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1.  Neural Progenitor Cell Survival in Mouse Brain Can Be Improved by Co-transplantation of Helper Cells Expressing bFGF under Doxycycline Control 
Experimental neurology  2013;247:73-79.
Cell-based therapy of neurological disorders is hampered by poor survival of grafted neural progenitor cells (NPCs). We hypothesized that it is possible to enhance the survival of human NPCs (ReNcells) by co-transplantation of helper cells expressing basic fibroblast growth factor (bFGF) under control of doxycycline (Dox). 293 cells or C17.2 cells were transduced with a lentiviral vector encoding the fluorescent reporter mCherry and bFGF under tetracycline-regulated transgene expression (Tet-ON). The bFGF secretion level in the engineered helper cells was positively correlated with the dose of Dox(Pearson correlation test; r=0.95 and 0.99 for 293 and C17.2 cells, respectively). Using bioluminescence imaging (BLI) as readout for firefly luciferase-transduced NPC survival, the addition of both 293-bFGF and C17.2-bFGF helper cells was found to significantly improve cell survival up to 6-fold in vitro, while wild-type (WT, non-transduced) helper cells had no effect. Following co-transplantation of 293-bFGF or C17.2-bFGF cells in the striatum of Rag2−/− immunodeficient mice, in vivo human NPC survival could be significantly improved as compared to no helper cells or co-transplantation of WT cells for the first two days after co-transplantation. This enhancement of survival in C17.2-bFGF group was not achieved without Dox administration, indicating that the neuroprotective effect was specific for bFGF. The present results warrant further studies on the use of engineered helper cells, including those expressing other growth factors injected as mixed cell populations.
PMCID: PMC3742733  PMID: 23570903
Neural progenitor cells; transplantation; cell survival; bFGF; bioluminescent imaging
2.  Metal Ion Sensing Using ion Chemical Exchange Saturation Transfer (iCEST)-19F MRI 
Journal of the American Chemical Society  2013;135(33):12164-12167.
Although metal ions are involved in a myriad of biological processes, a non-invasive means of detecting free metal ions in a deep tissue remains a formidable challenge. We present an approach for specifically sensing the presence of Ca2+ in which the amplification strategy of chemical exchange saturation transfer (CEST) is combined with the broad range in chemical shifts found in 19F NMR to obtain MR images of Ca2+. We exploit the chemical shift change (Δω) of 19F upon binding of Ca2+ to the difluoro derivative of [1,2,-bis(o-aminophenoxy) ethane-N,N,-N′,N′, tetra-acetic acid] (5F-BAPTA), by RF labeling at the bound-19F frequency, ω[Ca-5F-BAPTA], and detecting the label transfer to the free-19F frequency, ω5F-BAPTA. Through the substrate binding kinetics we were able to amplify the signal of Ca2+ onto free 5F-BAPTA and thus indirectly detect low Ca2+ concentrations with high sensitivity.
PMCID: PMC3786336  PMID: 23905693
3.  Immunomodulation by Transplanted Human Embryonic Stem Cell-Derived Oligodendroglial Progenitors in Experimental Autoimmune Encephalomyelitis 
Stem cells (Dayton, Ohio)  2012;30(12):2820-2829.
Transplantation of embryonic stem cells and their neural derivatives can lead to amelioration of the disease symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Oligodendroglial progenitors (OPs), derived from human embryonic stem cells (hESC, HES-1), were labeled with superparamagnetic iron oxide and transduced with luciferase. At 7 days following induction of EAE in C57/BL6 mice, 1 × 106 cells were transplanted in the ventricles of C57/BL6 mice and noninvasively monitored by magnetic resonance and bioluminescence imaging. Cells were found to remain within the cerebroventricular system and did not survive for more than 10 days. However, EAE mice that received hESC-OPs showed a significant improvement in neurological disability scores (0.9 ± 0.2; n = 12) compared to that of control animals (3.3 ± 0.4; n = 12) at day 15 post-transplantation. Histopathologically, transplanted hESC-OPs generated TREM2-positive CD45 cells, increased TIMP-1 expression, confined inflammatory cells within the subarachnoid space, and gave rise to higher numbers of Foxp3-positive regulatory T cells in the spinal cord and spleen. Our results suggest that transplantation of hESC-OPs can alter the pathogenesis of EAE through immunomodulation, potentially providing new avenues for stem cell-based treatment of MS.
