ventricular (LV) function, and inhibiting myocardial fibrosis [6]. Recently, Kocher et al. [7] attempted intravenous infusion of freshly isolated human CD34-positive MNCs (EPC-enriched fraction) into nude rats with myocardial ischemia. These experimental findings using immunodeficient animals suggest that both cultured and freshly isolated human EPCs have therapeutic potential in peripheral and coronary artery diseases [1]. In the above-mentioned studies, EPCs were always isolated by means of magnetic beads coated with antibody to the CD34 antigen [8]. The magnetic beads were not detached from the selected cells before transplantation. Previous data from our lab documents preserved viability and functionality of transplanted CD34-positive cells isolated by means of magnetic beads.

The development of progenitor and stem cell therapy in humans would be greatly enhanced by a technique to monitor their fate non-invasively thereby permitting serial assessment of the cellular biodistribution and migratory capacity [9]. To monitor the fate of transplanted cells, including their migration in vivo, cells are currently labeled ex vivo using a vital dye (e.g. a fluorochrome), a thymidine analog (e.g. BrdU), or a transfected gene (e.g. LacZ or green fluorescent protein, GFP), for later visualization using (immuno)histochemical procedures following tissue removal at a single time point. Considering the different delivery options for implementing myocardial cell transplantation (epicardial via intraoperative intramyocardial injection; endocardial, via catheter-based intramyocardial injection; intracoronary; retroperfusion, via cannulation of the coronary sinus; or intrapericardial, after transthoracic access to the pericardium) [10], a technique that could monitor the engraftment and localization of the transplanted cells is crucial to assess the safety and success of these procedures.

Most MR scanners used in clinical practice have magnetic fields equivalent to 1.5 T. Recent advances in the development of MR systems have allowed for scanners that operate at high magnetic fields. High-field MR scanners, including whole body clinical 3 T magnets for human use, provide excellent signal that allow for near microscopic resolution. The magnetic tagging of cells that facilitates separation also creates an opportunity to visualize these cells with magnetic resonance imaging (MRI).

MR signal results from the behaviour of protons in a magnetic environment. One behaviour is referred as the T2 of a tissue, an innate feature based upon its constituent molecules. The T2 characteristics of tissue are related to local interactions amongst protons. These so called ‘spin–spin’ interactions cause dephasing of the protons, which leads to the decay of MR signal over time. Disturbances in the local magnetic field affect the spin–spin interactions so that dephasing occurs more rapidly and therefore, the MR signal decay is also faster. This faster decay, or T2* decay, yields a lower signal intensity on the image. Thus, local magnetic field distortions caused by the tagged cells can be captured with MRI as a dark signal intensity particularly when T2* weighted sequences are used. An increase in the time between the excitation of the protons and the collection of the MR signal, or echo time (TE), allows for more dephasing of the protons. Hence, the longer the TE the greater decay in MR signal. By selecting gradually increasing echo times, the T2* effect resulting from the magnetic beads can be appreciated as an increase in the area of dark signal intensity or ‘blooming effect’ within the MR images. Gradient echo sequences (GRE) are a class of MRI strategies that are particularly sensitive to T2* effects. Finally, higher field strengths are more sensitive to T2* effects as compared with lower field strengths.

Conventional magnetic cell labeling techniques rely on surface attachment of magnetic beads ranging in size from several hundred nanometers to micrometers [11]. There are several prior reports describing magnetic resonance tracking of progenitor cells, in neural tissue, tumor or inflammatory tissue [9,11–14].

It has been established that bone marrow-derived EPCs present in the systemic circulation home to and incorporate into sites of neovascularization, and may be useful in therapeutic strategies of ‘supply-side angiogenesis’, for example, after myocardial ischaemia (MI) [2,10,15–20]. A technique that could monitor the engraftment and migration of intramyocardial injected EPCs, serially and non-invasive, could guide further advances for clinical application. We hypothesized that magnetic cell labeling would allow for intramyocardial visualization and localization of EPCs on high-field MRI.

