Human glial precursor cells (hGPs) have potential for remyelinating lesions and are an attractive cell source for cell therapy of multiple sclerosis (MS). To investigate whether transplanted hGPs can affect the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we evaluated the therapeutic effects of transplanted hGPs together with the in vivo fate of these cells using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). At 14 days post-EAE induction, mice (n = 19) were intracerebroventricularly (ICV) injected with 5 × 105 hGPs that were magnetically labeled with superparamagnetic iron oxide (SPIO) particles as MR contrast agent and transduced with firefly luciferase for BLI of cell survival. Control mice (n = 18) received phosphate buffered saline (PBS) vehicle only. The severity of EAE clinical disability in the hGP-transplanted group was significantly suppressed (P < 0.05) with concomitant inhibition of ConA and MOG-specific T cell proliferation in the spleen. Astrogliosis was reduced and a lower activity of macrophages and/or microglia was observed in the spinal cord (P < 0.05). On MRI, SPIO signal was detected within the lateral ventricle from 1 day post-transplantation and remained there for up to 34 days. BLI indicated that most cells did not survive beyond 5–10 days, consistent with the lack of detectable migration into the brain parenchyma and the histological presence of an abundance of apoptotic cells. Transplanted hGPs could not be detected in the spleen. We conclude that ICV transplantation of short-lived hGPs can have a remote therapeutic effect through immunomodulation from within the ventricle, without cells directly participating in remyelination.
multiple sclerosis; EAE; glial precursor cells; MR imaging; bioluminescence imaging
Cellular therapy can be defined as the transplantation of living cells for the treatment of medical conditions. Three main objectives of cellular therapy are regeneration of damaged tissue, replacement of function by secretion of biologically active molecules, and redirection of aberrant processes. Given the complex nature of these approaches, in vivo tracking of the transplanted cells is critical to evaluate their potential benefit and to optimize treatment strategies. Recent advances are reviewed that enable in vivo cell tracking as an important adjunct to implement cellular therapy in clinical practice.
in vivo imaging; tracking; cellular therapy; transplantation; regenerative medicine
Hepatocyte transplantation is currently being considered as a new paradigm for treatment of fulminant liver failure. Xeno- and allotransplantation studies have shown considerable success but the long-term survival and immunorejection of engrafted cells needs to be further evaluated. Using novel alginate-protamine sulfate-alginate microcapsules, we have co-encapsulated luciferase-expressing HepG2 human hepatocytes with superparamagnetic iron oxide nanoparticles to create magnetocapsules that are visible on MRI as discrete hypointensities. Magnetoencapsulated cells survive and secrete albumin for at least 5 weeks in vitro. When transplanted i.p. in immunocompetent mice, encapsulated hepatocytes survive for at least 4 weeks as determined using bioluminescent imaging, which is in stark contrast to naked, unencapsulated hepatocytes, that died within several days after transplantation. However, in vivo human albumin secretion did not follow the time course of magnetoencapsulated cell survival, with plasma levels returning to baseline values already at 1 week post-transplantation. The present results demonstrate that encapsulation can dramatically prolong survival of xenotransplanted hepatocytes, leading to sustained albumin secretion with a duration that may be long enough for use as a temporary therapeutic bridge to liver transplantation.
