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Keywords:

  • Heart diseases;
  • Bone marrow;
  • Cell homing;
  • Molecular imaging

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Bone marrow mononuclear cell (BMMC) therapy shows promise as a treatment for ischemic heart disease. However, the ability to monitor long-term cell fate remains limited. We hypothesized that molecular imaging could be used to track stem cell homing and survival after myocardial ischemia-reperfusion (I/R) injury. We first harvested donor BMMCs from adult male L2G85 transgenic mice constitutively expressing both firefly luciferase (Fluc) and enhanced green fluorescence protein reporter gene. Fluorescence-activated cell sorting analysis revealed ∼0.07% of the population to consist of classic hematopoietic stem cells (lin-, thy-int, c-kit+, Sca-1+). Afterward, adult female FVB recipients (n = 38) were randomized to sham surgery or acute I/R injury. Animals in the sham (n = 16) and I/R (n = 22) groups received 5 × 106 of the L2G85-derived BMMCs via tail vein injection. Bioluminescence imaging (BLI) was used to track cell migration and survival in vivo for 4 weeks. BLI showed preferential homing of BMMCs to hearts with I/R injury compared with sham hearts within the first week following cell injection. Ex vivo analysis of explanted hearts by histology confirmed BLI imaging results, and quantitative real-time polymerase chain reaction (for the male Sry gene) further demonstrated a greater number of BMMCs in hearts with I/R injury compared with the sham group. Functional evaluation by echocardiography demonstrated a trend toward improved left ventricular fractional shortening in animals receiving BMMCs. Taken together, these data demonstrate that molecular imaging can be used to successfully track BMMC therapy in murine models of heart disease. Specifically, we have demonstrated that systemically delivered BMMCs preferentially home to and are retained by injured myocardium.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Ischemic heart disease is the leading cause of morbidity and mortality in most industrial nations. Recent animal studies [1, 2] and clinical trials [3, [4]5] have shown that transplantation of bone marrow mononuclear cells (BMMCs) may improve heart function. Subsequently, it has been proposed that stem cells can release angiogenic factors, protect cardiomyocytes from apoptotic cell death, induce proliferation of endogenous cardiomyocytes, and recruit resident cardiac stem cells [6]. However, the retention rate of exogenously administered cells within the injured heart remains low, and the mechanistic processes underlying stem cell therapy remain unclear. In this regard, understanding the post-transplant homing, survival, and proliferation responses of transplanted cells represents a critical initial step toward a better elucidation of stem cell biology and physiology in living subjects.

Postmortem histology remains the most common technique to study engrafted cell fate in animal models. With this approach, cells are typically labeled with fluorescent dye (e.g., PKH2 or CM-Dil) [7] or genetically modified to express green fluorescence protein (GFP) or β-galactosidase prior to transplantation for later identification by fluorescence microscopy or enzyme staining of serial tissue sections [1, 2]. However, inherent in this method are sampling error and selection bias, as different sets of animals must be sacrificed at different time points to re-create a representative pattern of longitudinal stem cell survival [8, 9]. In addition, given the significant variability of transplanted cell behavior within individual subjects, the aforementioned invasive techniques are inadequate for studying the spatiotemporal kinetics of stem cell homing and engraftment.

In this study, we hypothesized that reporter gene-based imaging can be used to study transplanted BMMC homing, survival, and engraftment in the ischemic myocardium. Using donor BMMCs from transgenic animals that constitutively express both Fluc and enhanced green fluorescence protein (eGFP) in all tissues [10], the biodistribution of BMMCs was tracked for 4 weeks following systemic delivery into syngeneic wild-type mice. Although there are reports investigating such survival and homing kinetics using radiolabeling [11, [12]13] and iron particle labeling [13, 14] methodologies, those studies are limited by the inability to track long-term cell behavior in vivo. The technique used in the present study offers significant advantages in this regard, as we describe the homing and survival kinetics of cells up to 4 weeks following transplant.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Animals

Adult female FVB mice (n = 41; Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) and male L2G85 reporter transgenic mice (n = 14; Contag Laboratory, Stanford, CA, http://sci3.stanford.edu/lab) were used. Transgenic animals (L2G85) were created on the FVB background to constitutively express both firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP) in all tissues and organs, including bone marrow cell populations [10]. Animal care was provided in accordance with the Stanford University School of Medicine guidelines and policies for the use of laboratory animals.