The data presented in this study represent just a preliminary proof of principle where we try to define whether or not intramyocardial EPCs visualization and localization is possible with MRI.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
2.1. Animal models
All procedures were performed in accordance with St. Elizabeth's Institutional Animal Care Committee. We utilized male Sprague–Dawley rats (200–250 g weight) as our animal model.

2.2. Fresh isolation and intramyocardial transplantation of human EPCs
Human total peripheral blood MNCs were isolated from healthy volunteers by density-gradient centrifugation. CD34-positive mononuclear blood cells were isolated from total MNCs by means of colloidal super-paramagnetic beads (MACS-Microbeads) conjugated to monoclonal mouse anti-human CD34 antibody (isotype: mouse IgG1, clone: QBEND/10) (Miltenyi Biotec) as EPC-enriched fraction. Mononuclear cell isolation by density-gradient centrifugation is necessary for the initial step, because mononuclear cells in the systemic circulation contain a fraction capable of differentiation to endothelial lineage cells. After mononuclear cell isolation, total mononuclear cells are incubated with anti-CD34 antibodies coated with magnetic microbeads for fresh isolation. A magnetic column is used to collect only the cells binding to the antibodies with microbeads. After the isolation, CD34-negative MNCs were also collected. Both populations of CD34-positive and CD34-negative MNCs were labeled with fluorescent DiI-dye. Cells were counted using a haemocytometer and resuspended in 100 µl PBS. The magnetic beads were never detached from the isolated CD34-positive cells before transplantation. Non-ischemic (A) and ischemic (B) rat hearts were treated. (A) Non-ischemic: Sprague–Dawley rats (n=4) were anaesthetized with ketamine i.p. (0.6 ml/100 g) and intubated. Left parasternal longitudinal thoracotomy was performed. After pericardectomy, DiI-labeled CD34-positive MNCs in 100 µl of PBS were injected intramyocardially in the anterior and/or lateral wall of the LV using a 27G needle. These four rats were treated with intramyocardial injection of 105, 2x105, 1x106 or 2x106 CD34-positive cells, respectively. Two additional rats received 1x106 or 2x106 DiI-labeled CD34-negative MNCs in 100 µl of PBS as negative controls. (B) Ischemic: Two rats underwent ligation of the left anterior descending coronary artery (LAD). Ten minutes after the operation these rats were injected with 105 DiI-labeled CD34-positive MNCs in 100 µl of PBS. Cells were injected in two sites within the ischemic vascular territory of the LAD using a 27G needle. The ischemic zone was macroscopically identified by the pale color of the anterior and lateral walls after LAD ligation.

All rats were sacrificed 24 h after intramyocardial transplantation of CD34-positive EPCs or CD34-negative MNCs. From our experience, it takes about 24 h for the rat heart to absorb the injected saline and that is why 24 h was chosen as time point for sacrifice. The hearts were resected and fixed with 4% paraformaldehyde.

2.3. MR imaging and histopathological correlation
To obtain a completely dark background we embedded the specimens in a perfluoropolyether (Fomblin, Fluortek AB, Sweden) devoid of proton signals. This polymer was found to be inert, effectively sealing the specimens from dehydration, with no observed effects on tissue morphology. Three-dimensional spin echo MR images were obtained by using a 9 cm bore 8.5 T Bruker magnet (Bruker Biospin, Billerica, MA).

Specimens were imaged with a 20 mm birdcage coil. Scan parameters were: TR/TE=1700/25 ms, 256x128x128 matrix with an FOV=30x15x15 mm, affording a 117 µ3 resolution. Additionally, the hearts were scanned on a clinical 3 T magnet (General Electric Medical Systems, Milwaukee) using a 3-inch surface coil. Two-dimensional gradient echo MR images were obtained with the following parameters: TE=10 and 20 ms, TR=325, flip angle=30°, slice thickness of 1 mm, matrix 512x256, FOV=6 mm, NEX=1. The GRE sequence was performed with two echoes to facilitate an evaluation of the susceptibility effect of the magnetic beads, the longer echo time having greater sensitivity to this effect.