PMCID: PMC3638725  PMID: 22949039
Experimental autoimmune encephalomyelitis; Human embryonic stem cells; Oligodendrocyte progenitors; Immunomodulation; Cell tracking
4.  The Magnetic Appeal of Silencing Theranostics 
Diabetes  2012;61(12):3068-3069.
PMCID: PMC3501843  PMID: 23172955
5.  ICV-Transplanted Human Glial Precursor Cells Are Short-Lived Yet Exert Immunomodulatory Effects in Mice with EAE 
Glia  2012;60(7):1117-1129.
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.
PMCID: PMC3579214  PMID: 22499166
multiple sclerosis; EAE; glial precursor cells; MR imaging; bioluminescence imaging
6.  Use of Magnetocapsules for In Vivo Visualization and Enhanced Survival of Xenogeneic HepG2 Cell Transplants 
Cell medicine  2012;4(2):77-84.
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.
PMCID: PMC3534966  PMID: 23293747
Cell transplantation; fulminant liver failure; magnetic resonance imaging; iron nanoparticle contrast agent; bioluminescent imaging
7.  Imaging of Pancreatic Islet Cells 
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.
PMCID: PMC3218557  PMID: 22069256
Diabetes; islets; transplantation; magnetic resonance imaging; positron emission tomography; bioluminescent imaging
8.  Neural Precursors Exhibit Distinctly Different Patterns of Cell Migration Upon Transplantation During Either the Acute or Chronic Phase of EAE: A Serial MR Imaging Study 
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.
PMCID: PMC3190231  PMID: 21305597
neural stem cell; cell tracking; SPIO; multiple sclerosis; blood vessel
9.  Magnetovaccination as a Novel Method to Assess and Quantify Dendritic Cell Tumor Antigen Capture and Delivery to Lymph Nodes 
Cancer research  2009;69(7):3180-3187.
A major parameter limiting immune responses to vaccination is the number of activated antigen-presenting cells (APC) that capture antigen and migrate to draining lymph nodes (LN). Currently, a quantitative noninvasive technique for monitoring in vivo antigen capture and delivery is lacking. The use of cellular magnetic resonance (MR) imaging (MRI) is a promising approach for this purpose; however, cellular imaging currently requires ex vivo prelabeling of cells with contrast agents followed by reintroduction of cells into the subject being monitored. Here, we describe an in vivo labeling method, which relies upon cell-to-cell transfer of super-paramagnetic iron oxide (SPIO) from tumor cells to endogenous APCs, in situ, to quantify APC delivery to LNs in a tumor vaccine model. Mice were immunized with a tumor cell–based vaccine that was irradiated and labeled with SPIO. APCs that had captured SPIO were imaged over time as they accumulated in LNs. We show here that MRI is capable of monitoring, in vivo, the trafficking of magnetically labeled APCs inducing a tumor-specific immune response, and that these cells can be magnetically recovered ex vivo. Excellent correlation was observed between in vivo and ex vivo quantification of APCs, with resolution sufficient to detect increased APC trafficking elicited by an adjuvant. This study shows the potential of magnetovaccination and MRI cell tracking to systematically evaluate a key parameter relevant to the optimization of vaccine therapies through noninvasive MRI-based quantification of APC numbers.
PMCID: PMC3031988  PMID: 19276358
10.  Science to Practice: Can Decreased Lymph Node MR Imaging Signal Intensity Be Used as a Biomarker for the Efficacy of Cancer Vaccination? 
Radiology  2015;274(1):1-3.
