Dorota Kedziorek

Dynamic Imaging of Allogeneic Mesenchymal Stem Cells Trafficking to Myocardial Infarction

Background—Recent results from animal studies suggest that stem cells may be able to home to sites of myocardial injury to assist in tissue regeneration. However, the histological interpretation of postmortem tissue, on which many of these studies are based, has recently been widely debated. Methods and Results—With the use of the high sensitivity of a combined single-photon emission CT (SPECT)/CT scanner, the in vivo trafficking of allogeneic mesenchymal stem cells (MSCs) colabeled with a radiotracer and MR contrast agent to acute myocardial infarction was dynamically determined. Redistribution of the labeled MSCs after intravenous injection from initial localization in the lungs to nontarget organs such as the liver, kidney, and spleen was observed within 24 to 48 hours after injection. Focal and diffuse uptake of MSCs in the infarcted myocardium was already visible in SPECT/CT images in the first 24 hours after injection and persisted until 7 days after injection and was validated by tissue counts of radioactivity. In contrast, MRI was unable to demonstrate targeted cardiac localization of MSCs in part because of the lower sensitivity of MRI. Conclusions—Noninvasive radionuclide imaging is well suited to dynamically track the biodistribution and trafficking of mesenchymal stem cells to both target and nontarget organs.

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.

Positive contrast visualization of iron oxide‐labeled stem cells using inversion‐recovery with ON‐resonant water suppression (IRON)

In proton magnetic resonance imaging (MRI) metallic substances lead to magnetic field distortions that often result in signal voids in the adjacent anatomic structures. Thus, metallic objects and superparamagnetic iron oxide (SPIO)‐labeled cells appear as hypointense artifacts that obscure the underlying anatomy. The ability to illuminate these structures with positive contrast would enhance noninvasive MR tracking of cellular therapeutics. Therefore, an MRI methodology that selectively highlights areas of metallic objects has been developed. Inversion‐recovery with ON‐resonant water suppression (IRON) employs inversion of the magnetization in conjunction with a spectrally‐selective on‐resonant saturation prepulse. If imaging is performed after these prepulses, positive signal is obtained from off‐resonant protons in close proximity to the metallic objects. The first successful use of IRON to produce positive contrast in areas of metallic spheres and SPIO‐labeled stem cells in vitro and in vivo is presented. Magn Reson Med 58:1072–1077, 2007.

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.

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.

In vivo "hot spot" MR imaging of neural stem cells using fluorinated nanoparticles

To optimize 19F MR tracking of stem cells, we compared cellular internalization of cationic and anionic perfluoro‐15‐crown‐5‐ether (PFCE) nanoparticles using cell culture plates with different surface coatings. The viability and proliferation of anionic and cationic PFCE‐labeled neural stem cells (NSCs) did not differ from unlabeled cells. Cationic PFCE nanoparticles (19F T1/T2 = 580/536 ms at 9.4 Tesla) were superior to anionic particles for intracellular fluorination. Best results were obtained with modified polystyrene culture dishes coated with both carboxylic and amino groups rather than conventional carboxyl‐coated dishes. After injecting PFCE‐labeled NSCs into the striatum of mouse brain, cells were readily identified in vivo by 19F MRI without changes in signal or viability over a 2‐week period after grafting. These results demonstrate that neural stem cells can be efficiently fluorinated with cationic PFCE nanoparticles without using transfection agents and visualized in vivo over prolonged periods with an MR sensitivity of approximately 140 pmol of PFCE/cell. Magn Reson Med 60:1506–1511, 2008.

Noninvasive Detection of Macrophage-rich Atherosclerotic Plaque in Hyperlipidemic Rabbits using ‘Positive Contrast’ Magnetic Resonance Imaging

OBJECTIVES This study was designed to identify macrophage-rich atherosclerotic plaque noninvasively by imaging the tissue uptake of long-circulating superparamagnetic nanoparticles with a positive contrast off-resonance imaging sequence (inversion recovery with ON-resonant water suppression [IRON]). BACKGROUND The sudden rupture of macrophage-rich atherosclerotic plaques can trigger the formation of an occlusive thrombus in coronary vessels, resulting in acute myocardial infarction. Therefore, a noninvasive technique that can identify macrophage-rich plaques and thereby assist with risk stratification of patients with atherosclerosis would be of great potential clinical utility. METHODS Experiments were conducted on a clinical 3-T magnetic resonance imaging (MRI) scanner in 7 heritable hyperlipidemic and 4 control rabbits. Monocrystalline iron-oxide nanoparticles (MION)-47 were administrated intravenously (2 doses of 250 mumol Fe/kg), and animals underwent serial IRON-MRI before injection of the nanoparticles and serially after 1, 3, and 6 days. RESULTS After administration of MION-47, a striking signal enhancement was found in areas of plaque only in hyperlipidemic rabbits. The magnitude of enhancement on magnetic resonance images had a high correlation with the number of macrophages determined by histology (p < 0.001) and allowed for the detection of macrophage-rich plaque with high accuracy (area under the curve: 0.92, SE: 0.04, 95% confidence interval: 0.84 to 0.96, p < 0.001). No significant signal enhancement was measured in remote areas without plaque by histology and in control rabbits without atherosclerosis. CONCLUSIONS Using IRON-MRI in conjunction with superparamagnetic nanoparticles is a promising approach for the noninvasive evaluation of macrophage-rich, vulnerable plaques.

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. Magn Reson Med 63:1031–1043, 2010.

X‐Ray‐Visible Microcapsules Containing Mesenchymal Stem Cells Improve Hind Limb Perfusion in a Rabbit Model of Peripheral Arterial Disease

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. STEM CELLS2012;30:1286–1296

Microencapsulated cell tracking

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. Copyright