Images were obtained in the long and short axes of the resected hearts. The area of T2/T2* effect (marked hypointensity) on each short axis slice-image was measured for each heart, dividing each heart in 1 mm thick slice-images; this analysis was performed on a Macintosh computer using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). The marked hypointensity is presented as percentage of the left ventricular volume, excluding the intra-ventricular cavity, after measuring all short axis slices. Imaging analyses were always done by a blind observer (MR technician). Correlation analysis by Pearson was performed. After MR imaging, the (fixed) heart specimens were embedded in OCT compound (Miles Scientific) and snap-frozen in liquid nitrogen and stored at –80 °C until it was analyzed in cryosections under fluorescence microscopy.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A. Conference...
 References
 
Super-paramagnetic particles induce a T2/T2* effect that is detected by MR as regions with low signal intensity and appearing black relative to adjacent myocardium. Regions with CD34-positive cells were identified as intramyocardial areas of low signal intensity on T2*-weighted images at 8.5 and 3 T. Areas of susceptibility effect were localized within the anterior and lateral walls of the LV.

Susceptibility effect related to the presence of magnetic beads was confirmed by imaging the hearts with different echo times on the 3 T magnet. Intramyocardial areas of low signal intensity demonstrated increased magnetic susceptibility (blooming effect) with longer echo time. An increase in the echo time from 10 to 20 ms, resulted in a doubling of the area of low signal intensity, independently of the cell dosage injected. Computational planimetric analysis (NIH imaging software) demonstrated a proportional increase of the intramyocardial areas of low signal intensity on the MR images with the increase in the number of EPCs transplanted (cell dosage). Areas of low signal intensity related to transplanted EPCs increased proportionally to the cell dosage. MR images of each heart acquired with similar echo time demonstrated a linear relation between the number of cells injected and the area of low signal intensity (P<0.0001).

For each heart, transverse cryosections corresponding to the injection sites were analysed under a fluorescence microscope. MR images were visually correlated with the results from histopathological analysis by fluorescence microscopy. There was an excellent correlation between the location of the areas of low signal intensity on MR images and the identification of labeled cells in the injection sites by fluorescent microscopy (Fig. 2) .



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  		Fig. 2. T2-weighted images on 8.5 T Bruker magnet; representative transversal heart sections only. CD34-positive cells were identified as anterolateral intramyocardial areas of low signal intensity. (A) (Arrows) 105 CD34-positive cells. (B) (Arrows) 1x106 CD34-positive cells. (C) (Arrows) 2x106 CD34-positive cells. (D) The area of low signal intensity is proportional to the cell dosage. (LV) Left ventricle cavity. (RV) Right ventricle cavity. (Stars) Some areas of low signal intensity, far away from the injection sites, are usually seen in ex vivo preparations and are well known as haemosiderine precipitations after blood coagulation.

 
There was no difference in depiction of the labeled cells between ischemic and non-ischemic myocardium, since it was not possible to enhance the MR signal of ischemic myocardium within 24 h postoperative ex vivo (data not shown).

Areas engrafted with fluorescent DiI CD34-positive cells matched closely with the areas of low signal intensity seen on the MR images. The areas of low signal intensity on the MR images were slightly larger than the areas with CD34-positive cells on fluorescence microscopy. This was likely caused by the blooming effect secondary to an extended-range susceptibility effect on the magnetic particles (Fig. 3) .



