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Imaging Reporter Genes for Cell Tracking With PET and SPECT

Posted on: Friday, 17 March 2006, 06:00 CST

By Acton, P D; Zhou, R

Transplantation of cells into damaged tissue has tremendous therapeutic potential in a number of disorders, such as Parkinson's disease and myocardial infarction. Positron emission tomography (PET) and single photon emission tomography (SPECT) are highly sensitive imaging modalities, which can detect and track these cellular implants through a number of mechanisms. The stable transfection of cells with a reporter gene, which can be visualized using a radioactive PET or SPECT reporter probe, allows the repeated visualization of the migration and function of cells. These imaging techniques can be used to assess cell trafficking with methods that are easily translatable to humans. This review describes several reporter genes for PET and SPECT, and compares them against other techniques and imaging modalities.

KEY WORDS: Reporter gene * Cell tracking * Stem cell * PET * SPECT.

The reporter (or marker) gene concept has become standard in various molecular biology protocols. In general, the function of the promoter and other regulatory regions of a gene are assessed through the regulated expression of a reporter gene. Classic reporter genes include LacZ (encoding the enzyme β-galactosiclase) and green fluorescence protein (GFP). The reporter gene can either be fused with the gene of interest resulting in expression of a chimeric protein, or it can be expressed as a separate protein. In the latter case, the reporter may have its own promoter or it can share a common promoter sequence with the gene of interest. Expression of a hicistronic messenger RNA is followed by cap-dependent translation of one protein and cap-independent translation of the other protein via an internal ribosome entry site (1RES).1 Assays for these reporter genes require invasive methods (e.g. histology analysis of biopsy or necropsy samples) except for transparent or superficial tissues which may allow non-invasive GFP detection.

Transplantation of cells, such as stem cells or progenitor cells, into damaged tissue has tremendous potential for the treatment of a number of disorders, such as Parkinson's disease, Alzheimer's disease, and myocardial infarction. Following the fate of transplanted cells in vivo is a vital step in determining the efficacy of the implant. Non-invasive imaging techniques have been used to monitor cells, and can provide information on three important features of cellular implants; cell tracking (are the cells reaching the target tissue?), cell viability (once they get there are they alive?), and cell function (if they are alive are they actually functioning?).

Radionuclide imaging techniques, such as positron emission tomography (PET) and single photon emission tomography (SPECT), allow the imaging of radiolabeled markers and their interaction with biochemical processes in living animals. Due to their exquisite nanomolar (<10^sup -9^ M) sensitivity, PET and SPECT are able to measure biological processes at very low concentrations. The mass of radiotracer injected is extremely small, and generally does not impact the biological system under study. Technological developments of both PET and SPECT have led to the implementation of specialized systems for small animal imaging, with much higher spatial resolution (<2 mm),2-12 which has dramatically advanced the field of cell tracking in animal models in vivo.

Figure 1.-Schematic diagrams of three different types of reporter genes. A) HSV1-tk expression results in the production of the enzyme HSV1-TK. This enzyme phosphorylates the radiotracer ^sup 18^F-FHBG, causing it to become trapped inside the cells. B) The D^sub 2^R reporter gene produces the dopamine D2 receptor, which migrates to the cell surface. This seven-transmembrane receptor binds radioactive ligands, such as ^sup 18^F-FESP. C) The sodium-iodide symporter actively transports radioactive iodine, and other analogous tracers, into the cell. However, without subsequent organification, the trapping is incomplete, and some iodine may leak out.

PET and SPECT have sufficient sensitivity to detect and track cell implants through a number of mechanisms. Imaging plays a role in monitoring short-term cell tracking, longer-term cell survival and function, and as a surrogate marker of implant efficacy. Labeling implanted cells with relatively long-lived isotopes, such as ^sup 111^In for SPECT and ^sup 64^Cu for PET, allows shortterm, real-time cell tracking, to determine bioclistribution and availability in the target organ. However, these cell labeling techniques have some significant disadvantages, such as efflux of the tracer from the cells over time, and do not give any information about the function or survival of the cells. An alternative approach involves the stable transfection of cells with a reporter gene, such as herpes simplex virus type-1 thymidine kinase (HSV1-tk), whose expression can be visualized using a radioactive PET or SPECT reporter probe. Stem cells carrying reporter genes permit longitudinal monitoring of their survival and differentiation status within the context of physiologically authentic environments. For monitoring the survival of cells following their engraftment, a strong viral promoter would allow constitutive expression of the reporter gene whenever the cell is alive. An additional role for imaging involves measurement of functional recovery of the organ or tissue under study, such as assessing dopaminergic function in Parkinson's disease, which provides a surrogate marker of implant efficacy. PET and SPECT imaging can be used to assess cell trafficking, function, and efficacy, using methods which are easily translatable to humans. Reporter gene approaches are particularly valuable, as they provide information not only on cell trafficking, but also on cellular function and survival.

