Journal of Nuclear Medicine, published on January 25, 2013 as doi:10.2967/jnumed.112.108316 18F-FDG Labeling of Mesenchymal Stem Cells and Multipotent Adult Progenitor Cells for PET Imaging: Effects on Ultrastructure and Differentiation Capacity Esther Wolfs1, Tom Struys2,3, Tineke Notelaers4, Scott J. Roberts5, Abhishek Sohni4, Guy Bormans6, Koen Van Laere1, Frank P. Luyten5, Olivier Gheysens1, Ivo Lambrichts2, Catherine M. Verfaillie4, and Christophe M. Deroose1 1Division of Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium; 2Lab of Histology, Department of Functional Morphology, Biomedical Research Institute, Universiteit Hasselt, Diepenbeek, Belgium; 3Biomedical NMR Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium; 4Stem Cell Institute, Department of Development and Regeneration, KU Leuven, Leuven, Belgium; 5Laboratory for Skeletal Development and Joint Disorders, KU Leuven, Leuven, Belgium; and 6Laboratory for Radiopharmacy, KU Leuven, Leuven, Belgium influenced by 18F-FDG labeling. Small-animal PET studies con- Because of their extended differentiation capacity, stem cells firmed the intracellular location of the tracer and the possibility have gained great interest in the field of regenerative medicine. of imaging injected prelabeled stem cell types in vivo. There- For the development of therapeutic strategies, more knowledge fore, direct labeling of MSCs and MAPCs with 18F-FDG is a suit- on the in vivo fate of these cells has to be acquired. Therefore, able technique to noninvasively assess cell delivery and early stem cells can be labeled with radioactive tracer molecules retention with PET. such as 18F-FDG, a positron-emitting glucose analog that is Key Words: mesenchymal stem cell; multipotent adult progenitor taken up and metabolically trapped by the cells. The aim of this cell; 18F-FDG; radiotoxicity; PET study was to optimize the radioactive labeling of mesenchymal J Nucl Med 2013; 54:1–8 stem cells (MSCs) and multipotent adult progenitor cells DOI: 10.2967/jnumed.112.108316 (MAPCs) in vitro with 18F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). Methods: Mouse MSCs and rat MAPCs were used for 18F-FDG uptake kinetics and tracer retention studies. Cell metabolic ac- tivity, proliferation, differentiation and ultrastructural changes In the last few decades, adult stem cells have gained in- after labeling were evaluated using an Alamar Blue reagent, terest because they possess great potential for tissue engi- doubling time calculations and quantitative TEM, respectively. neering and regenerative medicine, with limited ethical Additionally, mice were injected with MSCs and MAPCs prela- concerns regarding their use (1,2). Nearly all human tissues beled with 18F-FDG, and stem cell biodistribution was investi- gated using small-animal PET. Results: The optimal incubation contain a source of adult stem cells, where they function in period for 18F-FDG uptake was 60 min. Significant early tracer tissue repair after injury or for natural tissue turnover. De- washout was observed, with approximately 30%–40% of the pending on their origin, these cells possess the ability to tracer being retained inside the cells 3 h after labeling. Cell differentiate into a variety of cell types of the body. viability, proliferation, and differentiation capacity were not se- Mesenchymal stem cells (MSCs) are self-renewing 18 verely affected by F-FDG labeling. No major changes at the multipotent stem cells that were first isolated from bone ultrastructural level, considering mitochondrial length, lysosome marrow (3); however, other body tissues such as muscle (4), size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed adipose tissue, (5) and dental pulp (6) were also proven to with TEM. Small-animal PET experiments with radiolabeled contain a population of MSCs. These cells have the ability MAPCs and MSCs injected intravenously in mice showed a pre- to differentiate into different cell types along the mesen- dominant accumulation in the lungs and a substantial elution of chymal lineage, with the ability to form bone, cartilage, and 18F-FDG from the cells. Conclusion: MSCs and MAPCs can be adipose tissue (7,8). They appear to be good candidates for 18 successfully labeled with F-FDG for molecular imaging pur- clinical use, because they can be expanded easily in vitro poses. The main cellular properties are not rigorously affected. and they lack immunogenicity, and they have significant TEM confirmed that the cells’ ultrastructural properties are not trophic effects on endogenous (stem) cells. Furthermore, Received May 7, 2012; revision accepted Sep. 25, 2012. MSCs possess immunity-modulating properties, through For correspondence or reprints contact: Christophe Deroose, UZ Leuven, the inhibition of immune cell function and proliferation, Division of Nuclear Medicine, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. and their use as immunomodulators is being explored clin- E-mail: [email protected] ically (9). Published online nnnn. COPYRIGHT ª 2013 by the Society of Nuclear Medicine and Molecular Multipotent adult progenitor cells (MAPCs) may repre- Imaging, Inc. sent another clinically useful source of adult stem cells. 