Cell isolation and culture

hMSCs were isolated from normal human placenta (38–40 weeks of gestation) obtained from informed consent mothers at the Research Center for Obstetrics, Gynecology and Perinatology (Moscow, Russian Federation) using a conventional procedure described elsewhere [31]. The study was approved by the local Ethical Committee of the Research Center for Obstetrics, Gynecology and Perinatology and the Pirogov Russian National Research Medical University. In brief, human placenta tissue samples were thoroughly minced with scissors, extensively washed with cold Hank’s solution and after gentle mechanical agitation incubated for 30 min at 37°C in Hank’s solution in the presence of 0.1% type I collagenase (Gibco). The suspension was then centrifuged at 300×g for 10 min at 4°C, supernatant was discarded and pellet resuspended in the complete culture medium comprising DMEM-F12, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% fetal bovine serum (all reagents from Gibco). Cells were plated in T75 culture flasks and cultured in humidified atmosphere under standard conditions (37°C, 5% CO₂). Cells were allowed to adhere for 3 days and non-adherent cells were removed by replacing the medium. Upon reaching 80–90% confluence, adherent hMSCs were harvested by trypsinization and subcultured at 1:3 ratio in T75 flasks.

Cell labeling

hMSCs were labeled with SPIO-containing microspheres (MC03F Bangs Laboratories, mean diameter 0.50–0.99 μm) carrying Dragon Green fluorescent dye (λex = 480 nm, λem = 520 nm). Cell culture grown to 80–90% confluence was maintained for 12 hours in a CO₂ incubator in the presence of SPIO microspheres (5 μl of the stock suspension supplied by the manufacturer per 1 ml of culture medium), washed twice with Hank’s solution to remove microspheres that had not been absorbed by the cells, harvested by trypsinization and used in the phantom and animal transplantation experiments. In the preliminary experiments different times of incubation of cells with MC03F microspheres and concentrations of microspheres were tested. If 5–10 μl of the stock suspension per 1 ml of the culture medium was added, labeling reached its maximum after 10 hours of incubation and remained unchanged for at least 36 hours. To verify cell labeling, fluorescent and transmitted light microscopy was performed the next day after procedure using an AxioPlan-2 (Zeiss) fluorescent microscope and Zeiss Axiovert 40CFL microscope. Prior to microscopic examination, cells were washed three times with PBS, fixed with 4% paraformaldehyde, and stained with DAPI to allow cell nuclei visualization with fluorescent microscopy. The efficacy of the uptake of MC03F microspheres was also evaluated by flow cytometry. hMSC culture grown to 80–90% confluence was labeled with MC03F microspheres as described above. Cells maintained without SPIO microspheres served as negative control. After incubation, the cells were washed twice with PBS to remove microspheres that had not been absorbed by the cell. Cells were then harvested with 0.25% trypsin-EDTA, centrifuged (1000 rpm for 5 min at 25°C) and resuspended in serum free DMEM F12 (Gibco) medium to a final cell concentration of 4x10⁵ cells/ml. The fluorescence intensity and light scattering of the Dragon Green fluorescent dye were measured by FACS-Aria flow cytometer/cell sorter (BD Bioscience).

In some experiments hMSCs labeled with SPIO microparticles were additionally labeled with PKH26 red fluorescent dye (Sigma-Aldrich) according to the manufacturer's protocol. Briefly, after labeling with SPIO microspheres cells were harvested and placed in a conical polypropylene tube at the concentration of 2x10⁶ cells per ml. Cells were washed with the serum-free medium, centrifuged for 5 minutes, and the supernatant was aspirated. hMSCs were resuspended in 1 ml of Diluent C. 1 ml of Diluent C was separately mixed with 4 μl of PKH26 and added to the hMSCs suspension. After incubation for 5 minutes, the labeling reaction was stopped by adding an equal volume of fetal bovine serum. Double-labeled cells were immediately transplanted into the animals.