Study Design

Female FVB mice were mechanically ventilated with a 2%–3% mixture of isoflurane and 100% O2. Animals were randomized into two groups: (a) ischemia-reperfusion (I/R) injury (n = 24) by occlusion of left anterior descending (LAD) coronary artery for 30 minutes, and (b) sham procedure with open thoracotomy and suture around the LAD coronary artery but no occlusion (n = 17). Surgery was performed by a single experienced surgeon (A.Y.S.). Three hours following surgery, I/R animals (n = 17) received 5 × 106 BMMCs (harvested from male L2G85 transgenic donors) via tail vein injection. A subset of the I/R animals (n = 5) received 100 μl of phosphate-buffered saline (PBS) via tail vein to serve as controls for functional echocardiography study. Cell therapy was monitored by optical bioluminescence imaging (BLI) on days 1, 2, 4, 6, 8, 10, 14, 21, and 28 using D-Luciferin (300 mg/g body weight, intraperitoneal) as the reporter probe [15]. BLI results were validated by ex vivo by histological evaluation and real-time polymerase chain reaction (RT-PCR) analysis for the male Sry gene. Echocardiography was performed preoperatively and at 1 and 4 weeks postoperatively to determine functional improvement in animals that received BMMCs compared with those that received PBS.

Preparation of BMMCs

Bone marrow cells were harvested from the long bones of male L2G85 transgenic mice and isolated by centrifugation in a density cell separation medium (Ficoll-Hypaque; GE Healthcare, Piscataway, NJ, http://www.gehealthcare.com) prior to cardiac injection [3].

Flow Cytometry Analysis

BMMCs (1 × 106) were incubated in 2% fetal bovine serum/PBS at 4°C for 30 minutes with 1 μl of monoclonal antibody specific for CD31, CD34, CD45, sca-1, or c-kit (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com) or left unstained for analysis by FACSCalibur with CellQuest software (Becton Dickinson).

Bioluminescence Imaging of BMMC Transplantation

BLI was performed using the Xenogen In Vivo Imaging System (Alameda, CA, http://www.xenogen.com). The system consists of a supersensitive, cooled (−90°C) charge-coupled device (CCD) camera mounted onto a light-tight imaging chamber. The CCD chip is 2.7 cm2 and consists of 2,048 × 2,048 pixels at 13.5 μm each. The camera is capable of detecting a minimum radiance of 100 photons per second per cm2 per steridian (photons/second/cm2/sr) and can achieve a minimal image pixel resolution of 50 μm [15]. The system does not allow for three-dimensional imaging, and hence spatial resolution is limited to a compressed, two-dimensional image for analysis. Images were acquired using 1–10-minute intervals until peak signal was observed. BLI was quantified by creation of polygonal regions of interest (ROIs) over the precordium by a blinded operator (F.C.). For ex vivo cardiac imaging, hearts were explanted and immediately immersed in 5-mm culture dishes containing 2–3 ml of 12 M D-Luciferin in PBS. Images were acquired using a 1–2-minute interval until peak signal was observed.

Tissue Fixation and Immunohistochemical Analysis

Following intubation, the chest was opened and the heart perfusion fixed for 2 minutes at 120 mmHg with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) in PBS via left ventricular stab (a right atrial defect provided the egress for blood and fluid). Fixed hearts were immersed in 30% sucrose overnight, embedded into optimal cutting temperature compound (Sakura Finetek, Torrence, CA, http://www.sakuraeu.com), frozen, and prepared into 10-μm-thick frozen sections. Anti-GFP (rabbit polyclonal conjugated to Alexa Fluor 488 [Invitrogen, Carlsbad, CA, http://www.invitrogen.com] and rabbit polyclonal anti-troponin I [Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com]) staining was carried out. Primary antibodies were used at dilutions of 1:200 (anti-GFP) and 1:100 (anti-troponin I). Secondary biotinylated anti-rabbit antibody (Invitrogen) was used for troponin I and visualized with streptavidin Alexa Fluor 555 (Invitrogen). Confocal microscopy was performed on a Leica SP5 confocal system (Leica, Wetzlar, Germany, http://www.leica.com).