Cell transplantation; fulminant liver failure; magnetic resonance imaging; iron nanoparticle contrast agent; bioluminescent imaging
Understanding how individual cells behave inside living systems will help enable new diagnostic tools and cellular therapies. Superparamagnetic iron oxide (SPIO) particles can be used to label cells and theranostic capsules for non-invasive tracking using MRI. Contrast changes from SPIO are often subtle relative to intrinsic sources of contrast, presenting a detection challenge. Here we describe a versatile post-processing method, called Phase map cross-correlation Detection and Quantification (PDQ), that automatically identifies localized deposits of SPIO, estimating their volume magnetic susceptibility and magnetic moment. To demonstrate applicability, PDQ was used to detect and characterize SPIO-labeled magnetocapsules implanted in porcine liver and suspended in agarose gel. PDQ was also applied to mouse brains infiltrated by MPIO-labeled macrophages following traumatic brain injury (TBI); longitudinal, in vivo studies tracked individual MPIO clusters over three days, and tracked clusters were corroborated in ex vivo brain scans. Additionally, we applied PDQ to rat hearts infiltrated by MPIO-labeled macrophages in a transplant model of organ rejection. PDQ magnetic measurements were SNR-invariant for images with SNR>11. PDQ can be used with conventional gradient-echo pulse sequences, requiring no extra scan time. The method is useful for visualizing biodistribution of cells and theranostic magnetocapsules, and for measuring their relative iron content.
SPIO; magnetic moment; MRI; cell tracking; magnetocapsules; susceptibility
A variety of (super)paramagnetic contrast agents are available for enhanced MR visualization of specific tissues, cells, or molecules. In order to develop alternative contrast agents without the presence of metal ions, liposomes were developed containing simple bioorganic and biodegradable compounds that produce diamagnetic Chemical Exchange Saturation Transfer (DIACEST) MR contrast. This DIACEST contrast is frequency-dependent, allowing the unique generation of “multi-color” images. The contrast can be turned on and off at will, and standard images do not show the presence of these agents. As an example, glycogen, L-arginine, and poly-L-Lysine were encapsulated inside liposomes and injected intradermally into mice to image the lymphatic uptake of these liposomes. Using a frequency-dependent acquisition scheme, it is demonstrated that multi-color MRI can differentiate between different contrast particles in vivo following their homing to draining lymph nodes. Being non-metallic and bioorganic, these DIACEST liposomes form an attractive novel platform for multi-color imaging in vivo.
Nanoparticles; poly(propylene fumarate) (PPF); magnetic resonance imaging (MRI); chemical exchange saturation transfer (CEST); Protamine sulfate (PS) drug release; doxorubicin
At present, the onset and progress of diabetes, and the efficacy of potential treatments, can only be assessed through indirect means, i.e., blood glucose, insulin, or C-peptide measurements. The development of non-invasive and reliable methods for 1) quantification of pancreatic beta islet cell mass in vivo, 2) determining endogenous islet function and survival, and 3) visualizing the biodistribution, survival, and function of transplanted exogenous islets are critical to further advance both basic science research and islet cell therapy in diabetes. Islet cell imaging using magnetic resonance (MR), bioluminescence, positron emission tomography (PET), or single photon emission computed tomography (SPECT) may provide us with a direct means to interrogate islet cell distribution, survival, and function. Current state-of-the-art strategies for beta cell imaging are discussed and reviewed here in context of their clinical relevance.
Diabetes; islets; transplantation; magnetic resonance imaging; positron emission tomography; bioluminescent imaging
Recent technological developments in biomedicine have facilitated the generation of data on the anatomical, physiological and molecular level for individual patients and thus introduces opportunity for therapy to be personalized in an unprecedented fashion. Generation of patient-specific stem cells exemplifies the efforts toward this new approach. Cell-based therapy is a highly promising treatment paradigm; however, due to the lack of consistent and unbiased data about the fate of stem cells in vivo, interpretation of therapeutic remains challenging hampering the progress in this field. The advent of nanotechnology with a wide palette of inorganic and organic nanostructures has expanded the arsenal of methods for tracking transplanted stem cells. The diversity of nanomaterials has revolutionized personalized nanomedicine and enables individualized tailoring of stem cell labeling materials for the specific needs of each patient. The successful implementation of stem cell tracking will likely be a significant driving force that will contribute to the further development of nanotheranostics. The purpose of this review is to emphasize the role of cell tracking using currently available nanoparticles.