Evaluation of the viability and proliferation rate of SPIO labeled cells

Cells were seeded in 24-well cell culture plates (30,000 cells per well), left overnight to attach to plastic, and labeled with fluorescent magnetic microspheres as described above. Labeled hMSCs were rinsed three times in DMEM/F12 medium to remove the unbound label, fresh DMEM/F12 added, and cells were cultured for seven days under standard conditions, with medium exchange every two days. The number of viable cells was assessed with CytoTox 96 assay kit (Promega, USA; the assay is based on the measurement of the activity of lactate dehydrogenase (LDH) in live cells) immediately after completion of the labeling procedure (day 0) and after 1, 3, 5 and 7 days of culture, following the slightly modified manufacturer’s protocol. Briefly, the wells with labeled cells were washed three times with PBS and filled with 300 μl of deionized water. Cell membranes were lysed by three rounds of freezing and thawing. 50 μl aliquots of cell lysate were transferred into a 96-well plate, mixed with equal volume of substrate reagent supplied in the kit, and incubated for 1h at room temperature in the dark. The reaction was quenched by the addition of 50 μl of stop reagent (supplied in the kit). Optical density was measured at 490 nm using TECAN MX300 Pro plate reader. Cells subjected to exactly the same procedures, except that they were kept in the culture medium without microspheres, served as controls.

Measurement of the iron content in SPIO-labeled cells

To determine the average content of iron per one SPIO-labeled cell, 5x10⁵ labeled hMSCs were pelleted by centrifugation and dissolved by incubation in 0.5 ml of the iron release reagent (0.5 M HCl, 2.25% w/v KMnO₄) for 2 hours at 60°C. 250 μl of the resulting solution were mixed with 125 μl of H₂O and 25 μl of the iron detection reagent (6.5 mM of the sodium salt of 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid, 6.5 mM neocuproine, 2.5 М ammonium acetate, 1 M ascorbic acid). Light absorbance at 490 nm was measured with Victor X3 spectrophotometer (PerkinElmer) and iron concentration determined using the calibration curve created utilizing standard solutions with known iron content and expressed in pg iron per cell. Three independent experiments with separate cell preparations were carried out and results were presented as means of 3 experiments ± standard deviation.

Cell transplantation into the rat brain

All manipulations with experimental animals were approved by the local Ethical Committee of the Pirogov Russian National Research Medical University (Protocol No 140 from December 15, 2014) and carried out in accordance with the directive 2010/63/EU on the protection of animals used for scientific purposes of the European Parliament and the Council of the European Union of September 22, 2010. Adult male Wistar rats weighing 250–300 g were obtained from AlCondi, Ltd animal breeding house, Moscow, Russian Federation. The animals (n = 39) were randomly attributed to one of the following seven experimental groups: transplantation of 10¹ SPIO-labeled hMSCs (n = 5); transplantation of 10² SPIO-labeled hMSCs (n = 8); transplantation of 10³ SPIO-labeled hMSCs (n = 4); transplantation of 10⁴ SPIO-labeled hMSCs (n = 7); transplantation of 10⁵ SPIO-labeled hMSCs (n = 8); saline injection (negative control, n = 4); transplantation of 10⁵ hMSCs double-labeled with SPIO and PKH26 (n = 3). For the surgery, rats were anesthetized with 3% isoflurane and maintained at artificial ventilation with the mixture of 2–2.5% isoflurane and 97.5–98% atmospheric air supplied by E-Z-7000 animal anesthesia system (E-Z Anesthesia® Systems). Throughout all surgical procedures, body temperature was maintained at 37°C with a heating pad. For cell transplantation, anesthetized rats were placed in the Angle Two computer-assisted stereotaxic system (Leica). After skin incision and removing of surface tissues, a hole was drilled in the skull and 20 μl of the suspension of SPIO-labeled hMSCs in saline or 20 μl of saline were slowly injected over the 5 min time interval using a 500 μl Hamilton microsyringe and an automatic syringe pump system (KD Scientific). Injection was made into the right striatum according to the following stereotactic coordinates: bregma +0.6 mm AP (anterior-posterior), +3.5 mm ML (medial-lateral), -4.5 mm VD (ventral-dorsal). After transplantation, the needle was left in place for 5 min to allow the cell suspension to distribute in the brain tissue and to minimize the reverse flow of transplanted material into the needle. The needle was slowly withdrawn and skin was closed with simple interrupted stitches.