Figure 2.-A) A representative lentivector plasmid is shown with a reporter gene (HSV1-tk) under a viral promoter, SV40. A drug selection marker. Neo (neomycin resistance gene) with its promoter also is present. Other features of this plasmid include the LTR (long terminal repeats) for viral packaging and reverse transcription. EM7 fur driving expression of Neo gene expression in bacteria for selection, ampicillin resistance gene also is for selection in bacteria, and RRE (Rev response element) for nuclear export of the unspliced viral mRNA. B) Stable transfection of cells with a reporter gene. A plasmid or a viral vector can lie used to transfert the cells, followed by selection of stable expression clones with an antibiotic.

A number of reporter genes have been developed for radionuclicle imaging, generally divided into three different classes, using either receptors, transporters, or enzymes (Figure 1). In receptor- based reporters, the radioactive tracer binds to a gene-encoded protein, such as the dopamine D2 receptor (D^sub 2^R). Alternatively, enzyme-based systems use reporter gene production of a specific enzyme, such as HSVl-TK. resulting in the accumulation of a radioactive metabolite of the tracer.

Figure 3.-PET image of ^sup 18^F-FHBG (in color, arrowed) overlaid on a gray-scale image ot ^sup 18^F-FDG uptake in a rat. The image is shown in a cardiac long axis view. Cells transfected with HSVl-tk were injected into the myocardium, and imaged 1 h after injection of ^sup 18^F-FHBG ( 10 min scan). Since FHBG is only taken up by engrafted cells, another tracer. FDG, was used to delineate the myocardium for localization of FHBG signal. Immediately following the ^sup 18^F-FHBG scan, the animal was injected with ^sup 18^F-Fl)G and scanned 1 h later ( 15 min scan). The ^sup 18^F-FDG image gave a useful indication of anatomy, and was in perfect spatial alignment with the image of HSV1-tk gene expression, indicating uptake of the tracer in the myocardium. The color image has had a threshold applied to remove some background signal.

This review summarizes the methods for labeling cells with reporter genes, and describes techniques for imaging each reporter system, and how they have been applied to cell tracking applications.

Transfecting cells with reporter genes

The approach of utilizing a reporter gene, whose expression can be quantified by the imaging signal, is attractive for the evaluation of survival simply because dead cells will not express the reporter. To ensure that every cell is accounted for and the reporter expression is not diluted upon cell division, it is necessary to establish cell clones that are stably expressing the reporter gene. As shown in Figure 2A, a molecular construct (plasmid) is made first that contains the reporter gene and its promoter; it should also contains a drug selection marker (here neomycin-resistant gene, Neo, is used as an example). The plasmid vector can be used directly to transfect the cells; their entry into cells are facilitated by cationic lipid based transfecting agents or by electroporation.13

Alternatively (shown in Figure 2B), the plasmid can be wrapped into a recombinant viral vector, which is then used to transfect the cells. After transfection, cells are subjected to a selection procedure in which only cells that have integrated the reporter gene in the genome will be selected. This is achieved by incubation of cells with a suitable antibiotic drug; cells not expressing the antibiotic-resistant gene (.e.g. Neo) will be killed by the drug (e.g. neomycin). Clones of surviving cell\s will be collected and amplified for further characterization. The level of reporter gene expression may vary among the clones; therefore, the clone that stably expresses the highest level of the reporter gene is chosen. The lentiviral vector is preferred over other viral vectors (e.g. adeno or retrovirus) for stable transfection of stem cells because the lentivirus can transduce both dividing and non-dividing cells and enables the stable integration of transgenes into the host cell genome. Furthermore silencing of the foreign gene, which is often observed in retroviral mediated transfection, occurs at a much lower frequency in lentivirus-mediated transfection.14, 15 Viral transfection. usually has higher efficiency than electroporation or transfecting agents. It has been shown that the expression of the foreign gene delivered by a lentiviral vector was maintained throughout differentiation into progeny of all three germ layers both in vitro (embryoid body) and in vivo (teratomas); thus, the transduced embryonic stem cells seemed to have retained the capability for self-renewal and their pluripotent potential.16 However, the effect of such genetic modification should be evaluated for a specific type of stem cells and the reporter gene involved.