In the study of Zhang et al (1), tumor-bearing mice were vaccinated with magnetically labeled, tumor antigen–primed dendritic cells (DCs). After homing of these antigen-presenting cells to the draining lymph node (LN), it was shown that the iron oxide–induced decrease in LN magnetic resonance (MR) imaging signal intensity correlated with the observed tumor growth delay, suggesting that the degree of hypointensity can serve as a surrogate marker for the efficacy of tumor vaccination.
PMCID: PMC4336745  PMID: 25531469
11.  Science to Practice: Highly Shifted Proton MR imaging—A Shift toward Better Cell Tracking? 
Radiology  2014;272(3):615-617.
A “hot spot” magnetic resonance (MR) imaging cell tracking technique has been developed that allows direct detection of dysprosium- or thulium-1,4,7,10-tetraazacyclododecane-α,α′,α″,α‴-tetramethyl-1,4,7,10-tetraacetic acid (DOTMA)–labeled protons inside cells. These highly shifted protons may allow specific detection of multiple cell types because it does not rely on acquiring the proton signal from bulk water.
PMCID: PMC4321787  PMID: 25153271
12.  Dual-Modality Monitoring of Targeted Intraarterial Delivery of Mesenchymal Stem Cells After Transient Ischemia 
Background and Purpose
In animal models of stroke, functional improvement has been obtained after stem cell transplantation. Successful therapy depends largely on achieving a robust and targeted cell engraftment, with intraarterial (IA) injection being a potentially attractive route of administration. We assessed the suitability of laser Doppler flow (LDF) signal measurements and magnetic resonance (MR) imaging for noninvasive dual monitoring of targeted IA cell delivery.
Transient cerebral ischemia was induced in adult Wistar rats (n = 25) followed by IA or intravenous (IV) injection of mesenchymal stem cells (MSCs) labeled with superparamagnetic iron oxide. Cell infusion was monitored in real time with transcranial laser Doppler flowmetry while cellular delivery was assessed with MRI in vivo (4.7T) and ex vivo (9.4T).
Successful delivery of magnetically labeled MSCs could be readily visualized with MRI after IA but not IV injection. IA stem cell injection during acute stroke resulted in a high variability of cerebral engraftment. The amount of LDF reduction during cell infusion (up to 80%) was found to correlate well with the degree of intracerebral engraftment, with low LDF values being associated with significant morbidity.
High cerebral engraftment rates are associated with impeded cerebral blood flow. Noninvasive dual-modality imaging enables monitoring of targeted cell delivery, and through interactive adjustment may improve the safety and efficacy of stem cell therapy.
PMCID: PMC2857730  PMID: 18323495
laser Doppler flow; MRI; stroke; stem cells; transplantation
13.  Gene Expression Profiling Reveals Early Cellular Responses to Intracellular Magnetic Labeling with Superparamagnetic Iron Oxide Nanoparticles 
With MRI (stem) cell tracking having entered the clinic, studies on the cellular genomic response toward labeling are warranted. Gene expression profiling was applied to C17.2 neural stem cells following superparamagnetic iron oxide/PLL (poly-L-lysine) labeling over the course of 1 week. Relative to unlabeled cells, less than 1% of genes (49 total) exhibited greater than 2-fold difference in expression in response to superparamagnetic iron oxide/PLL labeling. In particular, transferrin receptor 1 (Tfrc) and heme oxygenase 1 (Hmox1) expression was downregulated early, whereas genes involved in lysosomal function (Sulf1) and detoxification (Clu, Cp, Gstm2, Mgst1) were upregulated at later time points. Relative to cells treated with PLL only, cells labeled with superparamagnetic iron oxide/PLL complexes exhibited differential expression of 1399 genes. Though these differentially expressed genes exhibited altered expression over time, the overall extent was limited. Gene ontology analysis of differentially expressed genes showed that genes encoding zinc-binding proteins are enriched after superparamagnetic iron oxide/PLL labeling relative to PLL only treatment, whereas members of the apoptosis/ programmed cell death pathway did not display increased expression. Overexpression of the differentially expressed genes Rnf138 and Abcc4 were confirmed by quantitative real-time polymerase chain reaction. These results demonstrate that, although early reactions responsible for iron homeostasis are induced, overall neural stem cell gene expression remains largely unaltered following superparamagnetic iron oxide/PLL labeling.