Phantom for the in vitro MRI studies

In vitro MRI was carried out using phantoms made of 5 ml plastic syringes filled with Sphero®GEL (Biomir Service, Krasnoznamensk, Moscow region, Russia) and injected with 10¹ hMSCs in 20 μl saline utilizing the stereotaxic system described in the previous section. Sphero®GEL is a gel material made of processed bovine collagen and certified for medical use. We did not try to inject single cells, because we considered it impossible to reproducibly deliver a single cell into a phantom.

Magnetic resonance imaging (MRI)

MRI of rat brain for all experimental groups was performed immediately after cell transplantation with the 7T ClinScan system (Bruker BioSpin, USA), 30/12 cm bore size, 630 mT/m gradient strength, 6300 T/m/s maximum slew rate, with phased array four elements rat brain coil. During the procedure rats were maintained at inhalation anesthesia with the mixture of 2–2.5% isoflurane and 97.5–98% atmospheric air supplied by an EZ-7000 animal anesthesia system (E-Z Anesthesia® Systems). The following MRI protocols were used to visualize SPIO-labeled cells in the rat brain (the total duration was approximately 20 min.):

• T2 weighted imaging (T2WI; Turbo Spin Echo pulse sequence with restore magnetization pulse; turbo factor = 9; TR/TE = 4000/46 ms; averages = 2; spectral fat saturation; FOV = 37 x 29.6 mm; slice thickness = 0.5 mm; matrix size = 320 x 256; acquisition time = 4:44);
• T2* weighted imaging based on MEDIC pulse sequence (T2*WI based on MEDIC; MEDIC—Multi Echo Data Image Combination with RF spoiling pulse sequence; combined echoes = 5; TR/TE = 1540/30 ms; flip angle = 30; averages = 1; spectral fat saturation; FOV = 37x29.6 mm; slice thickness = 0.5 mm; matrix size = 320 x 256; acquisition time = 6:34);
• T2* weighted imaging based on FLASH 3D pulse sequence (T2*WI based on FLASH 3D; 3D Gradient Echo pulse sequence with RF spoiling and flow compensation; TR/TE = 50/19.1 ms; flip angle = 15; averages = 1; spectral fat saturation; FOV = 30 x 20.6 mm; slice thickness = 0.5 mm; matrix size = 256 x 176; acquisition time = 5:31);
• Susceptibility Weighted Imaging (SWI; 3D Gradient Echo with RF spoiling and flow compensation; TR/TE = 50/19.1 ms; flip angle = 15; averages = 1; spectral fat saturation; FOV = 30 x 20.6 mm; slice thickness = 0.5 mm; matrix size = 256 x 176; acquisition time = 5:31).

The same protocols, except the T2*WI based on MEDIC, were used for the in vitro MR visualization of SPIO-labeled cells injected into the phantoms.

Quantitative analysis of intracerebral SPIO-labeled cell transplantation MRI data

MRI data analysis was performed using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2015). The area of signal hypointensity at the site of stereotaxic SPIO-labeled cell transplantation was measured using semi-automatic region selection. The volume V of the hypointensity zone was calculated by the summation of areas measured on adjacent cross-sections using the following formula: V = (S1+…+Sn) × (h+d), where S1,…,Sn–is area measured on slice n, h is the slice thickness, and d is the interval between the slices. The ratio of minimum to mean signal was measured using points close to the straight line, drawn parallel to the coil and perpendicular to the direction of stereotaxic injection as shown in S1 Fig. The minimum signal intensity was measured at the site of injection, and the mean signal intensity was calculated using values obtained from intact surrounding tissue.