For reporting cell survival, a strong viral promoter, such as the CMV promoter, is usually chosen to drive the expression of the reporter gene since it usually leads to constitutive expression of the reporter. However, it has been reported that the CMV promoter is inactive or has reduced activity in undifferentiating embryonic stem cells compared to a mammalian cellular promoter, such as the polypeptide elongation factor 1 alpha (EF1α) promoter.17-19

The HSV1-tk reporter system

From suicide gene to reporter gene

The HSV1-tk gene expresses a viral thymidine kinase (note that in this article, HSV1-tk represents the gene while HSV1-TK represents the protein). TK phosphorylates a range of substrates, including thymidine (the natural substrate), analogs of pyrimidine and acycloguanosines. The resulting monophosphates are converted by cellular enzymes to di- and triphosphates, which are trapped inside the cells. These metabolites, if present at a sufficiently high concentration, will kill the cells, therefore, HSV1-tk has been used as a suicide gene in many gene therapy protocols 20-23 or as a negative selection marker to eliminate TK-expressing cells in standard molecular biology protocols. By taking advantage of the high sensitivity of PET or SPECT imaging modalities, it is possible to administer in tracer quantity the substrate that is labeled with a positron or single-photon emitting radioisotope for detection of HSVl-tk expression,24-38 and, as such, toxicity to tk expressing cells is negligible. If a target gene (e.g. a therapeutic gene) is linked to the HSV1-tk gene by molecular manipulations, the detection of HSV1-tk expression can report the location and level of the target gene expression, which may not be easily assessed. Various molecular biology methods can be employed to link the reporter and the target gene.39

Figure 4.-Pyrimidine derivatives for detection of HSV1-tk expression (adapted from Tjuvajev et al.38).

The concept of the reporter gene is applicable to reporting the survival status of grafted cells. Since a promoter of viral origin (such as cytomegalovirus, CMV) is thought to be constitutively active and minimally regulated by physiological processes in cells, HSVl-tk under the control of such a promoter is most commonly used for reporting the survival status of cells. PET imaging detection of H9c2 cells that are transiently transfected with CMV-HSVl-tk and were subsequently injected into rat myocardium has been reported.40 Figure 3 shows ^sup 18^F-FHBG signal (color) from HSVl-tk expressing cells grafted in the rat myocardium, overlaid on the ^sup 18^FDG image (grayscale), which delineates myocardial anatomy.

In order to quantify the survival of grafted cells, it is necessary that each cell carry the reporter gene and, furthermore, the reporter gene be stably integrated into host cell genome. The latter is to ensure that the reporter will not be lost or diluted upon cell division. In addition, accurate quantification of reporter gene expression is required.

Imaging tracers for HSV1-tk detection

A variety of uracil analogs and acycloguanosines have been used for imaging HSVl-tk expression and their structures are summarized in Figures 4 and 5 (adapted from Tjuvajev et al.).38 Cellular uptake of uracil derivatives and acycloguanosines are likely mediated by nucleoside transporters, which in mammalian cells, include two major families: the equilibrative nucleoside transporters (ENTs), and the concentrative nucleoside transporters (CNTs).41, 42 Therefore, the cellular retention of these tracers is dependent on the level of the transporters on the target cells, the efficiency of a tracer to utilize these transporters, and the level of HSV1-tk expression. Cells (RG2 or C6 glioma) that stably express HSV1-tk are found to have much higher retention of FIAU (a uracil analog) than FHBG, FHPG or PCV (acycloguanosines) 38, 43 suggesting less efficient uptake of acycloguanosines by the nucleoside transporters. In contrast, FHBG and PCV accumulation were much higher in cells that were transfected with adenovirus containing HSV1-tk suggesting that adenoviral transfection might have altered intracellular levels of thymidine. In addition, FHBG and PCV were found to accumulate to a significantly greater extent than FIAU in glioma cells stably expressing a mutant HSV1-tk (i.e., sr39tk).43 In murine embryonic stem cells that are stably express HSV1-tk, accumulation of FIAU was over 40-fold higher than the untransfected control cells, whereas accumulation of FHBG was about 5-8-fold higher than the control. Therefore, before an in vivo cell tracking study, in vitro comparisons of various tracers for their intracellular accumulation are highly recommended to achieve optimal tracer selection for a specific type of cells.