Ex Vivo Quantification of Intracardiac Surviving BMMCs

For ex vivo validation of BLI, a standard curve was first generated by correlating (a) cycle counts from RT-PCR probing for the Sry gene with (b) known numbers of male BMMCs injected into female hearts. Specifically, 10 female hearts from wild-type animals were excised and immediately injected with known amounts of male BMMCs ranging from 100 to 1 × 107 cells. Whole heart DNA was then isolated using DNAzol reagent (Invitrogen) according to the manufacturer's protocol. RT-PCR was performed on a 7900HT Sequence Detection System with TaqMan Assays-on-Demand gene expression probes (systems and probe from Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) for the SRY gene (Mm00441712_s1). Hearts were then collected from animals in both groups (shams, n = 11; I/R, n = 11) at day 2 and week 2 postoperatively, followed by DNA extraction and RT-PCR for SRY. Cycle count results were fit to the equation generated by the standard curve to determine number of male cells present in each sample.

Echocardiographic Determination of Left Ventricular Contractility

Echocardiography was performed using the General Electric Vivid 7 Dimension imaging system equipped with a 13-MHz linear probe (General Electric, Milwaukee, http://www.ge.com). Mice were assessed preoperatively and weekly thereafter post-BMMC or PBS infusion. Animals were induced with isoflurane, received continuous inhaled anesthetic (1.5%–2%) for the duration of the imaging session, and were imaged in the supine position. Echocardiography was performed by an independent operator (F.C.) blinded to the study conditions. M-mode short axis views of the left ventricle were obtained and archived. Analysis of the M-mode images was performed using GE built-in analysis software. Left ventricular end diastolic diameter (EDD) and end-systolic diameter (ESD) were measured and used to calculate fractional shortening (FS) by the following formula: FS = (EDD − ESD)/EDD [16].

Statistical Analysis

Experimental results are expressed as mean ± SEM. Linear regression analysis was performed to determine correlation between two variables. Repeated measures analysis of variance with post hoc testing and nonpaired Student's t test were used where appropriate. The level of significance was set at p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Flow Cytometry Analysis of BMMCs

To understand the characteristics of BMMCs isolated from L2G85 transgenic mice, we analyzed their cell surface markers. Based on the average of three fluorescence-activated cell sorting (FACS) analyses, ∼41% of BMMCs expressed CD31, an endothelial cell marker (Fig. 1A); ∼28% expressed stem cell marker c-kit, which is present on hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) (Fig. 1B) [17]; ∼99% expressed CD45, a hematopoietic and leukocyte marker (Fig. 1C); ∼16% expressed stem cell antigen-1 (Sca-1) marker (Fig. 1D); ∼10% expressed CD34, a myeloid progenitor cell antigen that is also present in endothelial cells and some fibroblasts (Fig. 1E) [18]; and ∼4.5% coexpressed both c-kit and Sca-1 (Fig. 1F). Expression of c-kit and Sca-1 was reciprocal; however, when the lineage marker-negative (Lin) population was gated, the c-kit and Sca-1 double-positive fraction was approximately 0.07% of the bone marrow, representing the “classic” HSC population as described by Okada et al. [17]. Overall, the surface marker patterns of L2G-derived BMMCs were consistent with those used in clinical trials [3, [4]5].

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Figure Figure 1.. Fluorescence-activated cell sorting analysis of bone marrow mononuclear cells (BMMCs) from L2G85 transgenic reporter mice revealed typical proportions of progenitor cells for the FVB strain. Green curves with corresponding percentages represent BMMC samples labeled with CD31 (A), c-kit (B), CD45 (C), Sca-1 (D), and CD34 (E). Blue curves and percentages demonstrate negative controls (no antibody). (F): Approximately 4.5% of the BMMCs stained positive for both c-kit and Sca-1, similar to preparations used in clinical studies of BMMC transplantation in humans. Abbreviations: APC, allophycocyanin; PE, phycoerythrin.

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Linear Correlation Between Cell Numbers and Ex Vivo BLI Signals or In Vitro Enzyme Assay

L2G85 mice constitutively express both Fluc and eGFP reporter genes in all tissues and organs, including harvested bone marrow cell populations [10]. Representative bioluminescence images of BMMCs are shown in Figure 2A. Ex vivo analysis showed a linear relationship between cell number and BLI signal (R2 = 0.99), as well as between cell number and Fluc enzyme activity (R2 = 0.99) (Fig. 2B). These data suggest that BLI of Fluc reporter gene can be used to accurately follow and quantify transplanted stem cells in small living animals. Fluorescence microscopy of BMMC cells demonstrated uniform eGFP expression within the cytosol (Fig. 2C). FACS analysis demonstrated that more than 87% of the BMMC fraction strongly expressed eGFP, as shown in Figure 2D.