Nanoparticles; Transplantation; Stem cells; Imaging; Theranostics
Chemical Exchange Saturation Transfer (CEST) is a new approach to generate magnetic resonance imaging (MRI) contrast that allows monitoring of protein properties in vivo. In this method, radiofrequency is used to saturate the magnetization of specific protons on a target molecule, where it is then transferred to water protons via chemical exchange and detected using MRI. One advantage of CEST imaging is that the magnetization of the different protons can be specifically saturated at different resonance frequencies. This enables the detection of multiple targets simultaneously in living tissue. We present here a CEST MRI approach to detect the activity of Cytosine Deaminase (CDase), an enzyme that catalyzes the deamination of cytosine to uracil. Our findings suggest that metabolism of two substrates of the enzyme, cytosine and 5-fluorocytosine (5FC), can be detected using saturation pulses targeted specifically to protons at +2 ppm and +2.4 ppm (with respect to water), respectively. Indeed, after deamination by recombinant CDase, the CEST contrast disappears. In addition, expression of the enzyme in three different cell lines, each with different expression levels of CDase, show good agreement with the CDase activity measured with CEST MRI. Consequently, CDase activity was imaged with high-resolution CEST-MRI. These data demonstrate the ability to detect enzyme activity based on proton exchange. Consequently, CEST MRI has the potential to follow the kinetics of multiple enzymes in real-time in living tissue.
In vivo imaging of engraftment and immunorejection of transplanted islets is critical for further clinical development, with 1H MR imaging of superparamagnetic iron oxide-labeled cells being the current premier modality. Using perfluorocarbon nanoparticles, we present here a strategy for non-invasive imaging of cells using other modalities. To this end, human cadaveric islets were labeled with rhodamine-perfluorooctylbromide (PFOB) nanoparticles, rhodamine-perfluoropolyether (PFPE) nanoparticles or Feridex® as control and tested in vitro for cell viability and c-peptide secretion for 1 week. 19F MRI, computed tomography (CT) and ultrasound (US) imaging was performed on labeled cell phantoms and on cells following transplantation beneath the kidney capsule of mice and rabbits. PFOB and PFPE-labeling did not reduce human islet viability or glucose responsiveness as compared with unlabeled cells or SPIO-labeled cells. PFOB- and PFPE-labeled islets were effectively fluorinated for visualization by 19F MRI. PFOB-labeled islets were acoustically reflective for detection by US imaging and became sufficiently brominated to become radiopaque allowing visualization with CT. Thus, perfluorocarbon nanoparticles are multimodal cellular contrast agents that may find applications in real-time targeted delivery and imaging of transplanted human islets or other cells in a clinically applicable manner using MRI, US or CT imaging.
cell tracking; molecular imaging; fluorine MRI; ultrasound imaging; X-ray imaging; islet cell; diabetes
Transplantation of glial progenitor cells results in transplant-derived myelination and improved function in rodents with genetic dysmyelination or chemical demyelination. However, glial cell transplantation in adult CNS inflammatory demyelinating models has not been well studied. Here we transplanted human glial-restricted progenitor (hGRP) cells into the spinal cord of adult rats with inflammatory demyelination, and monitored cell fate in chemically immunosuppressed animals. We found that hGRPs migrate extensively, expand within inflammatory spinal cord lesions, do not form tumors, and adopt a mature glial phenotype, albeit at a low rate. Human GRP-transplanted rats, but not controls, exhibited preserved electrophysiological conduction across the spinal cord, though no differences in behavioral improvement were noted between the two groups. Although these hGRPs myelinated extensively after implantation into neonatal shiverer mouse brain, only marginal remyelination was observed in the inflammatory spinal cord demyelination model. The low rate of transplant-derived myelination in adult rat spinal cord may reflect host age, species, transplant environment/location, and/or immune suppression regime differences. We conclude that hGRPs have the capacity to myelinate dysmyelinated neonatal rodent brain and preserve conduction in the inflammatory demyelinated adult rodent spinal cord. The latter benefit is likely dependent on trophic support and suggests further exploration of potential of glial progenitors in animal models of chronic inflammatory demyelination.