PMCID: PMC2858578  PMID: 20373404
superparamagnetic iron oxide; MR contrast agent; magnetic resonance imaging; iron metabolism; cell tracking; cell therapy; microarray
14.  MR Tracking of Transplanted Cells With “Positive Contrast” Using Manganese Oxide Nanoparticles 
Rat glioma cells were labeled using electroporation with either manganese oxide (MnO) or superparamagnetic iron oxide (SPIO) nanoparticles. The viability and proliferation of SPIO-labeled cells (1.9 mg Fe/ml) or cells electroporated with a low dose of MnO (100 μg Mn/ml) was not significantly different from unlabeled cells; a higher MnO dose (785 μg Mn/ml) was found to be toxic. The cellular ion content was 0.1−0.3 pg Mn/cell and 4.4 pg Fe/cell, respectively, with cellular relaxivities of 2.5−4.8 s−1 (R1) and 45−84 s−1 (R2) for MnO-labeled cells. Labeled cells (SPIO and low-dose MnO) were each transplanted in contralateral brain hemispheres of rats and imaged in vivo at 9.4T. While SPIO-labeled cells produced a strong “negative contrast” due to the increase in R2, MnO-labeled cells produced “positive contrast” with an increased R1. Simultaneous imaging of both transplants with opposite contrast offers a method for MR “double labeling” of different cell populations.
PMCID: PMC2575033  PMID: 18581402
manganese oxide; iron oxide; cellular imaging; contrast agent; transplantation; nanoparticles
15.  Diamagnetic chemical exchange saturation transfer (diaCEST) liposomes: physicochemical properties and imaging applications 
Chemical exchange saturation transfer (CEST) is a new type of magnetic resonance imaging (MRI) contrast based on labile spins which rapidly exchange with solvent, resulting in an amplification of signal which allows detection of solute protons at millimolar to micromolar concentrations. An additional feature of these agents is that natural organic and biodegradable compounds can provide strong CEST contrast, allowing the development of diamagnetic CEST (diaCEST) MRI contrast agents. The sensitivity of the CEST approach per unit of agent increases further when diaCEST contrast agents are loaded into liposomes to become diaCEST liposomes. In this review, we will discuss the unique and favorable features of diaCEST liposomes which are well suited for in vivo imaging. diaCEST liposomes are nanocarriers which feature high concentrations of encapsulated contrast material, controlled release of payload, and an adjustable coating for passive or active tumor targeting. These liposomes have water permeable bilayers and both the interior and exterior can be fine-tuned for many biomedical applications. Furthermore, a number of liposome formulations are used in the clinic including Doxil™, which is an approved product for treating patients with cancer for decades, rapid translation of these materials can be envisaged. diaCEST liposomes have shown promise in imaging of cancer, and monitoring of chemotherapy and cell transplants. The unique features of diaCEST liposomes are discussed to provide an overview of the applications currently envisioned for this new technology and to provide an overall insight of their potential.
PMCID: PMC3971518  PMID: 24339357
16.  Non-invasive imaging of infection after treatment with tumor-homing bacteria using chemical exchange saturation transfer (CEST) MRI 
To develop a non-invasive MRI method for determining the germination and infection of tumor-homing bacteria in bacteriolytic cancer therapy using endogenous CEST contrast.
The CEST parameters of the anaerobic gram-positive bacterium Clostridium novyi-NT (C. novyi-NT) were first characterized in vitro, then used to detect C. novyi-NT germination and infection in subcutaneous CT26 colorectal tumor-bearing mice (n=6) after injection of 300 million bacterial spores. Lipopolysacharide (LPS) injected mice were used to exclude that the changes of CEST MRI were due to inflammation.
CEST contrast was observed over a broad frequency range for bacterial suspensions in vitro, with the maximum contrast around 2.6 ppm from the water resonance. No signal could be detected for bacterial spores, demonstrating the specificity for germination. In vivo, a significant elevation of CEST contrast was identified in C. novyi-NT infected tumors as compared to those before bacterial germination and infection (p<0.05, n=6). No significant change was observed in tumors with LPS-induced sterile inflammation (p> 0.05, n=4).
