Personalized diagnosis and treatment with allogenic or autologous cells are becoming a reality in the field of medicine (1, 2). Cytotoxic or engineered T cells are under clinical trial for the treatment of hematopoietic or other malignant diseases (3). Contrast agent-tagged macrophages are used as cellular probes to image the early inflammatory processes in macrophage-rich conditions such as inflammation, atherosclerosis, and acute cardiac graft rejection (4). The roles of stem cells are under intensive investigation in the therapeutic and regenerative medicine such as regenerating cardiomyocytes, neurons, bone, and cartilage (1). Genetically modified cells are used to treat genetic disorders (5). With the promising results from these studies, a critical issue is how to monitor the temporal and spatial migration and the homing of these cells, as well as the engraftment efficiency and functional capability of the transplanted cells in vivo (6, 7). Histopathological techniques have only been used to obtain information on the fate of implanted cells at the time of animal euthanization or via biopsy or surgery. To track the real-time changes of cell location, viability, and functional status, cell imaging techniques have been introduced during the last few years. Cells of interest are labeled with reporter genes, fluorescent dyes, or other contrast agents that transform the tagged cells into cellular probes or imaging agents (2, 6, 7).
The ability to monitor superparamagnetic iron oxide particles (SPIO) by magnetic resonance imaging (MRI) has been utilized in animal models as well as in a few clinical settings to investigate the fate of labeled cells (6-9). The advantages of using MRI for cell tracking include the high spatial resolution with high anatomic background contrast, the lack of exposure to ionizing radiation, and the ability to follow the cells for months. SPIO particles provide a strong change in signal per unit of metal, in particular on T2-weight images. In addition, cell labeling with SPIO nanoparticles is generally nontoxic and does not affect the cell proliferation and differentiation capacity, although a few studies have reported that the stem cells labeled with SPIO lose part of their differentiation capacity in a SPIO concentration–dependent manner (10, 11). The important limitation of MRI is the fact that MRI signals cannot indicate whether cells are dead or alive. It is also unknown whether the MRI signal comes from targeted or labeled cells or from macrophages. Basically, SPIO particles are used to label the target cells by systemic application or by injecting into the tissue area of interest to monitor target cell migration after phagocytosis. SPIO are more frequently used to label the cells in vitro by incorporating into the cells directly. Furthermore, SPIO are usually encapsulated by organic polymers to increase their stability and bio-compatibility, and allow the chemical modification of their surfaces. The fact is that the uptake of different particles varies largely between different cell types (6, 7).
Mesenchymal stem cells (MSCs) represent a heterogeneous subset of pluripotent stromal cells that can be isolated from different adult tissues including adipose tissue, liver, muscle, amniotic fluid, placenta, umbilical cord blood, and dental pulp, although the bone marrow remains the principal source for most preclinical and clinical studies (1, 12, 13). Although MSCs account for only 0.01–0.001% of the total nucleated cells within isolated bone marrow aspirates, they can easily be isolated and expanded in vitro through as many as 40 population doublings in ~8–10 weeks of culture (1, 14). These cells exhibit the potential to differentiate into cells of diverse lineages, such as adipocytes, chondrocytes, osteocytes, myoblasts, cardiomyocytes, neurons, and astrocytes. In addition, MSCs possess remarkable immunosuppressive properties, and they have been shown to be effective against tumor cell growth (15, 16). Wang et al. developed amine-modified silica-coated polyhedral SPIO nanoparticles (SPIO@SiO2-NH2) for MRI real-time cell tracking of implanted MSCs (8). Using the nanoparticles, the investigators obtained a high labeling efficiency in labeling the rabbit bone marrow–derived MSCs, and they could monitor the labeled MSCs with MRI for up to 2–3 months in vivo.
Wang et al. described the synthesis procedure of SPIO@SiO2-NH2 in detail (8). They synthesized the polyhedral crystalline SPIO nanoparticles (6 nm in diameter) with co-precipitation of FeCl3 and FeSO4, followed by solvothermal treatment. The SPIO nanoparticles were then coated with a thin layer of silica with (SPIO@SiO2-NH2) or without (SPIO@SiO2) amine groups on the surface of the particles. SPIO@SiO2-NH2 and SPIO@SiO2 nanoparticles exhibited similar structural features except for their surface charges at certain pH values. Both were nearly monodispersed polyhedral crystals with an average diameter of 10.7 ± 2.6 nm for SPIO@SiO2-NH2 and 8.5 ± 3.0 nm for SPIO@SiO2. Under electron microscopy, the nanoparticles had a single iron oxide core with a single silica shell and were uniform in size. The iron contents of the SPIO@SiO2-NH2 and SPIO@SiO2 nanoparticles were 44.8 ± 0.2% and 21.6 ± 0.1%, respectively. The amounts of silicon, carbon, and nitrogen elements in the SPIO@SiO2-NH2 nanoparticles were 11%, 11%, and 5%, separately. The amine surface modification did not affect the original morphology or the crystallinity of the magnetite core. The saturation magnetizations were 52.5 and 43.5 emu/g Fe, and the MR relaxivities (r2) were 43.5 ± 9.1 and 18.9 ± 3.6 mmol-1s-1 for the SPIO@SiO2-NH2 and SPIO@SiO2 nanoparticles, respectively.