Magnetic resonance imaging (MRI) cell tracking has become an important non-invasive technique to interrogate the fate of cells upon transplantation. At least 6 clinical trials have been published at the end of 2010, all of which have shown that real-time monitoring of the injection procedure, initial engraftment, and short-term biodistribution of cells is critical to further advance the field of cellular therapeutics. In MRI cell tracking, cells are loaded with superparamagnetic iron oxide (SPIO) particles that provide an MRI contrast effect through microscopic magnetic field disturbances and dephasing of protons. Magnetic particle imaging (MPI) has recently emerged as a potential cellular imaging technique that promises to have several advantages over MRI, primarily linear quantification of cells, a higher sensitivity, and “hot spot” tracer identification without confounding background signal. Although probably not fully optimized, SPIO particles that are currently used as MRI contrast agent can be employed as MPI tracer. Initial studies have shown that cells loaded with SPIO particles can give a detectable MPI signal, encouraging further development of MPI cell tracking.
Magnetic resonance imaging; magnetic particle imaging; superparamagnetic iron oxide; cell tracking
Neural stem cell (NSC)-based therapy is actively being pursued in preclinical and clinical disease models. Magnetic resonance imaging (MRI) cell tracking promises to optimize current cell transplantation paradigms, however, it is limited by dilution of contrast agent during cellular proliferation, transfer of label from dying cells to surrounding endogenous host cells, and/or biodegradation of the label. Here, we evaluated the applicability of magnetic resonance imaging for long-term tracking of transplanted neural stem cells labeled with superparamagnetic iron oxide and transfected with the bioluminescence reporter gene luciferase. Mouse neural stem cells were transplanted into immunodeficient, graft-accepting Rag2 mice or immunocompetent, graft-rejecting Balb/c mice. Hypointense voxel signals and bioluminescence were monitored over a period of 93 days. Unexpectedly, in mice that rejected the cells, the hypointense MR signal persisted throughout the entire time-course, whereas in the nonrejecting mice, the contrast cleared at a faster rate. In immunocompetent, graft-rejecting Balb/c mice, infiltrating leukocytes, and microglia were found surrounding dead cells and internalizing superparamagnetic iron oxide clusters. The present results indicate that live cell proliferation and associated label dilution may dominate contrast clearance as compared with cell death and subsequent transfer and retention of superparamagnetic iron oxide within phagocytes and brain interstitium. Thus, interpretation of signal changes during long-term MR cell tracking is complex and requires caution.
superparamagnetic iron oxide; MR cell tracking; neural stem cells
Cell therapy has the potential to treat or cure a wide variety of diseases. Non-invasive cell tracking techniques are, however, necessary to translate this approach to the clinical setting. This protocol details methods to create microcapsules that are visible by X-ray, ultrasound (US ) or magnetic resonance (MR) for the encapsulation and immunoisolation of cellular therapeutics. Three steps are generally used to encapsulate cellular therapeutics in an alginate matrix: (i) droplets of cell-containing liquid alginate are extruded, using an electrostatic generator, through a needle tip into a solution containing a dissolved divalent cation salt to form a solid gel; (ii) the resulting gelled spheres are coated with polycations as a cross-linker; and (iii) these complexes are then incubated in a second solution of alginate to form a semipermeable membrane composed of an inner and an outer layer of alginate. The microcapsules can be rendered visible during the first step by adding contrast agents to the primary alginate layer. Such contrast agents include superparamagnetic iron oxide for detection by 1H MR imaging (MRI); the radiopaque agents barium or bismuth sulfate for detection by X-ray modalities; or perfluorocarbon emulsions for multimodal detection by 19F MRI, X-ray and US imaging. The entire synthesis can be completed within 2 h.