18F-FDG LABELING OF MSCS AND MAPCS • Wolfs et al. 1 jnm108316-pm n 1/24/13 Copyright 2013 by Society of Nuclear Medicine. MAPCs have been isolated from mouse, rat, and human Gibco) and incubated with a 0.74 MBq/mL solution of 18F-FDG bone marrow cells and from murine muscle and brain in glucose-free Dulbecco modified Eagle medium (Gibco) at 37°C. (10,11). In contrast to MSCs, they have broader differenti- After incubation, cells were washed 3 times with PBS, and tracer ation ability; in rats, for instance, they differentiate into concentration in the cell fraction was measured using a g-counter various cell types of the 3 different germ layers, including (Perkin Elmer). For uptake kinetics assessment (n 5 6), cells were incubated endothelial cells, smooth muscle cells, osteoblasts, adipo- with 18F-FDG for different periods (5, 15, 30, 45, 60, 90, 120, 150, cytes, hepatocytes, and neural stem cell-like cells (12,13). 180, 210, 240, 270, 300, and 360 min). Cellular tracer concentra- Additionally, MAPCs have immune modulatory properties tion was similarly measured after each time point. similar to MSCs (11). Tracer washout was measured by incubation of cells as Several preclinical studies have already shown the described above for 1 h, followed by 3 PBS washes and a second positive effects of grafted stem cells on the repair and incubation on cold PBS for different periods (0, 5, 15, 30, 60, 90, regeneration of various tissues in vivo, such as bone (14), 150, and 180 min). Cellular tracer concentration was similarly cartilage (15), and myocardium (16). Additionally, MSCs measured after each time point (n 5 6). and MAPCs injected intravenously have been shown to home to tumors in vivo, making them potential vehicles In Vitro Toxicity Assays for anticancer therapy (17,18). Moreover, both MSCs and For the determination of tracer radiotoxicity on MSCs and MAPCs, cells were seeded and incubated with a 0.74 MBq/mL MAPCs have gained interest for in vivo applications be- solution of 18F-FDG in glucose-free Dulbecco modified Eagle cause of their immunomodulatory properties (11), and both medium at 37°C for 30, 60, 90, 120, and 180 min. Different cell cell types are being tested in phase I and II clinical studies. type–specific properties were assessed, such as cell proliferation, Together with the development of new stem cell–based cell metabolic activity, differentiation capacity, and ultrastructural therapeutic approaches, the search for effective and non- properties (n 5 3). invasive imaging techniques for stem cell tracking in vivo Cell Proliferation. The effect of radiolabeling on cell pro- is crucial. In vivo imaging can provide information on cell liferation was assessed for 10 d by determining cell doubling biodistribution, quantification of targeted cells, in situ per- times (DTs), calculated from cell counts and interpassage time sistence, survival, and function (19). according to the following formulas (24): Nuclear imaging modalities such as PET can serve as CD 5 lnðNf=NiÞ=lnð2Þ noninvasive, sensitive, quantitative, and longitudinal ap- proaches for in vivo stem cell tracking. Stem cells can be DT 5 CT=CD; labeled with low doses of radioactive tracer molecules and 18 detected on decay. F-FDG has been used for the imaging where CT is the interpassage period, CD the cell doubling number, of initial stem cell biodistribution after injection into sub- Nf the number of calculated cells at the end of passage x, and Ni jects (20). the initial number of seeded cells for passage x. Data are presented 18F-FDG has already been used to image different cell as DT values relative to unlabeled control cells. types, such as dendritic cells, bone marrow–derived cells, Cellular Metabolic Activity. Cell metabolic activity after radio- or hematopoietic stem cells, both in preclinical models and labeling was measured using the Alamar Blue (Invitrogen) reagent. in humans (21–23). However, the effects of labeling stem After radiolabeling, cells were seeded, and 10% of Alamar Blue cells with 18F-FDG have not been extensively studied with was added to the culture medium for 2 h at 37°C. Fluorescence respect to their function and viability. was measured at an excitation wavelength of 570 nm, with an The aim of this study was thus to optimize and emission of 585 nm in a Victor 1420 plate reader (Perkin Elmer). The effect on cell metabolism was monitored for 10 d, and data are characterize radiolabeling of MSCs and MAPCs with 18F- presented as values relative to unlabeled control cells. FDG. We performed a thorough examination of stem cell MSC and MAPC Differentiations. Information about MSC and properties and function after labeling and presented for the MAPC differentiation is given in the supplemental data.