In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

Department of Radiology, Stanford University School of Medicine, USA.
Journal of Visualized Experiments (Impact Factor: 1.33). 02/2008; 2(14). DOI: 10.3791/740
Source: PubMed


The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

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Available from: Joseph C Wu, Mar 23, 2015
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    • "Using BLI, cells derived from genetically engineered mice with luciferase reporters, or modified cells with a luciferase gene can be detected in vivo. Furthermore, by utilizing fluorophores with different emission spectra, it is possible to monitor two different biologic processes in the same animal, such as stem cell homing and sites of tissue injury (Wilson et al., 2008; Luker and Luker, 2010). As radionuclide imaging techniques, such as SPECT and PET, allow the imaging of radiolabeled cells and their interaction with biological processes in living animals, a number of reporter genes have been developed for these imaging techniques (Sun et al., 2009). "
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    ABSTRACT: Studies on stem cell are rapidly developing since these cells have great therapeutic potential for numerous diseases and has generated much promise as well as confusion due to contradictory results. Major questions in this research field have been raised as to how and in which numbers stem cells home to target tissues after administration, whether the cells engraft and differentiate, and what their long-term fate is. To answer these questions, reliable in vivo tracking techniques are essential. In vivo molecular imaging techniques using magnetic resonance imaging, bioluminescence, and scintigraphy have been applied for this purpose in experimental studies. The aim of this review is to discuss various radiolabeling techniques for early stem cell tracking, the need for validation of viability and performance of the cells after labeling, and the routes of administration in experimental animal models. In addition, we evaluate current problems and directions related to stem cell tracking using radiolabels, including a possible role for their clinical implementation.
    Full-text · Article · Jun 2011 · Journal of Cellular Physiology
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    • "In small animal models, optical imaging (including BLI and fluorescence) is able to longitudinally detect transplanted cells sensitively and reliably which has significantly facilitated the understanding of the spatial-temporal kinetics of hESC engraftment, proliferation, and teratoma formation in living subjects [31]. In many studies, both BLI and fluorescence imaging are incorporated for different purposes which can be complementary to each other. "
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    ABSTRACT: Human embryonic stem cells (hESCs) hold tremendous therapeutic potential in a variety of diseases. Over the last decade, non-invasive imaging techniques have proven to be of great value in tracking transplanted hESCs. This review article will briefly summarize the various techniques used for non-invasive imaging of hESCs, which include magnetic resonance imaging (MRI), bioluminescence imaging (BLI), fluorescence, single-photon emission computed tomography (SPECT), positron emission tomography (PET), and multimodality approaches. Although the focus of this review article is primarily on hESCs, the labeling/tracking strategies described here can be readily applied to other (stem) cell types as well. Non-invasive imaging can provide convenient means to monitor hESC survival, proliferation, function, as well as overgrowth (such as teratoma formation), which could not be readily investigated previously. The requirement for hESC tracking techniques depends on the clinical scenario and each imaging technique will have its own niche in preclinical/clinical research. Continued evolvement of non-invasive imaging techniques will undoubtedly contribute to significant advances in understanding stem cell biology and mechanisms of action.
    Full-text · Article · May 2010 · Current pharmaceutical biotechnology
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    • "While bioluminescence imaging allows sensitive detection of firefly luciferase in whole animals, light from luciferase enzymes cannot be detected readily by microscopy or flow cytometry. This limitation of luciferase enzymes can be overcome by fusing a fluorescent protein, such as GFP, to luciferase (Wang et al., 2002; Wilson et al., 2008). Although not utilized in the mice described in this review, transgenic reporter constructs including both a luciferase enzyme and fluorescent protein would integrate microscopic imaging, cellular analyses, and whole animal imaging techniques. "
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    ABSTRACT: In vivo bioluminescence imaging offers the opportunity to study biological processes in living animals, and the study of viral infections and host immune responses can be enhanced substantially through this imaging modality. For most studies of viral pathogenesis and effects of anti-viral therapies, investigators have used recombinant viruses engineered to express a luciferase enzyme. This strategy requires stable insertion of an imaging reporter gene into the viral genome, which is not feasible for many RNA viruses, and provides data on the viral component of pathogenesis but not on the host. Genetically engineered mice with luciferase reporters for specific viral or host genes provide opportunities to overcome these limitations and expand applications of bioluminescence imaging in viral infection and therapy. We review several different types of reporter mice for bioluminescence imaging, including animals that permit in vivo detection of viral replication, trafficking of immune cells, activation of key genes in host immunity to viral infection, and response to tissue damage. By utilizing luciferase enzymes with different emission spectra and/or substrates, it is possible to monitor two different biologic processes in the same animal, such as pathogen replication and sites of tissue injury. Combining imaging reporter viruses with genetically engineered reporter mice is expected to substantially enhance the power of bioluminescence imaging for quantitative studies of viral and host factors that control disease outcome and effects of established and new therapeutic agents.
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