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1.  The Magnetic Appeal of Silencing Theranostics 
Diabetes  2012;61(12):3068-3069.
doi:10.2337/db12-1038
PMCID: PMC3501843  PMID: 23172955
2.  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.
doi:10.1016/j.biomaterials.2013.03.095
PMCID: PMC3653424  PMID: 23623429
Hyaluronic acid; hydrogel; transplantation; neural stem cells; bioluminescence imaging
3.  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.
doi:10.1038/jcbfm.2013.32
PMCID: PMC3677113  PMID: 23486296
glial-restricted progenitors; intracarotid injection; mesenchymal stem cells; stroke; transplantation
4.  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.
doi:10.1021/cb400617q
PMCID: PMC3985336  PMID: 24138139
5.  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.
doi:10.1002/mrm.24271
PMCID: PMC3404207  PMID: 22499503
6.  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.
doi:10.1021/ja312353e
PMCID: PMC3560322  PMID: 23289583
7.  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.
doi:10.1038/nri3531
PMCID: PMC3886235  PMID: 24013185
8.  Cell Motility of Neural Stem Cells is Reduced after SPIO-Labeling, which is Mitigated after Exocytosis 
MRI is used for tracking of superparamagnetic iron oxide (SPIO)-labeled neural stem cells (NSCs). Studies have shown that long-term MR tracking of rapidly dividing cells underestimates their migration distance. Time-lapse microscopy of random cellular motility and cell division was performed to evaluate the effects of SPIO-labeling on NSC migration. Labeled cells divided symmetrically, and exhibited no changes in cell viability, proliferation, or apoptosis. However, SPIO-labeling resulted in decreased motility of NSCs as compared to unlabeled controls. When SPIO-labeled NSCs and human induced pluripotent stem cells (iPSCs) were transplanted into mouse brain, rapid exocytosis of SPIO by live cells was observed as early as 48 hours post-engraftment, with SPIO-depleted cells showing the farthest migration distance. As label dilution is negligible at this early time point, we conclude that MRI underestimation of cell migration can also occur as a result of reduced cell motility, which appears to be mitigated following SPIO exocytosis.
doi:10.1002/mrm.24216
PMCID: PMC3366052  PMID: 22374813
superparamagnetic iron oxide; cell tracking; neural stem cell; exocytosis
9.  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.
doi:10.1002/stem.1218
PMCID: PMC3638725  PMID: 22949039
Experimental autoimmune encephalomyelitis; Human embryonic stem cells; Oligodendrocyte progenitors; Immunomodulation; Cell tracking
10.  MRI Biosensor for Protein Kinase A Encoded by a Single Synthetic Gene 
PURPOSE
Protein kinases including Protein Kinase A (PKA) underlie myriad important signaling pathways. The ability to monitor kinase activity in vivo and in real-time with high spatial resolution in genetically-specified cellular populations is a yet unmet need, crucial for understanding complex biological systems as well as for preclinical development and screening of novel therapeutics.
METHODS
Using the hypothesis that the natural recognition sequences of protein kinases may be detected using chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI), we designed a genetically encoded biosensor composed of eight tandem repeats of the peptide LRRASLG, a natural target of PKA.
RESULTS
This sensor displays a measurable change in CEST signal following phosphorylation by PKA. The natural PKA substrate LRRASLG exhibits a CEST-MRI contrast at +1.8 and +3.6 ppm, with a >50% change after phosphorylation with minutes-scale temporal resolution. Expression of a synthetic gene encoding eight monomers of LRRASLG yielded two peaks at these CEST frequencies.
CONCLUSION
Taken together, these results suggest that this gene may be used to assay PKA levels in a biologically relevant system. Importantly, the design strategy used for this specific sensor may be adapted for a host of clinically interesting protein kinases.
doi:10.1002/mrm.24483
PMCID: PMC3504643  PMID: 23023588
chemical exchange saturation transfer (CEST); biosensor; reporter gene; protein kinase A (PKA)
11.  Natural D-Glucose as a biodegradable MRI contrast agent for detecting cancer 
Purpose
Modern imaging technologies such as CT, PET, SPECT, and MRI employ contrast agents to visualize the tumor microenvironment, providing information on malignancy and response to treatment. Currently, all clinical imaging agents require chemical labeling, i.e. with iodine (CT), radioisotopes (PET/SPECT), or paramagnetic metals (MRI). The goal was to explore the possibility of using simple D-glucose as an infusable biodegradable MRI agent for cancer detection.
Methods
D-glucose signals were detected using chemical exchange saturation transfer (glucoCEST) MRI of its hydroxyl groups. Feasibility was established in phantoms as well as in vivo using two human breast cancer cell lines, MDA-MB-231 and MCF-7, implanted orthotopically in nude mice. PET and contrast-enhanced MRI were also acquired.