Recently, several protocols for labeling of stem cells with superparamagnetic iron oxides (SPIOs) have been developed, leading to an active and growing field aimed at visualizing stem cells using MRI (magnetic resonance imaging), including image-guided stem cell injections. This development occurred simultaneously with a significant rise in the number of cell therapy clinical trials for cardiovascular applications and their preceding pre-clinical studies in animal models. In this chapter, we will describe several labeling strategies that can be used to label cells with SPIO nanoparticles. This is followed by a discussion of current strategies for using MRI to visualize these cells in myocardial infarct.
Magnetic resonance imaging (MRI); stem cells; superparamagnetic iron oxide (SPIO); cellular labeling; cellular imaging; myocardial infarct
Editorials; atrial fibrillation; autonomic function; cardiovascular magnetic resonance imaging; electrophysiology; iron oxide labeling
Stem cell therapies offer great promise for many diseases, especially those without current effective treatments. It is believed that noninvasive imaging techniques, which offer the ability to track the status of cells after transplantation, will expedite progress in this field and help to achieve maximized therapeutic effect. Today’s biomedical imaging technology allows for real-time, noninvasive monitoring of grafted stem cells including their biodistribution, migration, survival, and differentiation, with magnetic resonance imaging (MRI) of nanoparticle-labeled cells being one of the most commonly used techniques. Among the advantages of MR cell tracking are its high spatial resolution, no exposure to ionizing radiation, and clinical applicability. In order to track cells by MRI, the cells need to be labeled with magnetic nanoparticles, for which many types exist. There are several cellular labeling techniques available, including simple incubation, use of transfection agents, magnetoelectroporation, and magnetosonoporation. In this overview article, we will review the use of different magnetic nanoparticles and discuss how these particles can be used to track the distribution of transplanted cells in different organ systems. Caveats and limitations inherent to the tracking of nanoparticle-labeled stem cells are also discussed.
As the complex pathogenesis of multiple sclerosis contributes to spatiotemporal variations in the trophic micromilieu of the central nervous system, the optimal intervention period for cell-replacement therapy must be systematically defined. We applied serial, 3D high-resolution magnetic resonance imaging to transplanted neural precursor cells (NPCs) labeled with superparamagnetic iron oxide nanoparticles and 5-bromo-2-deoxyuridine, and compared the migration pattern of NPCs in acute inflamed (n = 10) versus chronic demyelinated (n = 9) brains of mice induced with experimental allergic encephalomyelitis (EAE). Serial in vivo and ex-vivo 3D magnetic resonance imaging revealed that NPCs migrated 2.5 ± 1.3 mm along the corpus callosum in acute EAE. In chronic EAE, cell migration was slightly reduced (2.3 ± 1.3 mm) and only occurred in the lateral side of transplantation. Surprisingly, in 6/10 acute EAE brains, NPCs were found to migrate in a radial pattern along RECA-1+ cortical blood vessels, in a pattern hitherto only reported for migrating glioblastoma cells. This striking radial biodistribution pattern was not detected in either chronic EAE or disease-free control brains. In both acute and chronic EAE brain, Iba1+ microglia/macrophage number was significantly higher in central nervous system regions containing migrating NPCs. The existence of differential NPC migration patterns is an important consideration for implementing future translational studies in multiple sclerosis patients with variable disease.
neural stem cell; cell tracking; SPIO; multiple sclerosis; blood vessel
Cell tracking; Cell therapy; Magnetic resonance; MR contrast agent; Molecular imaging; MR microscopy; Cell labeling; Stem cell; Nanoparticle
Shortly after the introduction of 1H MRI, fluorinated molecules were tested as MR-detectable tracers or contrast agents. Many fluorinated compounds, which are nontoxic and chemically inert, are now being used in a broad range of biomedical applications, including anesthetics, chemotherapeutic agents, and molecules with high oxygen solubility for respiration and blood substitution. These compounds can be monitored by fluorine (19F) MRI and/or MRS, providing a noninvasive means to interrogate associated functions in biological systems. As a result of the lack of endogenous fluorine in living organisms, 19F MRI of ‘hotspots’ of targeted fluorinated contrast agents has recently opened up new research avenues in molecular and cellular imaging. This includes the specific targeting and imaging of cellular surface epitopes, as well as MRI cell tracking of endogenous macrophages, injected immune cells and stem cell transplants.