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Figure Figure 2.. Reporter gene activity correlates robustly with cell number. (A): BLI of increasing numbers of bone marrow mononuclear cells (BMMCs) in vitro (total cell count given above corresponding well, with color scale bar representing range of signal in p/s per cm2 per steridian). (B): Correlation of cell numbers (x-axis) with BLI signal (left) and Fluc enzyme activity (right) demonstrated linear relationships, with R2 values of 0.99. (C): Confocal laser microscopy of BMMCs demonstrated bright, cytosolic enhanced green fluorescence protein (eGFP) expression, with corresponding nuclei stained blue with 4,6-diamidino-2-phenylindole. Scale bar = 5 μm. (D): Fluorescence-activated cell sorting analysis of BMMCs from WT control FVB mouse (red) and L2G85 transgenic mouse (green) demonstrated robust eGFP expression by more than 87% of the cells. Abbreviations: BLI, bioluminescence imaging; GFP, green fluorescence protein; p/s, photons per second; WT, wild-type.

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Tracking BMMC Fate in Living Mice by In Vivo BLI

To assess homing and biodistribution, noninvasive imaging was performed on days 1, 2, 4, 6, 8, 10, 14, 21, and 28 (Fig. 3). ROIs were created over the precordium, and average radiance was measured. For the sham group, Fluc imaging signals were 1.92 ± 0.01 × 103 photons/second/cm2/sr on day 1, 3.51 ± 0.11 × 103 on day 2, 6.29 ± 0.93 × 103 on day 4, 7.19 ± 0.22 × 103 on day 6, 6.86 ± 0.78 × 103 on day 8, 4.97 ± 0.22 × 103 on day 10, 4.10 ± 0.97 × 103 on day 14, 3.77 ± 0.53 × 103 on day 18, 3.62 ± 0.36 × 103 on day 21, and 3.55 ± 0.13 × 103 on day 28 (Fig. 3). Background imaging signal (taken from ROIs created upon control nonrecipient animals [n = 5] given only D-Luciferin) ranged between 1.5 × 103 and 3.5 × 103 photons/second/cm2/sr. Migration of BMMCs to lung, spleen, and femurs could be visualized in both groups. Quantitative analysis also showed that the I/R group had significantly stronger chest Fluc activities at day 2 and day 6 compared with the sham group (p < .01) (Fig. 4A). As animals in both groups received lateral thoracotomy, the higher imaging signals in the I/R group indicate that more BMMCs are homing to the acutely injured hearts compared with the normal hearts. We further confirmed this observation by ex vivo imaging of explanted whole hearts on day 2 following BMMC transplant (Fig. 4B). Interestingly, the largest differences in signal activity between I/R and sham were observed within the first 10 days following transplantation, suggesting increased homing, proliferation, and/or retention within the ischemic myocardium during this period. Overall, the sham animals were also noted to have a relatively high signal compared with expected background levels from an unoperated mouse. This effect is most likely explained by the thoracotomy itself, as the inflammatory insult provided by the surgical wound serves as a stimulus for BMMC homing and retention.

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Figure Figure 3.. Bioluminescence imaging demonstrates that exogenously delivered bone marrow mononuclear cells (BMMCs) preferentially home to injured myocardium. Shown are images following the same animals (sham on left and I/R injury on right) for 4 weeks following i.v. delivery of L2G85-derived BMMCs (the maximum values for scale bars in p/s/cm2/sr are different in the three rows). Persistently elevated signal from the area overlying the heart can be observed through day 14, followed by relatively similar decreasing trend in signal intensity by day 28. Images at day 10 demonstrate entrapment of cells in extracardiac sites such as the spleen (yellow arrows) and long bones of the lower extremities (red arrows). Abbreviations: I/R, ischemia-reperfusion; p/s/cm2/sr, photons per second per cm2 per steridian.

