Assaf A. Gilad

Mesoporous Silica-Coated Hollow Manganese Oxide Nanoparticles as Positive T1 Contrast Agents for Labeling and MRI Tracking of Adipose-Derived Mesenchymal Stem Cells

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.

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. Methods— 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). Results— 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. Conclusions— 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.

Quantifying exchange rates in chemical exchange saturation transfer agents using the saturation time and saturation power dependencies of the magnetization transfer effect on the magnetic resonance imaging signal (QUEST and QUESP): pH calibration for poly-L-lysine and a starburst dendrimer

The ability to measure proton exchange rates in tissue using MRI would be very useful for quantitative assessment of magnetization transfer properties, both in conventional MT imaging and in the more recent chemical exchange saturation transfer (CEST) approach. CEST is a new MR contrast mechanism that depends on several factors, including the exchange rate of labile protons in the agent in a pH‐dependent manner. Two new methods to monitor local exchange rate based on CEST are introduced. The two MRI‐compatible approaches to measure exchange are quantifying exchange using saturation time (QUEST) dependence and quantifying exchange using saturation power (QUESP) dependence. These techniques were applied to poly‐L‐lysine (PLL) and a generation‐5 polyamidoamine dendrimer (SPD‐5) to measure the pH dependence of amide proton exchange rates in the physiologic range. Data were fit both to an analytical expression and to numerical solutions to the Bloch equations. Results were validated by comparison with exchange rates determined by two established spectroscopic methods. The exchange rates determined using the four methods were pooled for the pH‐calibration curve of the agents consisting of contributions from spontaneous (k0) acid catalyzed (ka), and base catalyzed (kb) exchange rate constants. These constants were k0 = 68.9 Hz, ka = 1.21 Hz, kb = 1.92 × 109 Hz, and k0 = 106.4 Hz, ka = 25.8 Hz, kb = 5.45 × 108 Hz for PLL and SPD‐5, respectively, showing the expected predominance of base‐catalyzed exchange for these amide protons. Magn Reson Med, 2006.

Instant MR labeling of stem cells using magnetoelectroporation

For cellular MR imaging, conventional approaches to intracellular magnetic labeling of nonphagocytic cells rely on the use of secondary compounds such as transfection agents and prolonged incubation of cells. Magnetoelectroporation (MEP) was investigated as an alternative method to achieve instant (<1 s) endosomal labeling with the FDA‐approved formulation Feridex, without the need for adjunct agents or initiating cell cultures. While MEP was harmful at higher voltages or pulse durations, the procedure could be properly calibrated using a pulse of 130 V and 17 ms. Labeling was demonstrated for stem cells from mice, rats, and humans; the uptake of iron was in the picogram range and comparable to values obtained using transfection agents. MEP‐labeled stem cells exhibited an unaltered viability, proliferation, and mitochondrial metabolic rate. Labeled mesenchymal stem cells (MSCs) and neural stem cells (NSCs) differentiated into adipogenic, osteogenic, and neural lineages in an identical fashion as unlabeled cells, while containing Feridex particles as demonstrated by double immunofluorescent staining. MEP‐labeled NSCs proliferated normally following intrastriatal transplantation and could be readily detected by MR imaging in vivo. As MEP circumvents the use of secondary agents, obviating the need for clinical approval, MEP labeling may be ideally suitable for bedside implementation. Magn Reson Med, 2005.

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. Magn Reson Med 60:1–7, 2008.

New "multicolor" polypeptide diamagnetic chemical exchange saturation transfer (DIACEST) contrast agents for MRI.

An array of 33 prototype polypeptides was examined as putative contrast agents that can be distinguished from each other based on the chemical exchange saturation transfer (CEST) mechanism. These peptides were chosen based on predictions of the chemical exchange rates of exchangeable amide, amine, and hydroxyl protons that produce this contrast, and tested at 11.7T for their CEST suitability. Artificial colors were assigned to particular amino acid units (lysine, arginine, threonine, and serine) based on the separate resonance frequencies of these exchangeable protons. The magnitude of the CEST effect could be fine‐tuned by altering the amino acid sequence, and these three exchangeable groups could be distinguished in an MR phantom based on their different chemical shifts (“colors”). These new diamagnetic CEST (DIACEST) agents possess a wide range of electrostatic charges, compositions, and protein stabilities in vivo, making them potentially suitable for a variety of biological applications such as designing MR reporter genes for imaging cells and distinguishing multiple targets within the same MR image. Magn Reson Med 60:803–812, 2008.

Applicability and limitations of MR tracking of neural stem cells with asymmetric cell division and rapid turnover: The case of the shiverer dysmyelinated mouse brain

LacZ‐transfected C17.2 neural stem cells (NSCs) were labeled with the superparamagnetic iron oxide formulation Feridex prior to ICV injection in shi/shi neonates. Feridex labeling did not alter cell differentiation in vitro and in vivo. Initially, MR images obtained at 11.7T correlated closely to NSC distribution as assessed with anti‐dextran and anti‐β‐galactosidase double‐fluorescent immunostaining. However, at 6 days postgrafting there was already a pronounced mismatch between the hypointense MR signal and the histologically determined cell distribution, with a surprisingly sharp cutoff rather than a gradual decrease of signal. Positive in vivo BrdU labeling of NSCs showed that significant cell replication occurred post‐transplantation, causing rapid dilution of Feridex particles between mother and daughter cells toward undetectable levels. Neural differentiation experiments demonstrated asymmetric cell division, explaining the observed sharp cutoff. At later time points (2 weeks), the mismatch further increased by the presence of non‐cell‐associated Feridex particles resulting from active excretion or cell death. These results are a first demonstration of the inability of MRI to track rapidly dividing and self‐renewing, asymmetrically dividing SCs. Therefore, MR cell tracking should only be applied for nonproliferating cells or short‐term monitoring of highly‐proliferative cells, with mitotic symmetry or asymmetry being important for determining its applicability. Magn Reson Med 58:261–269, 2007.

MRI-detectable pH nanosensors incorporated into hydrogels for in vivo sensing of transplanted-cell viability

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.

Long-Term MR Cell Tracking of Neural Stem Cells Grafted in Immunocompetent Versus Immunodeficient Mice Reveals Distinct Differences in Contrast Between Live and Dead Cells

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. Magn Reson Med, 2011.

MRI Reporter Genes

Noninvasive molecular imaging of dynamic processes has benefited tremendously from the use of reporter genes. These genes encode for proteins that emit light, bind radiolabeled probes, or, as covered in this review, modulate MRI contrast. Reporter genes play a pivotal role in monitoring cell trafficking, gene replacement therapy, protein–protein interactions, neuronal plasticity, and embryonic development. Several strategies exist for generating MRI contrast: using enzyme-catalyzed chemical modification of metal-based contrast agents or (phosphorus) metabolites, iron-binding and iron-storage proteins to accumulate iron as a contrast agent, and artificial proteins for imaging based on chemical exchange saturation transfer. MRI reporter genes have the advantage that the specific signal can be coregistered with soft-tissue anatomy and functional tissue information and have, therefore, become an active and growing area of scientific interest.