Results
Both tumor types exhibited significant glucoCEST signal enhancement during systemic sugar infusion (mild hyperglycemia), allowing their noninvasive visualization. GlucoCEST showed differences between types, while PET and CE-MRI did not. Data are discussed in terms of signal contributions from the increased vascular volume in tumors and especially from the acidic extracellular extravascular space (EES), where glucoCEST signal is expected to be enhanced due to a slow-down of hydroxyl proton exchange.
Conclusions
This observation opens up the possibility for using simple non-toxic sugars as contrast agents for cancer detection with MRI by employing hydroxyl protons as a natural label.
doi:10.1002/mrm.24520
PMCID: PMC3505108  PMID: 23074027
glucose; MRI; cancer detection; screening; contrast agent; biodegradable
12.  CEST phase mapping using a Length and Offset VARied Saturation (LOVARS) scheme 
Chemical exchange saturation transfer (CEST) MRI is a promising new technique for cellular and molecular imaging. This contrast allows the detection of tumors and ischemia without the use of gadolinium as well as the design of microenvironment-sensitive probes that can be discriminated based on their exchange contrast properties and saturation frequency. Current acquisition schemes to detect and analyze this contrast suffer from sensitivity to spatial B0 inhomogeneity and low contrast-to-noise-ratio (CNR), which is an obstacle to widespread adoption of the technology. A new method to detect CEST contrast is proposed here, termed “Length and Offset VARied Saturation” or “LOVARS”, which acquires a set of images with the saturation parameters varied so as to modulate the exchange contrast. Either fast fourier transform or the general linear model can be employed to decompose the modulation patterns into separate sources of water signal loss. After transformation, a LOVARS phase map is generated, which is insensitive to B0 inhomogeneity. When collected on live mice bearing 9L gliosarcomas, and compared to the conventional MTRasym map using offset increment correction, the results show that LOVARS phase mapping obtains about four times higher CNR and exhibits less B0 artifacts.
doi:10.1002/mrm.23312
PMCID: PMC3336046  PMID: 22246684
13.  MR Monitoring of Minimally Invasive Delivery of Mesenchymal Stem Cells into the Porcine Intervertebral Disc 
PLoS ONE  2013;8(9):e74658.
Purpose
Bone marrow stem cell therapy is a new, attractive therapeutic approach for treatment of intervertebral disc (IVD) degeneration; however, leakage and backflow of transplanted cells into the structures surrounding the disc may lead to the formation of undesirable osteophytes. The purpose of this study was to develop a technique for minimally invasive and accurate delivery of stem cells.
Methods
Porcine mesenchymal stem cells (MSCs) were labeled with superparamagnetic iron oxide nanoparticles (SPIO, Molday ION rhodamine) and first injected into the explanted swine lumbar IVD, followed by ex vivo 3T MRI. After having determined sufficient sensitivity, IVD degeneration was then induced in swine (n=3) by laser-evaporation. 3 x 106 SPIO-labeled cells embedded within hydrogel were injected in 2 doses using a transcutaneous cannula and an epidural anesthesia catheter. T2-weighted MR images were obtained at 3T before and immediately after cell infusion. Two weeks after injection, histological examination was performed for detection of transplanted cells.
Results
MSCs were efficiently labeled with Molday ION rhodamine. Cells could be readily detected in the injected vertebral tissue explants as distinct hypointensities with sufficient sensitivity. MR monitoring indicated that the MSCs were successfully delivered into the IVD in vivo, which was confirmed by iron-positive Prussian Blue staining of the tissue within the IVD.
Conclusion
We have developed a technique for non-invasive monitoring of minimally invasive stem delivery into the IVD at 3T. By using a large animal model mimicking the anatomy of IVD in humans, the present results indicate that this procedure may be clinically feasible.
doi:10.1371/journal.pone.0074658
PMCID: PMC3772957  PMID: 24058619
14.  MRI-detectable pH nanosensors incorporated into hydrogels for in vivo sensing of transplanted cell viability 
Nature materials  2013;12(3):268-275.
Biocompatible nanomaterials and hydrogels have become an important tool for improving cell-based therapies by promoting cell survival and protecting cell transplants from immune rejection. Although their potential benefit has been widely evaluated, it is currently not possible to determine, in vivo, if and how long cells remain viable following their administration without the use of a reporter gene. We here report a pH nanosensor-based magnetic resonance imaging (MRI) technique that can monitor cell death in vivo non-invasively. We demonstrate that specific MRI parameters that change upon cell death of microencapsulated hepatocytes are associated with the measured bioluminescence imaging (BLI) radiance. Moreover, the readout from this pH-sensitive nanosensor can be directly co-registered with high-resolution anatomical images. All the components of these nanosensors are clinical-grade and hence this approach should be a translatable and universal modification of hydrogels.
doi:10.1038/nmat3525
PMCID: PMC3578129  PMID: 23353626
15.  Microencapsulated cell tracking 
NMR in biomedicine  2012;26(7):850-859.