perfluorocarbon; MR contrast agent; lung ventilation; oxygen measurement; molecular imaging; cellular imaging
Mesoporous silica-coated hollow manganese oxide (HMnO@mSiO2) nanoparticles were developed as a novel T1 magnetic resonance imaging (MRI) contrast agent. We hypothesized that the mesoporous structure of the nanoparticle shell enables optimal access of water molecules to the magnetic core, and consequently, an effective longitudinal (R1) relaxation enhancement of water protons, which value was measured to be 0.99 (mM−1s−1) at 11.7 T. Adipose-derived mesenchymal stem cells (MSCs) were efficiently labeled using electroporation, with much shorter T1 values as compared to direct incubation without electroporation, which was also evidenced by signal enhancement on T1-weighted MR images in vitro. Intracranial grafting of HMnO@mSiO2-labeled MSCs enabled serial MR monitoring of cell transplants over 14 days. These novel nanoparticles may extend the arsenal of currently available nanoparticle MR contrast agents by providing positive contrast on T1-weighted images at high magnetic field strengths.
The administration of exogenous stem cells offers promise to regenerate many damaged organs. However, failures of these cellular therapies could be related to many issues, such as the type of stem cell, the dose of cellular therapeutic, dosing regime, and mode of delivery. The recent ability to directly label stem cells with magnetic resonance (MR) contrast agents provides a simple, straight-forward manner to monitor accurate cell delivery and track stem cells non-invasively in a serial manner. Provided here is an overview of the currently available MR-labeling methods, including direct non-specific labeling with contrast agents, indirect specific labeling with contrast agents, labeling with MRI reporter genes, and fluorine hot spot labeling. Several of these approaches have now been applied successfully in preclinical animal models of cardiovascular disease. Once properly implemented, future clinical trials may benefit greatly from imaging stem cells with MRI.
stem cells; magnetic resonance imaging; superparamagnetic iron oxides; myocardial infarction; reporter gene
Myelin oligodendrocyte glycoprotein (MOG) is commonly used as an immunogen to induce an immune response against endogenous myelin, thereby modeling multiple sclerosis in rodents. When MOG is combined with complete Freund's adjuvant (CFA), multifocal, multiphasic disease ensues; whereas when MOG is combined with incomplete Freund's adjuvant (IFA), clinical disease is usually absent. MOG–IFA immunized animals can be induced to have neurological disease after intraspinal injections of cytokines and ethidium bromide (EtBr). In this study, we investigated whether MOG–IFA immunized rats exhibited subclinical injury as defined by somatosensory evoked potential (SEP) recordings. The titration of anti-MOG-125 antibodies showed robust peripheral mounting of immune response against myelin in MOG-immunized rats. However the SEP measures showed no significant change over time. Upon injecting cytokine–EtBr in the spinal cord after MOG sensitization, the SEP recordings showed reduced amplitude and prolonged latency, suggestive of axonal injury and demyelination in the dorsal column, respectively. These findings were later confirmed using T2-weighted MRI and histological hematoxylin–eosin stain of the spinal cord. This report establishes that MOG–IFA immunization alone does not alter neuronal conduction in SEP-related neural-pathways and that longitudinal in-vivo SEP recordings provide a sensitive read-out for focal myelitis (MOG–IFA and intraspinal cytokine–EtBr) in rats.
Somatosensory evoked potential (SEP); Myelin oligodendrocyte glycoprotein (MOG); Freund's adjuvant; Sensitization/immunization; Cytokine; Ethidium bromide