Histology

Immediately after MRI, animals were sacrificed by intraperitoneal injection of a lethal dose of Chloral hydrate and transcardially perfused with 0.1 M phosphate buffered saline (PBS, pH 7.4), followed by ice-cold 4% paraformaldehyde (PFA) in 0.1M PBS. The brains were removed, post-fixed at 4°C overnight in the same fixative, washed three times with PBS. Сoronal 40 μm-thick sections were cut the HM 650 v vibratome (Microm GmbH, Berlin, Germany). Every second section through the transplantation site was used for fluorescence microscopy, every third section—for fluorescent confocal microscopy, and each fourth section—for conventional bright-field microscopy. Prior to fluorescence microscopy, slide-mounted tissue sections were counterstained by incubating for 10 min at 20–22°C with DAPI solution (2 μg/mL, Sigma) to visualize cell nuclei. The fluorescence histological images were acquired with the BZ-9000E fluorescence microscope (Keyence, Japan). Fluorescence confocal micrographs were captured with the Nikon A1R MP+ laser scanning confocal microscope equipped with a 405, 488, 561, 638 lasers and Plan Apo20x/0.75 Dic N, Apo IR 60x/1.27 WI and Apo TIRF 60x/1.49 oil Dic objective lenses. To visualize the iron microspheres, Perls’ Prussian blue staining was performed. Tissue sections were rinsed with deionized water and immersed in Perls’ solution containing 2% potassium ferrocyanide and 2% HCl for 10 minutes. Sections were then rinsed with deionized water and counterstained with Neutral Red (Sigma) for 3 minutes. Bright-field images were acquired using Keyence BZ-9000E microscope.

Statistical analysis

Statistical analysis of cell viability and proliferation data was performed by one-way ANOVA using GraphPad Prism software (GraphPad, San Diego, CA, USA).

Statistical analysis of MRI data was performed with Statistica 10 (StatSoft, USA) using Wilcoxon matched pairs test for dependent samples and Mann-Whitney U test for independent samples. For all the results statistical significance was set at p < 0.05.

Labeling of hMSCs with SPIO microparticles and its influence on cell viability and proliferation

As clearly seen on Fig 1A–1C, 24 hours after labeling the cytoplasm of virtually all cells of hMSC cultures contained vast amounts of fluorescent microspheres. DAPI-stained nuclei seem to contain no microparticles. SPIO microparticles could also be visualized in the cell cytoplasm with conventional transmitted light microscopy (Fig 1D). Since cell nuclei remained totally clear, Fig 1D leaves no doubts that SPIO microspheres concentrate within the cytoplasm. The efficacy of labeling accurately assessed with flow cytometry is shown on Fig 1E. As follows from flow cytometry data, no less than 96% of the cells emitted green fluorescence indicating the presence of MC03F microparticles in the cytoplasm.

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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.

https://doi.org/10.1371/journal.pone.0186717.g001

To quantitatively evaluate the influence of labeling on cell viability and proliferation, we compared the dynamics of growth of intact and labeled hMSCs. The number of live cells on different days (1, 3, 5, 7) after labeling was evaluated using the CytoTox 96 LDH (lactate dehydrogenase) assay, in which the absorbance at 490 nm is proportional to total LDH activity and, hence, to the number of living cells. The results are presented in Fig 1F. Obviously, the proliferation rates of hMSCs containing microspheres and control cells are not significantly different suggesting the absence of negative effects associated with labeling on cell viability and proliferation. Earlier we have shown that labeled hMSCs retained the SPIO microparticles for at least four weeks [33]. Moreover, labeling did not noticeably affect the cells’ ability to undergo osteogenic, chondrogenic, adipogenic, and neurogenic differentiation in vitro [33,34].

Importantly, optimum incubation times and microparticles concentrations for labeling of MSCs with SPIO microspheres from Bangs Laboratories reported by other research groups are similar to ours [14,35].

Iron content per cell and per injection

Average iron content per cell detected spectrophotometrically as described in the Materials and Methods section was 3.8±0.3 pg/cell. Therefore, the total amount of iron injected with 10¹, 10², 10³, 10⁴, and 10⁵ SPIO-labeled cells was 0.038 ng, 0.38 ng, 3.8 ng, 38 ng, and 380 ng, respectively.