Microencapsulation of therapeutic cells has been widely pursued to achieve cellular immunoprotection following transplantation. Initial clinical studies have shown the potential of microencapsulation using semi-permeable alginate layers, but much needs to be learned about the optimal delivery route, in vivo pattern of engraftment, and microcapsule stability over time. In parallel with noninvasive imaging techniques for ‘naked’ (i.e. unencapsulated) cell tracking, microcapsules have now been endowed with contrast agents that can be visualized by 1H MRI, 19F MRI, X-ray/computed tomography and ultrasound imaging. By placing the contrast agent formulation in the extracellular space of the hydrogel, large amounts of contrast agents can be incorporated with negligible toxicity. This has led to a new generation of imaging biomaterials that can render cells visible with multiple imaging modalities.
doi:10.1002/nbm.2894
PMCID: PMC3655121  PMID: 23225358
microcapsules; cell therapy; diabetes; islet cell transplantation; MRI; ultrasound imaging; computed tomography; contrast agent
16.  X-Ray-Visible Microcapsules Containing Mesenchymal Stem Cells Improve Hind Limb Perfusion in a Rabbit Model of Peripheral Arterial Disease 
Stem cells (Dayton, Ohio)  2012;30(6):1286-1296.
The therapeutic goal in peripheral arterial disease (PAD) patients is to restore blood flow to ischemic tissue. Stem cell transplantation offers a new avenue to enhance arteriogenesis and angiogenesis. Two major problems with cell therapies are poor cell survival and the lack of visualization of cell delivery and distribution. To address these therapeutic barriers, allogeneic bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in alginate impregnated with a radiopaque contrast agent (MSC-Xcaps.) In vitro MSC-Xcap viability by a fluorometric assay was high (96.9% ± 2.7% at 30 days postencapsulation) and as few as 10 Xcaps were visible on clinical x-ray fluoroscopic systems. Using an endovascular PAD model, rabbits (n = 21) were randomized to receive MSC-Xcaps (n = 6), empty Xcaps (n = 5), unencapsulated MSCs (n = 5), or sham intramuscular injections (n = 5) in the ischemic thigh 24 hours postocclusion. Immediately after MSC transplantation and 14 days later, digital radiographs acquired on a clinical angiographic system demonstrated persistent visualization of the Xcap injection sites with retained contrast-to-noise. Using a modified TIMI frame count, quantitative angiography demonstrated a 65% improvement in hind limb perfusion or arteriogenesis in MSC-Xcap-treated animals versus empty Xcaps. Post-mortem immunohistopathology of vessel density by anti-CD31 staining demonstrated an 87% enhancement in angiogenesis in Xcap-MSC-treated animals versus empty Xcaps. MSC-Xcaps represent the first x-ray-visible cellular therapeutic with enhanced efficacy for PAD treatment.
doi:10.1002/stem.1096
PMCID: PMC3653421  PMID: 22438076
Mesenchymal stem cells; Cone-beam computed tomography; Angiogenesis inducing agents; Peripheral arterial disease; Barium sulfate
17.  Design of Targeted Cardiovascular Molecular Imaging Probes 
Molecular imaging relies on the development of sensitive and specific probes coupled with imaging hardware and software to provide information about the molecular status of a disease and its response to therapy, which are important aspects of disease management. As genomic and proteomic information from a variety of cardiovascular diseases becomes available, new cellular and molecular targets will provide an imaging readout of fundamental disease processes. A review of the development and application of several cardiovascular probes is presented here. Strategies for labeling cells with superparamagnetic iron oxide nanoparticles enable monitoring of the delivery of stem cell therapies. Small molecules and biologics (e.g., proteins and antibodies) with high affinity and specificity for cell surface receptors or cellular proteins as well as enzyme substrates or inhibitors may be labeled with single-photon–emitting or positron-emitting isotopes for nuclear molecular imaging applications. Labeling of bispecific antibodies with single-photon–emitting isotopes coupled with a pretargeting strategy may be used to enhance signal accumulation in small lesions. Emerging nanomaterials will provide platforms that have various sizes and structures and that may be used to develop multimeric, multimodal molecular imaging agents to probe one or more targets simultaneously. These platforms may be chemically manipulated to afford molecules with specific targeting and clearance properties. These examples of molecular imaging probes are characteristic of the multidisciplinary nature of the extraction of advanced biochemical information that will enhance diagnostic evaluation and drug development and predict clinical outcomes, fulfilling the promise of personalized medicine and improved patient care.