Despite the promising results obtained using this reporter system in cell tracking, monitoring cells in the brain is hampered by the fact that the PET and SPECT reporter probes for HSVl-tk expression do not penetrate the intact blood-brain barrier (BBB).44, 45 Imaging with this reporter system in the brain currently is not possible, although disruptions in the BBB, such as those resulting from cerebral hemorrhage or tumors, may allow influx of the tracer into the brain.

Figure 5.-Acydoguanosine derivatives for detection of HSVl-tk expression (adapted from Tjuvajev et al.38).

Quantification of HSV1-tk expression

Quantification of HSV1-tk expression in most studies is obtained by estimation of %ID (injected dose) from signal intensities on static images. However, the intensity represents the amount of tracer that has been metabolized by the HSV1-TK enzyme. It has been recognized that the amount of a product (metabolites) generated from an enzymatic reaction does not represent the concentration of the enzyme; a kinetic study, therefore, is necessary to calculate the flux, which is proportional to the enzyme concentration, a true measure of the reporter enzyme expression.46 In the radionuclide imaging field, tracer kinetic models have played a vital role in quantification of glucose metabolism, regional blood flow, or concentrations of receptors or other binding sites. Kinetic model based quantification generally requires knowledge of the activity concentrations in arterial blood, which provides the input function to the model. Absolute quantification of the expression of the HSV1- tk reporter gene is complicated by the fact that the amount of tracer in a ceil represents not only HSV1-TK enzyme activity, but also transport of the probe across the cell membrane.47 This makes it difficult to quantify, and more dependent on other mechanisms which are not related to gene expression.

Recent work 30 demonstrated that dynamic PET imaging combined with tracer kinetic models provides estimation of the rate of phosphorylation by HSV1-TK and is strongly correlated with TK activity measured by standard but invasive methods. Their work also suggested that %ID is a poor indicator of the HSV1-tk expression.

Other reporter systems

Receptor-based reporters

Alternative methods for imaging gene expression utilize the expression of receptor or transporter proteins. One such system, using the dopamine D2 receptor (D^sub 2^R), has been developed which uses the widely available PET D^sub 2^R tracer [^sup 18^F]fluoroethylspiperone (FESP) as a reporter probe. FESP binds with high affinity to the D^sub 2^R, and has been shown to provide a quantitative measure of D^sub 2^R expression in living animals.48 However, this technique also has potential problems, such as occupancy of the ectopic D^sub 2^R by the endogenous agonist. When a ligand activates the D^sub 2^R, cellular levels of cyclic adenosine monophosphate (cAMP) may be affected which could lead to physiologic consequences on the tissue under study.47 However, mutant strains of the D^sub 2^R which do not activate the signal pathway have been developed and studied with PET.49

The D^sub 2^R reporter is one of the few systems which could be used for cell tracking in the brain, since the reporter probes for PET and SPECT readily cross the intact BBB. However, in the brain, the presence of endogenous D^sub 2^R, particularly in the striatum, would provide a large background signal, and make it difficult to resolve the presence of the D^sub 2^R reporter system. The same problem applies to the dopamine transporter (DAT), which has been used as a reporter gene for SPECT in tissue outside the brain.50

A regulatable reporter system using the D^sub 2^R has been developed, in which the reporter gene is under the transcriptional control of a tetracycline-responsive element.51 Cells stably transfected \with this inclucible system have been injected into rats and imaged using the PET reporter probe FLB 457. Doxycycline- induced reporter gene activity, measured with PET, correlated with D,R-expressing cell fraction in the xenograft tumors.