In vitro MRI of the phantoms

Representative MR images of a phantom after injection of 10¹ SPIO-labeled hMSCs obtained using T2WI, T2*WI based on FLASH 3D, and SWI are shown in Fig 2. Due to the ability of SPIO microspheres to create local disturbance of the magnetic field and to reduce T2* relaxation time, injected SPIO-labeled cells cause MR signal reduction and can be visualized at MR images as dark areas of hypointensity. Images obtained using all three pulse sequences are presented in axial projection. In addition, susceptibility weighted image is given in coronal projection. Obviously, SWI allowing easy detection of just several cells injected into a phantom, is the most sensitive of the tested pulse sequences.

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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.

https://doi.org/10.1371/journal.pone.0186717.g002

MRI of SPIO-labeled cells transplanted into the rat striatum

Fig 3 demonstrates characteristic MR images of normal live rat brain obtained immediately after stereotaxic injection of 20 μl saline or varying quantities of SPIO-labeled hMSCs (from 10¹ to 10⁵ cells) in 20 μl saline into the right striatum. Images were taken using four different pulse sequences—T2WI, T2*WI based on MEDIC, T2*WI based on FLASH 3D and SWI. Similar to phantoms, the hypointensity areas at MR images of brains injected with labeled cells correspond to hMSCs transplantation sites. The size of the hypointensity regions correlates with the number of transplanted hMSCs and depends on the pulse sequence used. Large enough quantities of labeled hMSCs can create disturbance of the local magnetic field within a volume by far surpassing the volume occupied by the cells—a feature called «blooming effect». Small hypointensity areas on the SWI and T2*WI based on FLASH 3D images of the right striatum of saline-injected rat were probably produced by traumatic hemorrhages caused by the needle.

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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.

https://doi.org/10.1371/journal.pone.0186717.g003

The sensitivities obtained using different pulse sequences were quantitatively estimated by measuring the ratio of the minimum signal intensity at the site of transplantation to mean signal intensity in the adjacent brain tissue (Fig 4A). Ratios of minimum to mean signal calculated from SWI data were significantly lower than those assessed using alternative pulse sequences (Fig 4A), demonstrating higher sensitivity of SWI in SPIO-labeled cells detection.

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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.

https://doi.org/10.1371/journal.pone.0186717.g004

We also studied the ability of SWI to distinguish between signals produced by traumatic microhemorrhages inflicted by the syringe needle and different numbers of transplanted hMSCs. Comparison of the volumes of the hypointense zones and the values of the ratio of minimum to mean signal are shown in Fig 4B and Fig 4C. Data presented in Fig 4B and 4C indicate that after transplantation of 10² or higher numbers of hMSCs hypointense areas captured using SWI were significantly different from those obtained from saline-injected rats. However, the signal from 10¹ hMSCs could not be statistically distinguished from that from traumatic brain microhemorrhage inflicted by the syringe needle.

Correlation between MRI and histology data in SPIO-labeled hMSCs brain injection experiments

Cells used in this study were labeled with microspheres consisting of a SPIO core covered with a shell containing the Dragon Green fluorescent dye. They can be visualized in different ways on histological sections. Three microscopic techniques, namely, conventional fluorescent microscopy, confocal fluorescent microscopy, and transmitted light microscopy of Perls’ Prussian blue stained sections were applied to expose transplanted hMSCs in rat brain tissue in order to verify MRI results. Fig 5 shows histological images of the sections across the sites of injection of varying numbers of labeled hMSCs (three lower panel rows) in comparison with corresponding MR images taken utilizing T2WI (upper panel row) and SWI (second from top panel row) pulse sequences. At the lower left panel iron is visible due to hemoglobin biodegradation inside a microhemorrhage. Injections made directly into the live brain tissue cause microhemorrhages of varying severity at the injection site. Perls’ Prussian blue stain is unable to distinguish between iron in hemorrhages and SPIO label. And because hemorrhages may vary in size, the site of saline injection sometimes looks even darker than the site of injection of a small number of SPIO labeled cells. Bigger cell numbers (10³−10⁴ on Fig 5) mask magnetic field disturbances caused by microhemorrhages. Accordingly, staining for iron content with Perls’ Prussian blue could reveal no less than 10³ labeled cells or about 3.8 ng of iron (lower panel row), and so did conventional fluorescent microscopy (middle panel row). If the number of cells was under 10³, microhemorrhages at the injection site could be mistaken for labeled cells, because both produced magnetic field disturbances of similar intensity. Confocal fluorescent microscopy allowed detection of 10¹−10² cells (second from bottom panel row). However, in some cases detection of 10¹ cells by confocal fluorescent microscopy was somewhat obstructed by the interfering low intensity autofluorescence present after saline injection.

