Fig 1.
The efficacy of hMSCs labeling with MC03F microparticles and the effects of labeling on cell viability and proliferation.
(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.
Fig 2.
MR images of a phantom injected with 20 μl of saline containing 101 hMSCs labeled with MC03F microspheres.
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.
Fig 3.
MR images of live rat brains after stereotaxic injection of 20 μl saline or different quantities (from 101 to 105) of SPIO-labeled hMSCs in 20 μl saline into the right striatum.
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.
Fig 4.
Quantitative evaluation of MRI data.
(A) The medians of the values of the ratio of minimum to mean signal calculated from data obtained using different pulse sequences after injection of 101, 102 or 103 of SPIO-labeled hMSCs or saline into the rat striatum. Data for cell concentrations higher than 103 are not shown on the graph, because for them the minimum signal was zero for most MRI pulse sequences. Whiskers on the plot represent interquartile range. (B) Volumes of the hypointensity zones calculated from SWI data obtained after injection of varying quantities of SPIO-labeled hMSCs or saline into the rat striatum (box and whisker plot). (C) Values of the ratio of minimum to mean signal calculated from SWI data obtained after injection of 101, 102 or 103 of SPIO-labeled hMSCs or saline into the rat striatum (box and whisker plot). Greek letters show statistical significance of differences between pulse sequences (A), or saline-injected and SPIO-labeled cell-transplanted rats (B, C): α –p<0.05; β –p<0.01; γ –p<0.001.
Fig 5.
MR and histological images of the sites of stereotaxic injection of 20 μl of saline or varying numbers of SPIO-labeled hMSCs suspended in 20 μl of saline into the right striatum of rats.
Top panel row: T2WI. Second from top panel row: SWI. Middle panel row: conventional fluorescence microscopy. Cell nuclei stained with DAPI look blue. Transplanted hMSCs containing MC03F microparticles in their cytoplasm emit green fluorescence. Second from bottom panel row: confocal fluorescence microscopy. Bottom panel row: conventional microscopy of Perls’ Prussian Blue stained sections. The scale bars represent 500 μm in panoramic views of coronal sections and 50 μm in high-magnification images.
Fig 6.
MR and histological images of a rat brain taken after intra-arterial administration of 5x105 hMSCs labeled with MC03F microparticles via the right internal carotid artery.
MRI was performed immediately after cell transplantation and euthanasia–immediately after MRI. Top panel row, left to right: T2WI, T2*WI based on MEDIC, T2*WI based on FLASH 3D, and SWI images. Rectangles indicate the area of labeled cells distribution. Bottom panel, left to right: conventional fluorescence microscopy (low and high magnification), bright-field microscopy of Perls’ Prussian Blue stained sections, confocal fluorescence microscopy (low and high magnification). The scale bars in panoramic views of coronal sections represent 500 μm and at high-magnification pictures they correspond to 50 μm.
Fig 7.
SWI and high-magnification confocal fluorescence microscopy of normal rat brain immediately after intra-arterial injection of 5x105 hMSCs labeled with MC03F microparticles.
SWI allows detection of small groups and even single hMSCs found in brain tissue close or around cerebral blood vessels. The scale bars represent 50 μm.
Fig 8.
MR and confocal fluorescence microscopy images of rat brain after intracerebral and intra-arterial transplantation of hMSCs double-labeled with SPIO microparticles and PKH26.
(A) MRI of rat brain (T2WI and SWI) immediately after intracerebral injection of 105 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 105 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.
Table 1.
MRI detection thresholds of SPIO-labeled stem cells in phantom studies.
Table 2.
MR detection thresholds of SPIO-labeled stem cells in live animal brain.