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Figure Figure 4.. Ex vivo imaging confirms the presence of transplanted cells within the myocardium. (A): Quantification of signals from regions of interest over the thorax demonstrated significantly increased cell numbers in animals with I/R injury (white bars) compared with sham (gray bars) at days 2–6 following transplant (*, p < .05). Mean baseline log10BLI (p/s/cm2/sr) of unoperated, uninjected mice was 3.3 ± 2.1 (n = 5 animals imaged at different time points). This was consistently 1–2 orders of magnitude lower than that of the highest signals attained in the animals receiving cells and I/R injury. (B): Ex vivo imaging of hearts 2 days following I/R injury with i.v. PBS (left), sham surgery with bone marrow mononuclear cells (BMMCs) (middle), or I/R injury with BMMCs (right) confirmed homing of intravenously delivered BMMCs to the heart. Scale bars = 5 mm. Abbreviations: BLI, bioluminescence imaging; I/R, ischemia-reperfusion; p/s/cm2/sr, photons per second per cm2 per steridian; PBS, phosphate-buffered saline.

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Histological Evaluation of BMMC Homing to the Heart

Fluorescence microscopy revealed clear evidence of eGFP+ BMMC homing to the injured heart and further validated our BLI measurements. In keeping with the native, postinfarct inflammatory response, transplanted cells were observed in the infarct border zones (Fig. 5A, Fig. 5B). Confocal laser microscopy confirmed the presence of transplanted cells within the myocardium 2 days following delivery (Fig. 5C).

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Figure Figure 5.. Histological evaluation confirms bone marrow mononuclear cells (BMMCs) homing to the infarcted heart. Shown are fluorescence microscopy images of a representative heart 2 days following 30 minutes of ischemia-reperfusion injury and injection of 5 × 106 BMMC cells via tail vein. All panels stained with anti-troponin (red), anti-green fluorescence protein (anti-GFP) (green), and 4,6-diamidino-2-phenylindole (blue). (A): Infarcted area is demonstrated by lack of bright troponin stain, with numerous GFP-positive cells in the infarct border zone. Scale bar = 50 μm. (B): High-power view demonstrating numerous GFP-expressing cells within the myocardium. Scale bar = 10 μm. (C): Confocal laser microscopy image confirming the presence of GFP-expressing cells within infarcted areas of the myocardium. Scale bar = 5 μm.

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Ex Vivo Confirmation of BMMC Homing into Ischemic Myocardium by Probing Sry Gene

The Sry gene, a target DNA sequence located in the motif of Y-linked testis-determination gene, was originally amplified to study human sex determination at the single-cell level [19]. Since this test is highly precise, it has been adapted to monitor the clinical outcome of allogeneic hematopoietic transplantation [20], as well as that of animal study focused on cardiac stem cell therapy [21]. To confirm our imaging results of cell homing, we first established a correlation between RT-PCR (TaqMan) amplification cycle number and injected BMMC number by probing Sry gene from freshly isolated female heart samples directly injected (ex vivo) with known numbers of male BMMCs. Figure 6A shows the robust correlation between BMMC number and cycle number of real-time PCR (r2 = 0.99) using this approach. The Sry gene test was then performed on hearts harvested from our experimental animals at different time points following BMMC injection. Quantitative real-time PCR showed that significantly more BMMCs homed in to the myocardium in the I/R group compared with the sham group on day 2 (245 ± 68 vs. 2,580 ± 421; p < .01), consistent with our in vivo imaging analysis (Fig. 6B). There was also a trend for increased BMMC numbers in the I/R group at week 2 (1,540 ± 470 vs. 2,796 ± 585), but this did not reach statistical significance (p = .078).

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Figure Figure 6.. Real-time polymerase chain reaction quantification of surviving male transplanted cells within female hearts. (A): Plot of TaqMan-based Sry quantification versus number of male cells in female hearts demonstrated a robust correlation between cycle count and known cell number (R2 = 0.99). (B): Ex vivo TaqMan analysis of hearts undergoing sham surgery (gray bars) versus I/R injury (white bars) demonstrated statistically significant increase of bone marrow mononuclear cells homing in to injured hearts compared with sham at day 2 following transplant. This trend persisted at week 2 but did not achieve statistical significance (n = 5–6 per group) (*, p < .05). Both results mirror findings generated by longitudinal bioluminescence imaging in vivo, as shown in Figures 3 and 4. Abbreviation: I/R, ischemia-reperfusion.