Sodium-iodide symporter

The sodium-iodide symporter (NIS) has been suggested as a possible reporter gene.52-54 The NIS occurs naturally in high concentrations in the thyroid, with lower concentrations in salivary glands, stomach, thymus, breast, and other tissues. The symporter is a membrane glycoprotein, and provides an active transport mechanism for sodium and iodine ions into cells. The expression of the symporter can be imaged with radioactive iodine, such as ^sup 123^I for SPECT or ^sup 124^I for PET, or other tracers which bind to the same site, such as ^sup 99m^Tc-pertechnetate. A broad range of cell types and delivery mechanisms have been used with the NIS reporter. The NIS is not immunogenic, and its endogenous expression is limited to a few tissues, allowing it to be used in a variety of imaging applications. However, the use of NIS as a reporter system has been hampered somewhat by the efflux of the radiotracers out of non- thyroid tissue. In the thyroid, organification of the iodine occurs, catalyzed by the enzyme thyroperoxidase, which effectively traps the iodine after it is transported into the tissue. Co-expression of this enzyme with the NIS reporter gene may be required to improve the retention of radioactive iodine in transfectecl cells.55

The NIS reporter gene has been used with a tissuespecific promoter to develop a transgenic mouse model in which the reporter is active only in cardiomyocytes.56 This enables PET or SPECT imaging of cardiacspecific reporter gene expression and cellular differentiation after progenitor or stem cell implantation. Similarly, neural stem cells have been labeled with the NIS reporter, and used to image cell trafficking in vivo.57 The same group addressed the issues of reporter gene silencing, in which expression of NIS decreases over time with progressive cell differentiation, by pharmacologie intervention before implantation.57 However, it should be stressed that, like the HSV1- tk system, imaging NIS expression in the brain is not possible as the tracers do not cross the intact BBB. limiting its use to cell trafficking studies outside the brain, or where the barrier has been disrupted.

Tracking of tumor cells with the NIS reporter demonstrated accurate quantification of cell number, confirmed with a dual reporter system in which the NIS was coupled to a luciferase optical reporter.58 SFECT imaging of metastatic lung tumor nodules resulting from systemic injection of tumor cells transfected with NIS, showed excellent correlation with histology, and the ability to detect lesions as small as 3 mm.59

Other reporters

The somatostatin receptor is overexpressed in a number of neuroendocrine tumors, and has been used as a reporter system for SPECT using somatostatin analogs such as [^sup 111^In]octreotide and other radiolabeled peptides.60, 61 This reporter system holds tremendous promise for cell tracking, and should be translatable relatively easily to humans, since the SPECT reporter probes already are used routinely in clinical practice.

Comparison with other imaging modalities

Magnetic resonance imaging/spectroscopy (MRI/S)

In the past decade, there has been significant effort in the development of marker genes that can be detected in vivo by various imaging modalities. For ^sup 1^H MR imaging, an engineered transferrin receptor62 and ferritin 63-64 have been proposed as reporter genes. Their expression was detected by the signal loss due to T^sub 2^/T^sub 2^* decrease induced by the delivery of engineered transferrin or accumulation of ferritin molecules, respectively. A caged Gd compound has been introduced that is screened from contact with water by a galactopyranose residue that can be removed by (galactosidase. This reaction produces an analog of Gd-DTPA, a well known MRI contrast agent, which is detected with T^sub 1^-weighted imaging.65 For MRS, creatine kinase has been introduced as a liver specific marker gene 46 and Drosophila melanogaster arginine kinase as a marker for gene therapy of muscle diseases.66 Cytosine deaminase converts 5-flourocytosine into the anti-neoplastic agent 5- fluorouracil (5-FU) and has been used as a marker to report the accumulation of 5-FU by F-19 MRS.67

Super-paramagnetic iron oxide (SPIO) particles in the form of Feridex (or ferumoxides) are an FDA approved MRI contrast agent, although it is used "off-label" in cell tracking applications. Feridex labeling of stem cells 68, 69 has been applied for tracking stem cells in the brain,68, 70 heart71 and other organs. The addition of agents such as poly-L-lysine or protamine sulfate 72 in the labeling solution greatly improves the labeling efficiency. The relatively low toxicity observed in Feridex labeled cells makes it attractive for clinical application. The sensitivity of detection can be increased to achieve even single cell detection by using a much larger (micron size) particles.73, 74 Superior spatial resolution achieved by cardiac MRI allows delineation of myocardial landmarks for detailed mapping of spatial distribution or migration of grafted cells. However, this method suffers major limitations of any labeling approach; label release as the consequence of cell lysis and label dilution as the result of cell division. Both events limit its ability to quantify cell survival. For example, this method detects the presence of SPIO but seems not to distinguish labels contained in stem cells versus in macrophages that accumulate to the site of injection.75