Endogenous bacterial CEST contrast (bacCEST) can be used to monitor the germination and proliferation of the therapeutic bacterium C. novyi-NT without a need for exogenous cell labeling probes.
PMCID: PMC3857583  PMID: 24123389
17.  Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI 
Nature protocols  2013;8(12):10.1038/nprot.2013.140.
In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.
PMCID: PMC3870049  PMID: 24177294
18.  Fused X-ray and Mr imaging guidance of intrapericardial Delivery of Microencapsulated human Mesenchymal stem cells in immunocompetent swine 
Radiology  2014;272(2):427-437.
To assess intrapericardial delivery of microencapsulated, xenogeneic human mesenchymal stem cells (hMSCs) by using x-ray fused with magnetic resonance (MR) imaging (x-ray/MR imaging) guidance as a potential treatment for ischemic cardiovascular disease in an immunocompetent swine model.
Materials and Methods
All animal experiments were approved by the institutional animal care and use committee. Stem cell microencapsulation was performed by using a modified alginate-poly-l-lysine-alginate encapsulation method to include 10% (wt/vol) barium sulfate to create barium-alginate microcapsules (BaCaps) that contained hMSCs. With x-ray/MR imaging guidance, eight female pigs (approximately 25 kg) were randomized to receive either BaCaps with hMSCs, empty BaCaps, naked hMSCs, or saline by using a percutaneous subxiphoid approach and were compared with animals that received empty BaCaps (n = 1) or BaCaps with hMSCs (n = 2) by using standard fluoroscopic delivery only. MR images and C-arm computed tomographic (CT) images were acquired before injection and 1 week after delivery. Animals were sacrificed immediately or at 1 week for histopathologic validation. Cardiac function between baseline and 1 week after delivery was evaluated by using a paired Student t test.
hMSCs remained highly viable (94.8% ± 6) 2 days after encapsulation in vitro. With x-ray/MR imaging, successful intrapericardial access and delivery were achieved in all animals. BaCaps were visible fluoroscopically and at C-arm CT immediately and 1 week after delivery. Whereas BaCaps were free floating immediately after delivery, they consolidated into a pseudoepicardial tissue patch at 1 week, with hMSCs remaining highly viable within BaCaps; naked hMSCs were poorly retained. Follow-up imaging 1 week after x-ray/MR imaging–guided intrapericardial delivery showed no evidence of pericardial adhesion and/or effusion or adverse effect on cardiac function. In contradistinction, BaCaps delivery with xray fluoroscopy without x-ray/MR imaging (n = 3) resulted in pericardial adhesions and poor hMSC viability after 1 week.
Intrapericardial delivery of BaCaps with hMSCs leads to high cell retention and survival. With x-ray/MR imaging guidance, intrapericardial delivery can be performed safely in the absence of preexisting pericardial effusion to provide a novel route for cardiac cellular regenerative therapy.
PMCID: PMC4197996  PMID: 24749713
19.  The Survival of Engrafted Neural Stem Cells Within Hyaluronic Acid Hydrogels 
Biomaterials  2013;34(22):5521-5529.
Successful cell-based therapy of neurological disorders is highly dependent on the survival of transplanted stem cells, with the overall graft survival of naked, unprotected cells in general remaining poor. We investigated the use of an injectable hyaluronic acid (HA) hydrogel for enhancement of survival of transplanted mouse C17.2 cells, human neural progenitor cells (ReNcells), and human glial-restricted precursors (GRPs). The gelation properties of the HA hydrogel were first characterized and optimized for intracerebral injection, resulting in a 25 min delayed-injection after mixing of the hydrogel components. Using bioluminescence imaging (BLI) as a non-invasive readout of cell survival, we found that the hydrogel can protect xenografted cells as evidenced by the prolonged survival of C17.2 cells implanted in immunocompetent rats (p<0.01 at day 12). The survival of human ReNcells and human GRPs implanted in the brain of immunocompetent or immunodeficient mice was also significantly improved after hydrogel scaffolding (ReNcells, p<0.05 at day 5; GRPs, p<0.05 at day 7). However, an inflammatory response could be noted two weeks after injection of hydrogel into immunocompetent mice brains. We conclude that hydrogel scaffolding increases the survival of engrafted neural stem cells, justifying further optimization of hydrogel compositions.