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Assessment of Cardiac Function by Echocardiography

To determine whether intravenously delivered BMMCs improved cardiac contractility, we performed echocardiography preoperatively and weekly postoperatively. Representative M-mode images of mice injected with PBS as control or BMMCs at week 4 are shown in Figure 7A. At 1 week, fractional shortening decreased comparably in both groups. By 4 weeks, there was a trend toward better functional improvement in the BMMC group compared with control PBS group (38.5% ± 1.8% vs. 35.2% ± 1.3%), but this did not reach statistical significance (Fig. 7B). Data from intermittent assessment of FS between week 1 and week 4 demonstrated a similar, nonsignificant trend and are not shown on the graph to preserve clarity.

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Figure Figure 7.. Ventricular function following BMMC therapy. (A): Representative M-mode images of hearts at 4 weeks following ischemia-reperfusion (I/R) injury with administration of PBS (left) versus BMMCs (right). Scale bars = 5 mm. (B): Quantification of left ventricular function demonstrated a trend toward improved functional recovery at 4 weeks post-I/R in animals receiving BMMCs compared with PBS (n = 5–6 per time point) but did not achieve statistical significance. Abbreviations: BMMC, bone marrow mononuclear cell; PBS, phosphate-buffered saline; Preop, preoperative.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

In this study, we present evidence that reporter gene imaging can be used to track BMMC homing, retention, and survival in a mouse model of acute cardiac I/R injury. Our data can be summarized as follows: (a) a robust correlation existed between cell numbers and reporter gene imaging signals; (b) there was preferential homing of BMMCs within the first week of I/R injury; (c) <0.1% of the total BMMCs delivered eventually engrafted into the heart by 2 weeks after i.v. delivery; and (d) i.v. delivery of BMMCs did not confer significant improvement of ischemia-induced left ventricular contractility.

The success of stem cell therapy will likely require novel methods to determine the dynamic biodistribution and long-term fate of transplanted cells without reliance on postmortem histology. In recent years, several imaging techniques have been developed to better understand stem cell fate in vivo. In general, they can be divided into two broad methodologies: direct labeling and indirect reporter gene-based imaging [8, 9]. The former uses a detectable probe (e.g., radioactivity or iron particles) that can be loaded into cells prior to delivery. Aicher et al. first demonstrated that tissue distribution of endothelial progenitor cells incubated with radioactive [111In]-oxine could be successfully monitored by scintigraphic imaging [11]. As [111In]-oxine has a half-life of 67.3 hours, only ∼2% of the radioactivity remained in the infarcted heart after 96 hours. A follow-up study by Kraitchman et al. injected porcine mesenchymal stem cells labeled with [111In]-oxine intravenously and showed cardiac engraftment up to 7 days by single-photon emission computed tomography [13]. More recently, Hofmann et al. injected human bone marrow cells labeled with 2-[18F]-fluoro-2-deoxy-d-glucose ([18F]-FDG) via both intracoronary and i.v. routes [12]. Since [18F]-FDG has a half-life of 110 minutes, positron emission tomography (PET) imaging needed to be performed within 2 hours after cell delivery. The authors observed 1.3% to 2.6% of [18F]-FDG-labeled bone marrow cells present in the myocardium after intracoronary delivery, and interestingly, only background activity was detected after i.v. delivery. Taken together, these studies suggest that radiolabeling techniques are suitable for immediate, short-term tracking of delivered cells but less apt for long-term follow-up [8, 9].

In contrast to the short half-life of radioactive probes, iron oxide particles can be tracked for long periods of time. Amado et al. showed that porcine mesenchymal stem cells labeled with Feridex can be delivered by endomyocardial injection and tracked by magnetic resonance imaging (MRI) for 8 weeks [14]. However, the main limitation of such direct iron-labeling techniques is that the MRI signals do not necessarily reflect cell viability, because the iron particles might persist within dead cells, leak into intercellular space, and/or be engulfed by resident macrophages [22]. These factors might explain why quantitative analysis of the iron-labeled retention showed that >40% of the iron-labeled mesenchymal stem cells were still present 8 weeks after delivery in the study by Amado et al. [14]. Indeed, it is well recognized that adult stem cells have poor post-transplant viability, with an estimated 99% of mesenchymal stem cells dying within 4 days after injection into healthy mouse hearts [23].

