| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TRANSLATIONAL AND CLINICAL RESEARCH |
aDepartment of Cardiothoracic Surgery,
bMolecular Imaging Program,
cDepartment of Comparative Medicine, and
dDivision of Cardiology, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA
Key Words. Heart diseases • Bone marrow • Cell homing • Molecular imaging
Correspondence: Joseph C. Wu, M.D., Ph.D., Stanford University School of Medicine, Edwards Building R354, Stanford, California 94305-5344, USA. Telephone: 650-736-2246; Fax: 650-736-0234; e-mail: joewu{at}stanford.edu
Received on January 15, 2007;
accepted for publication on July 7, 2007.
First published online in STEM CELLS EXPRESS July 12, 2007.
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosures of Potential...AcknowledgmentsReferences |
|---|
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 x 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosures of Potential...AcknowledgmentsReferences |
|---|
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 [11–13] 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosures of Potential...AcknowledgmentsReferences |
|---|
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 x 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 x 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 x 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 x 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosures of Potential...AcknowledgmentsReferences |
|---|
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 [3–5].
|
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosures of Potential...AcknowledgmentsReferences |
|---|
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 [11–13]), 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-1
, 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosures of Potential...AcknowledgmentsReferences |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosures of Potential...AcknowledgmentsReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosures of Potential...AcknowledgmentsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. L. Kraitchman and J. W.M. Bulte In Vivo Imaging of Stem Cells and Beta Cells Using Direct Cell Labeling and Reporter Gene Methods Arterioscler. Thromb. Vasc. Biol., July 1, 2009; 29(7): 1025 - 1030. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Li, A. Lee, M. Huang, H. Chun, J. Chung, P. Chu, G. Hoyt, P. Yang, J. Rosenberg, R. C. Robbins, et al. Imaging survival and function of transplanted cardiac resident stem cells. J. Am. Coll. Cardiol., April 7, 2009; 53(14): 1229 - 1240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Wu Molecular Imaging: Antidote to Cardiac Stem Cell Controversy J. Am. Coll. Cardiol., November 11, 2008; 52(20): 1661 - 1664. [Full Text] [PDF]
|
|
|
|
 |
|
 P. Leucht, J.-B. Kim, R. Amasha, A. W. James, S. Girod, and J. A. Helms Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration Development, September 1, 2008; 135(17): 2845 - 2854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Zhang and J. C. Wu Comparison of Imaging Techniques for Tracking Cardiac Stem Cell Therapy J. Nucl. Med., December 1, 2007; 48(12): 1916 - 1919. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