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  		Fig. 3. T2-weighted image on 8.5 T Bruker magnet and fluorescent microscopy analysis. Excellent agreement between the areas of MR low signal intensity (black) (A) and histopathological fluorescence DiI staining for CD34-positive transplanted cells (red) (B) and (negative; black) (C). The cell location area in the MR image shows a blooming effect compared to the corresponding histological section caused by an extended-range susceptibility effect of the magnetic particles. (LV) Left ventricular cavity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

 
Two hearts transplanted with CD34-negative cells demonstrated no areas of low signal intensity within the myocardium. Fluorescence microscopy confirmed the presence of the fluorescence DiI CD34-negative cells in areas of myocardium that showed no susceptibility effect on MR (Fig. 4) . No areas of susceptibility effect were noted at the sites of the intramyocardial injection. The lack of susceptibility effect in these two hearts confirmed that the areas of low signal intensity were related to the magnetic beads attached to CD34-positive cells.



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  		Fig. 4. No low signal intensity areas in hearts transplanted with 2x106 CD34-negative cells. (A) Image with 3 T magnet. (B and C) Histopathological fluorescence DiI staining for 2x106 CD34-negative cells. (D) Image of sagittal section, 3D T2-weighted, on 8.5 T magnet. (Arrow) Tube with PBS solution including CD34-positive cells as positive control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A. Conference...
 References
 
Several clinical trials are being conducted that utilize exogenous stem or progenitor cells transplanted into damaged myocardium to augment myocardial performance and/or neovascularization after infarction or heart failure. Recently, Stamm et al. (EACTS 2002) reported results from ongoing clinical studies for intramyocardial transplantation of bone marrow progenitor cells isolated using antibodies coated with magnetic beads. These studies point to a need for techniques that permit an evaluation of the efficiency of transplantation and an evaluation of the potential postoperative migration of the transplanted cells. Such a technique would provide an important tool for assessing patients following treatment. The recent occurrence of postoperative arrhythmic complications in patients post-cell-transplant underscores the requirement for careful evaluation of cell location and fate.

The technique we investigate in this study, could be one such method for tracking the location of these cells in treated myocardium. It has the advantage of using magnetic beads both to isolate EPCs from the mononuclear cell fraction of peripheral blood (a standard technique) and to serve as the magnetic label for MRI.

Further studies will be needed to determine whether cell surface magnetic labeling, previously used only for cell selection, is stable enough to allow mid- and long-term in vivo imaging studies [11]. Magnetically selected cells can be cultured with attached beads and the cells grow and adhere normally. In previous studies from our lab and others [1,5,6], EPCs were also always isolated by means of magnetic beads coated with antibody to the CD34 antigen [8].

In these studies the magnetic beads were never de-attached from the selected cells before transplantation. Therefore, based on our previous data we have established that there is a preserved cell viability and functionality of CD34-positive cells isolated by means of magnetic beads.

However, some authors [21,22] claim that after approximately three passages in culture the beads are diluted out. Others suggest that after in vivo administration there is a more rapid reticuloendothelial recognition and clearance of cells thus surface-labeled [11]. Conversely, there is some evidence that the magnetic coated antibodies might be internalised to the cytoplasm and remain intracellular.

There are several works reporting the possibility of intracellular labeling with superparamagnetic ironoxide nanoparticles or magnetodendrimers [9,11,23] using fluid-phase or receptor-mediated endocytosis. These methods allow in vivo cell tracking and the magnetic beads are stably retained intracellularly over time (up to 6 months has been reported). Unfortunately, labeling efficiency is generally low and cells need to be exposed to culture media for long incubation periods. This methodology would not be compatible with the transplantation of autologous freshly isolated cells, a consideration that avoids any contact with potential immunogenic agents.

In the present study we limited our resources and goals to realize whether it is possible or not to identify the magnetic labeled cells by means of MRI just to establish a proof of principle. The selected cell dosages were established aiming previous functional studies in a range from low to relatively high dosage. We first used a small bore 8.5 T magnet, which is currently used for experimental purposes only, still far from clinical use, but allowed us to draw on the full potential of MRI. In a next step we were able to corroborate these initial results with the 3 T magnets currently in clinical use. This is especially encouraging taking into account that we were measuring small rat hearts with a machine that is used for human adults.