Notwithstanding the technical limitations of the aforementioned direct imaging techniques, the ideal cellular imaging platform should provide information regarding the following: (a) real-time, dynamic cell biodistribution kinetics; (b) long-term cell survival; and (c) rates of cellular proliferation. At present, both methodologies described above lack these characteristics. An alternative approach—reporter gene imaging—is playing an increasingly prominent role in monitoring stem cell fate, as demonstrated by this study and other studies reviewed elsewhere [8, 9]. Because reporter genes are DNA sequences that encode reporter proteins, one can follow the signal for as long as the transplanted cells and their progeny are viable. If, for example, the stem cells are dead or apoptotic, there will be no transcription and translation of the reporter gene and thus no imaging signal. Similarly, if cells are actively migrating away from a particular ROI, signal strength will also decrease. Likewise, if the transplanted stem cells proliferate in vivo, or migrate into a particular ROI, there will be an increase in the imaging signal detected from that area. Using this elegant reporter gene approach, we have been able to monitor BMMC homing over a relatively protracted time period (compared with radiolabeling technique [11, [12]13]), as well as to quantify BMMC survival more accurately (compared with iron labeling technique [14, 22]). However, the low-energy photons (2–3 eV) from BLI can become attenuated within deeper tissues (e.g., heart) compared with more superficial locations (e.g., skeletal muscles). In our experience, the lower detection limit of BMMCs within the heart is approximately on the order of 1,000 cells compared with 100 cells in the subcutaneous tissue over the leg (unpublished data).

In our study, bioluminescence imaging of the I/R group showed significantly higher cell signal activity in the heart compared with the sham group during the first week. This difference is likely due to activation of cytokines that promotes homing of BMMCs to the ischemic sites [6]. A previous study using gene expression analysis has shown that stromal cell-derived factor-1α, vascular endothelial growth factor, matrix metalloproteinase-9, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 are activated after myocardial infarction [24]. The exact chemoattractant factors responsible for stem cell homing remain unclear, and the process itself may be inefficient, as shown by our in vivo imaging, histological analysis, and ex vivo real-time PCR. In fact, by 2 weeks following transplant, less than 0.1% of the total injected cells remained engrafted in the recipient hearts. The low rate of cell engraftment may also explain the lack of improvement in cardiac function observed in our study. It is possible that the observed trend might have achieved statistical significance with a larger cohort of animals. Moreover, our study remains limited in that we did not follow animals out for longer than 4 weeks to observe whether the trend in functional improvement persisted or diminished. Critical evaluation of functional improvement as a function of cell dosage and time remains an area that requires further study.

We believe that in future studies, in vitro identification (e.g., by transcriptional profiling) and in vivo validation (e.g., by reporter gene imaging) of factors important to homing and cell retention will be an attractive approach to coax exogenously administered stem cells to home in to the heart and promote long-term functional improvement. However, one of the main drawbacks of bioluminescence imaging is its restriction to small animal preclinical validation studies, because the low-energy photons (2–3 eV) become attenuated and scattered within deep tissues [25]. In addition, the inability to perform three-dimensional (3D) BLI impairs the ability to accurately localize signal sources from deep tissues (e.g., heart), as discussed above. Specifically, one of the resultant challenges from compressing 3D data into a two-dimensional picture is that of increased noise-to-signal ratio. In the chest, for example, the summation of lung background might obscure a relatively low cell signal emitted from the heart and measured through an ROI designated over a two-dimensional space. Thus, ongoing development of a PET-based reporter gene and reporter probe technique that uses high-energy photons (511 keV) and has 3D imaging capabilities will be necessary for clinical application in the future [26].

In conclusion, our study suggests that reporter gene imaging can be a valuable tool for studying stem cell fate in vivo. The same imaging platform can be adopted to investigate basic mechanisms underlying myocardial cell therapy and optimize the key variables involved, such as the most efficacious cell type(s), appropriate cell dosing, and best routes of delivery (e.g., intracoronary vs. i.v.). We hope that carefully designed studies using the reporter gene imaging techniques developed here and in future investigations will lead not only to advancement of stem cell research but also to useful novel therapies and diagnostic tools for clinicians and patients.

Disclosures of Potential Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

This work was supported in part by grants from the American Heart Association, American College of Cardiology Fellowship/General Electric, American Society of Nuclear Cardiology (J.C.W.) and the National Heart, Lung, and Blood Institute (J.C.W., A.Y.S.) and by a Society of Nuclear Medicine Student Fellowship (S.-A.L.). A.Y.S. and S.-A.L. contributed equally to this work.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures of Potential Conflicts of Interest
  8. Acknowledgements
  9. References