7 research outputs found
Methodological aspects of MRI of transplanted superparamagnetic iron oxide-labeled mesenchymal stem cells in live rat brain
<div><p><i>In vivo</i> tracking of transplanted mesenchymal stem cells (MSCs) migration and homing is vital for understanding the mechanisms of beneficial effects of MSCs transplantation in animal models of diseases and in clinical trials. Transplanted cells can be labeled with superparamagnetic iron oxide (SPIO) particles and visualized in vivo using a number of iron sensitive MRI techniques. However, the applicability of those techniques for SPIO-labeled MSCs tracking in live brain has not been sufficiently investigated. The goal of this study was to estimate the efficiency of various MRI techniques of SPIO-labeled cell tracing in the brain. To achieve that goal, the precision and specificity of T2WI, T2*WI and SWI (Susceptibility-Weighted Imaging) techniques of SPIO-labeled MSCs tracing <i>in vitro</i> and in live rat brain were for the first time compared in the same experiment. We have shown that SWI presents the most sensitive pulse sequence for SPIO-labeled MSCs MR visualization. After intracerebral administration due to limitations caused by local micro-hemorrhages the visualization threshold was 10<sup>2</sup> cells, while after intra-arterial transplantation SWI permitted detection of several cells or even single cells. There is just one publication claiming detection of individual SPIO-labeled MSCs in live brain, while the other state much lower sensitivity, describe detection of different cell types or high resolution tracing of MSCs in other tissues. This study confirms the possibility of single cell tracing in live brain and outlines the necessary conditions. SWI is a method convenient for the detection of single SPIO labeled MSCs and small groups of SPIO labeled MSCs in brain tissue and can be appropriate for monitoring migration and homing of transplanted cells in basic and translational neuroscience.</p></div
MR images of live rat brains after stereotaxic injection of 20 μl saline or different quantities (from 10<sup>1</sup> to 10<sup>5</sup>) of SPIO-labeled hMSCs in 20 μl saline into the right striatum.
<p>The images were taken immediately after cell transplantation utilizing different MRI pulse sequences—T2WI, T2*WI based on MEDIC, T2*WI based on FLASH 3D, and SWI.</p
MR and confocal fluorescence microscopy images of rat brain after intracerebral and intra-arterial transplantation of hMSCs double-labeled with SPIO microparticles and PKH26.
<p>(A) MRI of rat brain (T2WI and SWI) immediately after intracerebral injection of 10<sup>5</sup> hMSCs double-labeled with MC03F SPIO microparticles and PKH26. Hypointense regions indicate the location of SPIO labeled cells or probably extracellular SPIO microspheres. (B) High-magnification confocal micrographs of the hMSCs injection site. The same rat brain as in fig A. White arrowhead points to a double-labeled hMSC (membrane dye PKH26 is red, SPIO microparticles in the cytoplasm are green, and cell nucleus stained with DAPI is blue). Single clusters of extracellular iron can also be visualized (white arrows). The scale bars represent 10 μm. (C) Confocal panoramic micrographs of the hMSCs injection site (the same rat brain as in fig A). Labeled hMSCs are located along the track of the injection needle and in corpus callosum. The scale bars represent 500 μm. (D) MRI of rat brain (T2WI and SWI) after intra-arterial transplantation of 10<sup>5</sup> hMSCs labeled with SPIO-microparticles and PKH26. The white arrows on the SWI picture indicate the location of SPIO labeled cells. (E) High-magnification confocal micrographs (the same rat brain as in fig D) of transplanted hMSCs. White arrowhead points at double-labeled cells (membrane dye PKH26 is red, SPIO microparticles in the cytoplasm are green, and cell nuclei stained with DAPI is blue). The scale bars represent 20 μm.</p
MR images of a phantom injected with 20 μl of saline containing 10<sup>1</sup> hMSCs labeled with MC03F microspheres.
<p>The images were taken utilizing different MRI pulse sequences—T2WI, T2*WI based on FLASH 3D and SWI. Labeled hMSCs are visualized as hypointense areas in the isointense Spherogel milieu. Images captured at all three pulse sequences are presented in axial projection (three left panels). In addition, the coronal projection of the SWI is presented (right panel). The scale bars represent 1 mm.</p
The efficacy of hMSCs labeling with MC03F microparticles and the effects of labeling on cell viability and proliferation.
<p>(A)-(C): fluorescent microscopy of a hMSC culture 24 hours after labeling with MC03F microparticles. (A) MC03F microparticles (Dragon Green fluorescence). (B) Cell nuclei (DAPI blue fluorescence). (C) A and B merged. (D) Transmitted light microscopy of unstained hMSC culture (the same area as in A-C) 24 hours after labeling with MC03F microparticles. SPIO microparticles are visualized in the cytoplasm as brown spots around clear nuclei. (E) Flow cytometry analysis of cells 24 hours after labeling. The solid line presents data for labeled hMSCs and the dotted line—for unlabeled, control hMSCs. X-axis shows fluorescence intensity and Y-axis—cell counts. The plot demonstrates that about 96% of the cells contained Dragon Green fluorescent microparticles. (F) Influence of the labeling with MC03F microparticles on hMSCs viability and proliferation. Optical density (Y axis) is proportional to lactate dehydrogenase (LDH) activity in the cells and, hence, to the number of living cells. The presented histograms show that the numbers of living cells were not significantly different in labeled and control cultures indicating the absence of negative effects associated with labeling on cell viability and proliferation. The scale bars on all microphotographs mark 100 μm.</p
MR detection thresholds of SPIO-labeled stem cells in live animal brain.
<p>MR detection thresholds of SPIO-labeled stem cells in live animal brain.</p
MRI detection thresholds of SPIO-labeled stem cells in phantom studies.
<p>MRI detection thresholds of SPIO-labeled stem cells in phantom studies.</p