PMCID: PMC3653424  PMID: 23623429
Hyaluronic acid; hydrogel; transplantation; neural stem cells; bioluminescence imaging
20.  Long-Term MRI Cell Tracking after Intraventricular Delivery in a Patient with Global Cerebral Ischemia and Prospects for Magnetic Navigation of Stem Cells within the CSF 
PLoS ONE  2014;9(6):e97631.
The purpose of the study was to evaluate the long-term clinical tracking of magnetically labeled stem cells after intracerebroventricular transplantation as well as to investigate in vitro feasibility for magnetic guidance of cell therapy within large fluid compartments.
After approval by our Institutional Review Board, an 18-month-old patient, diagnosed as being in a vegetative state due to global cerebral ischemia, underwent cell transplantation to the frontal horn of the lateral ventricle, with umbilical cord blood-derived stem cells labeled with superparamagnetic iron oxide (SPIO) contrast agent. The patient was followed over 33 months with clinical examinations and MRI. To evaluate the forces governing the distribution of cells within the fluid compartment of the ventricular system in vivo, a gravity-driven sedimentation assay and a magnetic field-driven cell attraction assay were developed in vitro.
Twenty-four hours post-transplantation, MR imaging (MRI) was able to detect hypointense cells in the occipital horn of the lateral ventricle. The signal gradually decreased over 4 months and became undetectable at 33 months. In vitro, no significant difference in cell sedimentation between SPIO-labeled and unlabeled cells was observed (p = NS). An external magnet was effective in attracting cells over distances comparable to the size of human lateral ventricles.
MR imaging of SPIO-labeled cells allows monitoring of cells within lateral ventricles. While the initial biodistribution is governed by gravity-driven sedimentation, an external magnetic field may possibly be applied to further direct the distribution of labeled cells within large fluid compartments such as the ventricular system.
PMCID: PMC4053317  PMID: 24919061
21.  Cell size and velocity of injection are major determinants of the safety of intracarotid stem cell transplantation 
Intracarotid transplantation has shown potential for efficient stem cell delivery to the brain. However, reported complications, such as compromised cerebral blood flow (CBF), prompted us to perform further safety studies. Glial-restricted precursors (GRPs) and mesenchymal stem cells (MSCs) were transplanted into the internal carotid artery of rats (n=99), using a microcatheter. Magnetic resonance imaging was used to detect post-transplantation complications, including the development of stroke, for the following experimental variables: cell size, cell dose, cell infusion velocity, delay between artery occlusion and cell infusion, discordant versus concordant xenografting, and intracarotid transplantation with preserved versus compromised blood flow. Immunocompatibility and delayed infusion did not affect the number of complications. An infusion velocity over ⩾1 mL/minute often resulted in stroke (27 out of 44 animals), even with an infusion of vehicle, whereas a lower velocity (0.2 mL/minute) was safe for the infusion of both vehicle and smaller cells (GRPs, diameter=15 μm). Infusion of larger cells (MSCs, diameter=25 μm) resulted in a profound decrease (75±17%) in CBF. Stroke lesions occurred frequently (12 out of 15 animals) when injecting 2 × 106 MSCs, but not after lowering the dose to 1 × 106 cells. The present results show that cell size and infusion velocity are critical factors in developing safe protocols for intracarotid stem cell transplantation.
PMCID: PMC3677113  PMID: 23486296
glial-restricted progenitors; intracarotid injection; mesenchymal stem cells; stroke; transplantation
22.  Human Protamine-1 as an MRI Reporter Gene Based on Chemical Exchange 
ACS chemical biology  2013;9(1):134-138.