Limitations of this study are the lack of experiments with big animals and, of course, the lack of measurements in living animals. These are surely the next steps to follow, being in vivo MRI of the heart a challenging issue because of the need for cardiac and respiratory gating in order to trigger the physiologic heart beats and lung movements.

In vivo imaging of animals would allow the investigator to follow the migration of transplanted cells in the heart and would provide considerable data related to the safety of cell transplantation procedures. Knowledge of the migration pattern of haematopoietic progenitor cells in vivo after homing to ischemic areas would be of considerable importance to understand their physiological impact.

In the present study, we demonstrate that magnetically labeled EPCs intramyocardially transplanted for therapeutic neovascularization can be accurately visualized with ex vivo MRI at high-field strengths. This can be achieved with an experimental, small bore 8.5 T magnet, as well as with the 3 T magnets currently in clinical use. We observed an excellent agreement between the areas demonstrating MR susceptibility effect and histopathological fluorescence DiI staining for CD34-positive transplanted cells. This study introduces MR tracking as a technique to monitor, non-invasively, the localization of magnetically labeled cells after intramyocardial transplantation.

Hence, this ex vivo experiment constitutes a proof of principle of the utility of cellular magnetic tags for tracking cellular transplants currently being evaluated for use in cardiovascular medicine.


   Acknowledgments
 
This study was supported by a grant from the Swiss National Research Foundation. This work was supported by NIH grants (HL-53354, HL-57515, HL-60911, HL-63414, HL-63695, HL-66957).


   Footnotes
 
Presented at the 16th Annual Meeting of the European Association for Cardio-thoracic Surgery, Monte Carlo, Monaco, September 22–25, 2002.


   Appendix A. Conference discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
References
 
Dr Hoerstrup (Zurich, Switzerland): My question is concerning the clinical applicability of this concept. You mentioned that you have sacrificed the animals after 24 hours, and you said that for future clinical application you might use intracellular labeling with magnetic particles. How long will it be possible of tracing down these cells in an in vivo model?

Dr Weber: Well, there are several studies, mainly at Johns Hopkins, already tracing these cells in with intracellular labeling; this group even started clinical studies recently. The imaging of these cells appears to be much better than the surface tracing, and the in vivo tracking seems to be possible for long term.

Dr Hoerstrup: What would be a time frame seen from these other studies?

Dr Weber: The last studies point out that the intracellular magnetic labeling is still available 6 months after implantation. However, these are neural stem cells which don't divide as fast as EPCs, so we don't know, but in our case the tracking follow up might be a little shorter.

Dr C. Stamm (Rostock, Germany): Am I right to say that you do see the iron particle, not the cell? And if I am right, can you think of a way to image a cell to say something about the viability of the cell instead of simply saying that the iron particle that you injected is there?

Dr Weber: In this case, this experiment constitutes proof of principle of the utility of cellular magnetic tags for tracking cells in their localization after intramyocardial injection. The next step will be to evaluate the follow-up and see, in the case of migration, if the correlation between cells and magnetic beads persists. However, in vitro experiments suggested that the magnetic-coated antibodies might be internalized to the cytoplasm and remain intracellular, mimicking the ex vivo intracellular labeling techniques. These methods, as I said before, demonstrated that the magnetic beads are stably retained intracellularly over time (up to 6 months, as reported by Johns Hopkins).

Dr Stamm: And once you have injected the cells, what you do see is the iron particle in the myocardium, right, you can't say this is a living cell inside the myocardium that you can image?

Dr Weber: In all our previous functional studies (see papers by Drs Isner and Asahara) the magnetic beads were never detached from the selected cells before transplantation. Therefore, based on our previous data we have established that there is preserved in vivo cell viability and functionality of CD34-positive cells isolated by means of magnetic beads.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 

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M. Oostendorp, M. J. Post, and W. H. Backes
Vessel Growth and Function: Depiction with Contrast-enhanced MR Imaging
Radiology, May 1, 2009; 251(2): 317 - 335.
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