Imaging of Mesenchymal Stem Cell Transplant by Bioluminescence and PET

Department of Nuclear Medicine/Radiology, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Journal of Nuclear Medicine (Impact Factor: 6.16). 01/2008; 48(12):2011-20. DOI: 10.2967/jnumed.107.043166
Source: PubMed


Dynamic measurements of infused stem cells generally require animal euthanasia for single-time-point determinations of engraftment. In this study, we used a triple-fusion reporter system for multimodal imaging to monitor human mesenchymal stem cell (hMSC) transplants.
hMSCs were transduced with a triple-fusion reporter, fluc-mrfp-ttk (encoding firefly luciferase, monomeric red fluorescent protein, and truncated herpes simplex virus type 1 sr39 thymidine kinase) by use of a lentiviral vector. Transduced cells were assayed in vitro for the expression of each functional component of the triple-fusion reporter. Transduced and control hMSCs were compared for their potential to differentiate into bone, cartilage, and fat. hMSCs expressing the reporter were then loaded into porous, fibronectin-coated ceramic cubes and subcutaneously implanted into NOD-SCID mice along with cubes that were loaded with wild-type hMSCs and empty cubes. Mice were imaged repeatedly over 3 mo by bioluminescence imaging (BLI), and selected animals underwent CT and PET imaging.
Osteogenic, adipogenic, and chondrogenic potential assays revealed retained differentiation potentials between transduced and wild-type hMSCs. Signals from the cubes loaded with reporter-transduced hMSCs were visible by BLI over 3 mo. There was no signal from the empty or wild-type hMSC-loaded control cubes. PET data provided confirmation of the quantitative estimation of the number of cells at one spot (cube). Cubes were removed from some animals, and histologic evaluations showed bone formation in cubes loaded with either reporter-transduced or wild-type hMSCs, whereas empty controls were negative for bone formation.
The triple-fusion reporter approach resulted in a reliable method of labeling stem cells for investigation in small-animal models by use of both BLI and small-animal PET imaging. It has the potential for translation into future human studies with clinical PET.

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Available from: Amad Awadallah, Oct 05, 2015
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    • "According to Lebond et al. (2009), the mean engraftment rate after systemic delivery is in a range of 0.01–0.1 %. However, imaging studies of labeled mesenchymal stem cells show that following intravenous delivery most of the cells home into the lung (Love et al. 2007). Yan et al. (2007) conclude that systemic administration of mesenchymal stem cells shortly after lung injury results in adequate engraftment, but their application at a later time might initiate fibrotic changes. "
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    ABSTRACT: Cystic fibrosis (CF) is a life-threatening autosomal recessive multi-organ disorder with the mean incidence of 0.737 per 10,000 people worldwide. Despite many advances in therapy, patients fail to have a satisfactory quality of life. The end-stage lung disease still accounts for significant mortality and puts patients in the need of lung transplantation. Even though the disease is monogenic, the trials of topical gene transfer into airway epithelial cells have so far been disappointing. It is proven that stem cells can be differentiated into type II alveolar epithelial cells. Wharton's jelly-derived mesenchymal stem cells (MSC) from non-CF carrier third-party donors could be an effective alternative to bone marrow or embryonic stem cells. The harvesting process is an easy and ethically uncontroversial procedure. The MSC cell should be applied through repetitive infusions due to rapid lung epithelial cell turnover. However, the low stem cell incorporation remains a problem. Pre-clinical studies imply that even 6-10 % of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) expression could be enough to restore chloride secretion. The route of administration, the optimal dose, as well as the intervals between infusions have yet to be determined. This review discusses the clinical potential of mesenchymal stem cell in CF patients.
    Advances in Experimental Medicine and Biology 09/2014; 833. DOI:10.1007/5584_2014_17 · 1.96 Impact Factor
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    • "It is noteworthy that there are no experimental data using stem cells transduced with an fl-mrfp-ttksr39 gene construct driven by a constitutive cytomegaloviral (CMV) promoter. Several studies have reported epigenetic silencing of CMV promoter after longitudinal passage of stem cells [16] [17]. Thus, we speculated whether the lentiviral-mediated overexpression of fl-mrfp-ttksr39 TF driven by the CMV promoter would induce aberrant characteristics or alter differentiation capacity of stem cells. "
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    ABSTRACT: Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.
    04/2014; 2014:605358. DOI:10.1155/2014/605358
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    • "Because stem cell transplantation is a valid treatment for ischemic heart disease, non-invasive molecular imaging methods have been actively pursued to monitor transplanted stem cells. First, PET reporter gene imaging is one of the most promising non-invasive molecular imaging tools, which is reliable and objective for locating transplanted stem cells in the myocardium of small animals and for quantitative analysis [12], [19], [20]. Willmann et al applied clinical PET to image large animals such as pigs, in which transplantation of human mesenchymal stem cells into the pig myocardium showed the feasibility of reporter gene imaging [21]. "
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    ABSTRACT: Non-invasive techniques to monitor the survival and migration of transplanted stem cells in real-time is crucial for the success of stem cell therapy. The aim of this study was to explore multimodality molecular imaging to monitor transplanted stem cells with a triple-fused reporter gene [TGF; herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescence protein (eGFP), and firefly luciferase (FLuc)] in acute myocardial infarction rat models. Rat myocardial infarction was established by ligating the left anterior descending coronary artery. A recombinant adenovirus carrying TGF (Ad5-TGF) was constructed. After transfection with Ad5-TGF, 5×106 bone marrow mesenchymal stem cells (BMSCs) were transplanted into the anterior wall of the left ventricle (n = 14). Untransfected BMSCs were as controls (n = 8). MicroPET/CT, fluorescence and bioluminescence imaging were performed. Continuous images were obtained at day 2, 3 and 7 after transplantation with all three imaging modalities and additional images were performed with bioluminescence imaging until day 15 after transplantation. High signals in the heart area were observed using microPET/CT, fluorescence and bioluminescence imaging of infarcted rats injected with Ad5-TGF-transfected BMSCs, whereas no signals were observed in controls. Semi-quantitative analysis showed the gradual decrease of signals in all three imaging modalities with time. Immunohistochemistry assays confirmed the location of the TGF protein expression was the same as the site of stem cell-specific marker expression, suggesting that TGF tracked the stem cells in situ. We demonstrated that TGF could be used as a reporter gene to monitor stem cells in a myocardial infarction model by multimodality molecular imaging.
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