Genetically engineered reporters have revolutionized the understanding of many biological processes. MRI-based reporter genes can dramatically improve our ability to monitor dynamic gene expression and allow coregistration of subcellular genetic information with high-resolution anatomical images. We have developed a biocompatible MRI reporter gene based on a human gene, the human protamine-1 (hPRM1). The arginine-rich hPRM1 (47% arginine residues) generates high MRI contrast based on the chemical exchange saturation transfer (CEST) contrast mechanism. The 51 amino acid-long hPRM1 protein was fully synthesized using microwave-assisted technology, and the CEST characteristics of this protein were compared to other CEST-based contrast agents. Both bacterial and human cells were engineered to express an optimized hPRM1 gene and showed higher CEST contrast compared to controls. Live cells expressing the hPRM1 reporter gene, and embedded in three-dimensional culture, also generated higher CEST contrast compared to wild-type live cells.
PMCID: PMC3985336  PMID: 24138139
23.  NOrmalized MAgnetization Ratio (NOMAR) Filtering for Creation of Tissue Selective Contrast Maps 
An MRI segmentation technique based on collecting two additional saturation transfer images is proposed as an aid for improved detection of CEST agents. In this approach, the additional images are acquired at saturation frequencies of −12.5 and −50ppm. Use of the ratio of these images allows differentiation of voxels with low MT contrast (such as fat, CSF, edema or blood) from target tissue voxels using a global threshold determined by histogram analysis. We demonstrate that this technique can reduce artifacts, in vitro, in a phantom containing tubes with CEST contrast agent embedded in either cross-linked BSA or buffer, and in vivo for detecting DIACEST liposomes injected into mice.
PMCID: PMC3404207  PMID: 22499503
24.  Transforming thymidine into a magnetic resonance imaging probe for monitoring gene expression 
Synthetic chemistry has revolutionized the understanding of many biological systems. Small compounds that act as agonists and antagonists of proteins, and occasionally as imaging probes, have contributed tremendously to the elucidation of many biological pathways. Nevertheless, the function of thousands of proteins is still elusive, and designing new imaging probes remains a challenge. Through screening and characterization we identified thymidine analog as probe for imaging the expression of the Herpes Simplex Virus type-1 thymidine kinase (HSV1-TK). To detect the probe, we used chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI), in which a dynamic exchange process between an exchangeable proton and the surrounding water protons is used to amplify the desired contrast. Initially, five pyrimidine-based molecules were recognized as putative imaging agents, since their exchangeable imino protons resonate at 5–6ppm from the water proton frequency and their detection is therefore less affected by endogenous CEST contrast or confounded by direct water saturation. Increasing the pKa value of the imino proton by reduction of its 5,6-double bond results in a significant reduction of the exchange rate (kex) between this proton and the water protons. This reduced kex of the dihydropyrimidine nucleosides fulfills the “slow to intermediate regime” condition for generating high CEST-MRI contrast. Consequently, we identified 5-methyl-5,6-dihydrothymidine as the optimal probe and demonstrated its feasibility for in vivo imaging of the HSV1-TK. In light of these findings, this new approach can be generalized for designing specific probes for the in vivo imaging of a variety of proteins and enzymes.
PMCID: PMC3560322  PMID: 23289583
25.  Tracking immune cells in vivo using magnetic resonance imaging 
Nature reviews. Immunology  2013;13(10):10.1038/nri3531.
The increasing complexity of in vivo imaging technologies, coupled with the development of cell therapies, has fuelled a revolution in immune cell tracking in vivo. Powerful magnetic resonance imaging (MRI) methods are now being developed that use iron oxide- and 19F-based probes. These MRI technologies can be used for image-guided immune cell delivery and for the visualization of immune cell homing and engraftment, inflammation, cell physiology and gene expression. MRI-based cell tracking is now also being applied to evaluate therapeutics that modulate endogenous immune cell recruitment and to monitor emerging cellular immunotherapies. These recent uses show that MRI has the potential to be developed in many applications to follow the fate of immune cells in vivo.
PMCID: PMC3886235  PMID: 24013185

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