Multimodality imaging

Imaging modalities such as MRI can achieve higher spatial resolutions for better delineation of tissue anatomy, despite lower sensitivity than radionuclide imaging approaches. It is likely that one optimal imaging modality could be used for monitoring each aspect of stem cell grafting, such that multiple image modalities can be used in concert to obtain a complete picture of stem cell survival, differentiation, and therapeutic benefit.

Preliminary efforts utilizing radionuclide imaging for cell tracking 76 and high resolution MR imaging for evaluation of global and regional cardiac function 77, 78 suggest that this approach is very promising to accomplish the challenging task of in vivo monitoring of stem cell grafting.

An important recent development is the multimodality fusion reporter system, which can be studied using both optical and radionuclide imaging.79, 80 This gene expresses a tri-fusion protein, which contains coding regions for red fluorescent protein, Renilla luciferase, and the HSV1-TK enzyme. Cellular fluorescence expression can be monitored using microscopic techniques, while in vivo monitoring can be performed using both optical and PET or SPECT methods. This fusion reporter offers the possibility of using the particular imaging technique that best suits the application; fluorescence for studying individual cells or for cell sorting, bioluminescence for high sensitivity in vivo imaging in small animals, and PET or SPECT when quantitative accuracy is important, or for the translation to humans.

Further developments of optical imaging techniques, such as quantum dots,81 diffuse optical tomography,82 and optical coherence tomography,83, 84 offer tremendous enhancements to currently available technology. Coupled with multi-modality reporter systems, optical imaging has an important role to play in gene therapy and cell tracking.

Comparison with other radionuclide imaging techniques

Direct cell labeling

Direct cell labeling with a radioactive isotope has been used for many years to track cells in vivo. In general, a radioisotope with a relatively long decay half-life is used to enable the tracking of cells over periods of several hours, or even days, such as ^sup 111^In (T^sub 1/2^=2.8 days) for SPECT or ^sup 64^Cu (T^sub 1/2^= 12.7 h) for PET. The isotope is carried into the cells using a lipophilic chelator, which governs the initial extraction of the tracer into the cells. Once inside the cells, a trapping mechanism reduces the lipophilicity of the molecule, and the isotope is retained.

The most attractive aspect of direct cell labeling with compounds such as ^sup 111^In-oxine or ^sup 64^Cu-pyruvaldehyde-bis(N4- methylthiosemicarbazone) (^sup 64^Cu-PTSM) is the simplicity. Cells are incubated with the radioactive tracer, allowing the lipophilic molecules to diffuse across the cell membrane, and the isotope becomes trapped. Following incubation, the cells are washed to remove any unbound activity, and the cells injected into the host. Labeling efficiencies close to 100% are not uncommon, although this depends critically on the incubation time and environment.

Numerous cell tracking experiments have been performed using cells labeled with a radioactive marker. Trafficking of systemically injected glioma cells in mice was monitored with PET using ^sup 64^Cu-PTSM, indicating that significant uptake occurred in lungs and liver.85 Labeling of the cells did not interfere with cell viability or proliferation, but efflux of the activity out of the cells occurred, indicating an incomplete trapping mechanism. Pinhole SPECT imaging of lymphocyte trafficking in a mouse model of colitis, using cells labeled with ^sup 111^In-oxine, demonstrated the homing of cells to areas of inflammation.86 Hematopoietic progenitor cells were labeled with nlln-oxine, and injected locally into the heart in a rat model of myocardial infarct.87 While gamma imaging revealed homing of the progenitor cells to infarcted myocardium, the labeling procedure had significant negative effects on cellular function, impairing proliferation and function. A dual isotope SPECT study of ^sup 111^In-labeled cardiomyoblasts and ^sup 99m^Tc-sestamibi enabled the imagin\g of both cell trafficking and cardiac function simultaneously.76 Significant washout of the ^sup 111^In radioisotope occurred over a period of a few days, which illustrates one of the major weaknesses of the direct labeling process(the inability to distinguish between loss of tracer and loss of cells. If the radioactive label diffuses out of the cells, the imaging signal will reduce over time. However, this is indistinguishable from the loss of labeled cells from the target tissue, and gives no information on cell viability or function.