doi:10.2967/jnumed.109.068130
PMCID: PMC3629978  PMID: 20395345
molecular imaging; bispecific antibodies; multimeric molecular imaging agents; nanomaterials; superparamagnetic iron oxide; nanoparticles
18.  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.
doi:10.1002/glia.22339
PMCID: PMC3579214  PMID: 22499166
multiple sclerosis; EAE; glial precursor cells; MR imaging; bioluminescence imaging
19.  In Vivo Tracking Techniques for Cellular Regeneration, Replacement, and Redirection 
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.
doi:10.2967/jnumed.112.106146
PMCID: PMC3579215  PMID: 23143090
in vivo imaging; tracking; cellular therapy; transplantation; regenerative medicine
20.  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.
doi:10.3727/215517912X653337
PMCID: PMC3534966  PMID: 23293747
Cell transplantation; fulminant liver failure; magnetic resonance imaging; iron nanoparticle contrast agent; bioluminescent imaging
21.  Automated detection and characterization of SPIO-labeled cells and capsules using magnetic field perturbations 
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.
doi:10.1002/mrm.22998
PMCID: PMC3170691  PMID: 21656554
SPIO; magnetic moment; MRI; cell tracking; magnetocapsules; susceptibility
22.  Using C-Arm X-Ray Imaging to Guide Local Reporter Probe Delivery for Tracking Stem Cell Engraftment 
Theranostics  2013;3(11):916-926.
Poor cell survival and difficulties with visualization of cell delivery are major problems with current cell transplantation methods. To protect cells from early destruction, microencapsulation methods have been developed. The addition of a contrast agent to the microcapsule also could enable tracking by MR, ultrasound, and X-ray imaging. However, determining the cell viability within the microcapsule still remains an issue. Reporter gene imaging provides a way to determine cell viability, but delivery of the reporter probe by systemic injection may be hindered in ischemic diseases.
In the present study, mesenchymal stem cells (MSCs) were transfected with triple fusion reporter gene containing red fluorescent protein, truncated thymidine kinase (SPECT/PET reporter) and firefly luciferase (bioluminescence reporter). Transfected cells were microencapsulated in either unlabeled or perfluorooctylbromide (PFOB) impregnated alginate. The addition of PFOB provided radiopacity to enable visualization of the microcapsules by X-ray imaging. Before intramuscular transplantation in rabbit thigh muscle, the microcapsules were incubated with D-luciferin, and bioluminescence imaging (BLI) was performed immediately. Twenty-four and forty-eight hours post transplantation, c-arm CT was used to target the luciferin to the X-ray-visible microcapsules for BLI cell viability assessment, rather than systemic reporter probe injections. Not only was the bioluminescent signal emission from the PFOB-encapsulated MSCs confirmed as compared to non-encapsulated, naked MSCs, but over 90% of injection sites of PFOB-encapsulated MSCs were visible on c-arm CT. The latter aided in successful targeting of the reporter probe to injection sites using conventional X-ray imaging to determine cell viability at 1-2 days post transplantation. Blind luciferin injections to the approximate location of unlabeled microcapsules resulted in successful BLI signal detection in only 18% of injections. In conclusion, reporter gene probes can be more precisely targeted using c-arm CT for in vivo transplant viability assessment, thereby avoiding large and costly systemic injections of a reporter probe.
doi:10.7150/thno.6943
PMCID: PMC3879108  PMID: 24396502
mesenchymal stem cells; reporter gene; microencapsulation; bioluminescence; c-arm CT; probe targeting.
23.  In Vivo Multi-Color Molecular MR Imaging Using DIACEST Liposomes 
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.
doi:10.1002/mrm.23100
PMCID: PMC3522097  PMID: 22392814
24.  MULTIMODAL IMAGING OF SUSTAINED DRUG RELEASE FROM 3-D POLY(PROPYLENE FUMARATE) (PPF) SCAFFOLDS 
doi:10.1016/j.jconrel.2011.06.035
PMCID: PMC3202643  PMID: 21763735
Nanoparticles; poly(propylene fumarate) (PPF); magnetic resonance imaging (MRI); chemical exchange saturation transfer (CEST); Protamine sulfate (PS) drug release; doxorubicin
25.  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.
doi:10.1002/dmrr.1248
PMCID: PMC3218557  PMID: 22069256
Diabetes; islets; transplantation; magnetic resonance imaging; positron emission tomography; bioluminescent imaging

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