MSCs were isolated from the bone marrow of 20-week-old New Zealand white rabbits and expanded with culture for 5–7 days before labeling. To label the MSCs, Wang et al. tested different concentrations of the nanoparticles, different labeling times, and various transfection agents, and they determined the optimal condition of labeling as an iron concentration of 4.5 µg/ml, an incubation time of 16 h at ambient temperature, and no use of transfection agents. Transfection agents poly-L-lysine and lipofectamine 2000 did not increase the labeling efficiency. On the basis of Prussian blue staining, the labeling efficiency was close to 100%. The iron contents of the labeled MSCs for the SPIO@SiO2 and SPIO@SiO2-NH2 were 17.4 ± 3.9 and 68.7 ± 11.2 pg/cell, respectively, immediate after labeling, and these values decreased to 4.2 ± 1.5 and 13.7 ± 2.1 pg/cell, respectively, 33 days after labeling. The nanoparticles were located in the lysosomes and vesicles, and there were no nanoparticles in the nuclei or other structures up to 33 days after labeling.
In Vitro Studies: Testing in Cells and Tissues
Cell pellet MRI showed that a pellet with ≥5,000 SPIO@SiO2-labeled MSCs or with ≥1,000 SPIO@SiO2-NH2-labeled MSCs was detectable immediate after labeling, and increased to ≥5 × 104 SPIO@SiO2-labeled MSCs or ≥104 SPIO@SiO2-NH2-labeled MSCs on day 33 of culture after labeling. These results indicate that both SPIO@SiO2 and SPIO@SiO2-NH2 nanoparticles had durable MSC-labeling capacity under in vitro culture conditions in which very fast cell division usually occurs under the stimulus of fetal calf serum. The results also demonstrated that the surface amine modification enhanced the labeling efficiency. At physiological pH, SPIO@SiO2 nanoparticles were negatively charged, whereas SPIO@SiO2-NH2 nanoparticles were positively charged because of amine modification. The cell labeling enhancement was thought to be due to the electrostatic interaction between the positively charged SPIO@SiO2-NH2 and the negatively charged MSC cell membrane at physiological pH (8).
After cell labeling with the particles (iron concentration in medium, 4.5 µg/ml) for 16 h, cytotoxicity analysis with trypan blue showed that the cell viability was 95.7 ± 2% and 94.2 ± 3% after labeling with SPIO@SiO2 and SPIO@SiO2-NH2, respectively. MTT assessments before labeling and at 16 h and 120 h after labeling did not reveal altered cell proliferation capacity. The labeled MSCs retained the osteogenic, adipogenic, and chondrogenic differentiation potentials.
To test the feasibility of tracking MSCs with MRI, Wang et al. injected SPIO@SiO2-NH2-labeled MSCs into the right hemisphere (1 cm depth) of the New Zealand male white rabbits’ brain (1 × 105 cells, n = 2) or into the left erector spinae muscle at a depth of 6 mm (5 × 104 cells, n = 2) (1 × 107 cells are commonly used for stem cell–based therapy in the clinical setting) (8). The first MRI was performed with a 3.0-T clinical whole-body MRI unit 2 days after MSC implantation, and then every 2 weeks until the eighth (n = 2) or twelfth week (n = 2). The parameters for imaging were described in detail by Wang et al. SPIO@SiO2-NH2-labeled MSCs induced decreased signal presenting as signal void areas at 2 days in the rabbit brain and in the erector spinae muscle. At 8 weeks, the signal void areas were still visible but became wider, indicating MSC migration. At 12 weeks, the signal void was still visible, but there was a strong decrease in the sizes of areas. Histological sections with Prussian blue staining suggested that the SPIO nanoparticles were predominantly located within the MSCs and MSC-derived cells. No apparent phagocytic cells existed at the implanted sites in either the brain or the erector spinae muscle. These results indicate that the SPIO@SiO2-NH2-labeled MSCs can be tracked with MRI for up to 3 months.
Other Non-Primate Mammals
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Created: December 11, 2009; Last Update: January 28, 2010.
National Center for Biotechnology Information (US), Bethesda (MD)
Shan L. Amine-modified silica-coated polyhedral superparamagnetic iron oxide nanoparticle–labeled rabbit bone marrow–derived mesenchymal stem cells. 2009 Dec 11 [Updated 2010 Jan 28]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.