While direct cell labeling has many attractive features, the drawbacks include radiotoxicity effects, loss of label from cells, dilution of signal from cell division, and lack of information on cell function or viability. In particular, the incomplete information regarding cell survival with direct lateling has teen the impetus tehind developing reporter genes to track cells. The same cardiomyoblasts which were monitored with ^sup 111^In-oxine labeling,76 have been transfected with the HSV1-sr39tk reporter gene and imaged with a PET reporter, ^sup 18^F-FHBG.32, 88 While ostensibly demonstrating the same cell tracking information as direct cell labeling, the PET signal can now confidently be ascribed to surviving cells. If the cells die, the reporter gene switches off, and the probe does not accumulate in the cells. Further, cell division does not dilute the signal. However, without stable transfection of the gene, in vivo reporter protein activity tends to decrease over time because the reporter DNA remains episomal (not integrated into the host cell genome) and will be lost upon cell division.

An additional benefit of the reporter gene approach is the possibility of linking a therapeutic gene to the reporter, allowing the system to monitor both cell trafficking and gene therapy simultaneously. For example, skeletal myoblasts have been used to deliver a therapeutic gene, such as vascular endothelial growth factor (VEGF), which may help the generation of new vessels in the infracted region being regenerated by the grafted myoblasts.89 Coupling a reporter gene to stem or progenitor cells would allow tracking of not only cell delivery and survival, but also the expression of the therapeutic gene.

Cell function

While imaging of cell trafficking is an important tool, it is vital to appreciate that none of these methods give direct information on cell function. Tracking methods, such as reporter genes, monitor viable cells, but these are not necessarily functional cells. Functionality tests are required to ensure the grafts are leading to genuine recovery, and imaging plays a vital role in the noninvasive assessment of cellular function. For example, simply tracking stem cells implanted in the brain in Parkinson's disease would give no information on whether those cells had acquired the necessary specialization, and were producing clopamine. However, measurement of dopamine synthesis, using ^sup 18^F-FDOPA and PET, would provide a surrogate marker of the recovery of clopaminergic function. Indeed, longitudinal imaging of ^sup 18^F- FDOPA would provide vital information on changes in dopamine- producing cells over time, and should correlate with any improvement in clinical symptoms.90 Clearly, assessing die efficacy of cell transplants requires both cell tracking and cell function measurements.

Conclusions

Cell transplants offer tremendous potential for the treatment of a wide variety of diseases, such as Parkinson's disease, heart disease, cancer, etc. Tracking implanted cells is vital to monitor the delivery and viability of the grafts over extended periods of time. Non-invasive imaging of cell trafficking can be used to monitor the delivery of implants in tissue, using a variety of techniques and imaging modalities. Information on transplanted cells can be derived from radioactive cell labeling, or tagging with MRI contrast agents(however, these techniques do not offer any information on cell viability. Reporter genes, coupled with PET or SPECT reporter probes, offer both cell tracking and viability measurements with extremely high sensitivity.

Acknowledgements.-The authors are indebted to Drs. Datta Ponde, Hui Qiao and Daniel Thomas for their help with the studies of gene expression in rat myocardium.

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P. D. ACTON 1, R. ZHOU 2

1 Department of Radiology, Thomas Jefferson University

Philadelphia, PA, USA

2 Department of Radiology, University of Pennsylvania

Philadelphia, PA, USA

This work was supported by National Institutes of Health grants R01-EB001809 (PDA). R01-NS048315 (PDA), and R21-EB002473 (RZ), and by the Pennsylvania State Department of Health Tobacco Block Grant (RZ).

Address reprint requests to: Dott. Acton P. D., Department of Radiology, Thomas Jefferson University, 796 G Main Building. 132 South 10th Street, Philadelphia. PA 19107-5244, USA. E-mail: paul.acton@iefferson.eclu

Copyright Edizioni Minerva Medica Dec 2005

Source: Quarterly Journal of Nuclear Medicine, The

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