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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.

https://doi.org/10.1371/journal.pone.0186717.g005

All three histological methods used showed the presence of labeled cells at the sites of hMSCs transplantation in the same locations as revealed by MRI. Taken together, histological data provided complete verification of MRI results. The sensitivity of the confocal fluorescent microscopy by far surpassed the detection limits of other histological techniques and was similar to that of SWI.

MRI of SPIO-labeled cells after intra-arterial transplantation

Immediately after intra-arterial injection of 5x10⁵ hMSCs via the right internal carotid artery labeled cells could be visualized with applicable MRI techniques and were distributed mainly in the basal ganglia area and in the cerebral cortex of the right hemisphere (Fig 6, top row). SPIO-labeled hMSCs could be visualized as hypointense spots on images taken using T2*WI based on MEDIC, T2*WI based on FLASH 3D, and SWI. As evident from Fig 6, SWI and T2*WI based on FLASH 3D were most sensitive permitting detection of small groups of labeled cells, probably even single cells. T2WI, because of its low sensitivity, revealed no SPIO-labeled cells at all. MRI data were confirmed by the results of histological examination (Fig 6, bottom row). All three histological techniques used (conventional fluorescence microscopy, bright-field microscopy of Prussian Blue stained tissue sections, and confocal fluorescence microscopy) showed the presence of the SPIO label exactly at the same sites as found by MRI.

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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.

https://doi.org/10.1371/journal.pone.0186717.g006

As seen at Fig 7, some labeled hMSCs were localized around or close to blood vessels, suggesting rapid migration of transplanted cells from circulation into the brain parenchyma through the intact blood-brain barrier. Fig 7 clearly demonstrates the possibility of MRI visualization of single SPIO-labeled transplanted cells in the recipient’s brain.

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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.

https://doi.org/10.1371/journal.pone.0186717.g007

MRI and histological visualization of hMSCs double-labeled with MC03F microparticles and PKH26

To confirm that SPIO microparticles remain localized inside hMSCs after transplantation, we performed additional labeling of cells with lipophilic membrane cell tracking dye PKH26. MR images of the injection site taken after intracerebral transplantation of 3x10⁵ double-labeled cells transplantation show the “blooming effect” (Fig 8A). As seen at the high resolution confocal fluorescent microscopy images (Fig 8B), SPIO-labeled cells (white arrowhead) carry two fluorescent labels: the Dragon Green label associated with SPIO particles inside the cytoplasm and the red membrane tracker PKH26. Co-localization of the two labels demonstrates that after intracerebral transplantation at least part of the SPIO label remains associated with hMSCs. However, small clusters of extracellular green fluorescent SPIO microspheres are also found (white arrows at Fig 8B). Extracellular SPIO can be mistaken for SPIO-labeled hMSCs on MR images. Panoramic confocal fluorescent microscopy images of the injection site (Fig 8C) demonstrate close co-localization of the two fluorescent labels. After intra-arterial transplantation double-labeled MSCs can be observed in vivo with SWI, while T2WI is not sensitive enough to visualize single hMSCs or small groups of transplanted cells (Fig 8D). It should be noted, that T2*WI based on FLASH 3D can also provide single cells detection, but the quality of images is poorer compared to SWI. The comparison of T2*WI based on FLASH 3D and SWI images is presented at S2 Fig in Supporting information). High resolution confocal microscopy images (Fig 8E) show double-labeled hMSCs (white arrowhead), but no extra-cellular SPIO microparticles.

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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.

https://doi.org/10.1371/journal.pone.0186717.g008