Efficient Tracking of Non-Iron-Labeled Mesenchymal Stem Cells With Serial MRI in Chronic Stroke Rats
Background and Purpose— Although stem cell-based treatments for neurodegenerative diseases have advanced rapidly, there is currently no clinically available method to monitor the fate of transplanted cells in the brain.
Methods— To use magnetic resonance imaging for tracking transplanted stem cells in the ischemic rat brain, we used the cellular labeling substance Effectene to transfect a standard contrast agent (Gd-DTPA) into immortalized human bone marrow stromal cells.
Results— The transfection efficiency of this method was up to 90%, which is substantially better than pure spontaneous endocytosis or other transfection agents. In addition, cellular uptake of Gd-DTPA in vitro was maintained for >28 days. Therefore, we could follow transplanted stem cell migration and homing into the penumbric area. Using double immunofluorescence, the transplanted cells were seen to differentiate into glial cells, neurons and vascular endothelial cells. Cortical neurochemical activity as evaluated by proton magnetic resonance spectroscopy (1H-MRS) also increased considerably after immortalized human bone marrow stromal cell transplantation.
Conclusion— This method of tracking immortalized human bone marrow stromal cells is highly efficient and allows for nontoxic labeling of cells.
- immortalized human bone marrow stromal cells
- in vivo monitoring
- magnetic resonance imaging
Stem cells have been used to treat neurological diseases in which neuronal death is the major pathogenetic mechanism, including cerebrovascular and other neurodegenerative diseases.1 Because the application of stem cell-based therapies is potentially wide-ranging, specific methods are needed to continuously and noninvasively monitor stem cell survival. Three-dimensional imaging and in vivo cell tracking capabilities allow magnetic resonance imaging (MRI) to provide high-resolution visualization of the fate of cells after transplantation, and the migration of cells after injection.2 To visualize stem cells in the brain using MRI, recent advances in cell-labeling techniques with iron oxide and paramagnetic particles (gadolinium-diethylene triamine penta-acetic acid [Gd-DTPA]) have also been developed.3 However, pure cell endocytosis and lipofectamine-mediated methods of transfection have low labeling efficiencies,4 and supraparamagnetic substances, such as iron oxide, have been demonstrated to have harmful effects on cell signaling and function.5
Here, we present a method whereby immortalized human bone marrow stromal cells (IhMSCs), labeled with Gd-DTPA using Effectene and transplanted into the rat ischemic brain, are tracked with 3.0-Tesla MRI. We also demonstrated that transplanted IhMSCs preserved their differentiation potential in the ischemic brain after being labeled with the MRI contrast agent Gd-DTPA.
Materials and Methods
IhMSC Preparation and Labeling
IhMSCs, donated by Dr Toguchida,6 were cultured and expanded as described.6 Gd-DTPA (Megnevist, Germany), which has a molecular weight of 547 Da, is the standard MR contrast medium for clinical use. Effectene (Qiagen) was used to transfect Gd-DTPA into IhMSCs (Gd-hMSCs) as previously described.7 Immortalized hMSCs (6×105 cells) were labeled with 50 μL 0.5 mol/L Gd-DTPA using 10 μL Effectene in 6-well plates with serum-containing DMEM medium following the manufacturers’ instructions. After discarding the supernatant, the labeled cells were recovered in original medium and incubated with 1 μg/mL bis-benzimide (Hoechst 33342; Sigma) for 24 hours at 37°C.
Cell Viability and Longevity of Contrast Medium Maintenance
After Gd-DTPA labeling, the viability of cells (1×105) was determined by trypan blue exclusion assays. To evaluate the longevity of Gd-DTPA maintenance in stem cells, labeled cells were cultured and propagated under standard conditions, and MRI was performed 3, 7, 14, and 28 days after the initial labeling procedure. Before MRI, cells were washed 3 times with phosphate-buffered saline to eliminate residual contrast agent particles in the supernatant.
Spectrophotometric Analysis of Cell Labeling Efficacy
The Gd-DTPA concentration within labeled cells was investigated by spectrophotometric measurement of the cellular uptake of Gd-DTPA particles using an atomic absorption spectrometer (Zeeman spectrometer model Z-8200; Japan) as previously described.4,5
Animal Brain Ischemia/Reperfusion Model
Adult male Sprague-Dawley rats (weighing 250 to 300 grams; Experimental Animal Center, Tzu-Chi University, Hualien, Taiwan) were subjected to 3-vessel ligation. Ligation of the right middle cerebral artery and bilateral common carotids were performed by modified methods as described previously.8
Before transplantation, Gd-hMSCs with bis-benzimide were trypsinized and resuspended in phosphate-buffered saline. One week after ischemia, adult male Sprague-Dawley rats were anesthetized with chloral hydrate (0.4 g/kg, intraperitoneally). They were then injected stereotactically with ≈1×106 Gd-hMSC cells into 3 cortical areas adjacent to the right middle cerebral artery,9 3.5 to 5.0 mm below the dura, as described previously.10 The approximate coordinates were l.0 to 2.0 mm anterior to the bregma and 3.5 to 4.0 mm lateral to the midline, 0.5 to l.5 mm posterior to the bregma and 4.0 to 4.5 mm lateral to the midline, and 3.0 to 4.0 mm posterior to the bregma and 4.5 to 5.0 mm lateral to the midline. Rat hosts did not receive any immunosuppressive medication.11
Neurological Behavioral Measurement
Behavioral assessments were performed as described previously.12
Gd-hMSCs Tracing With MRI
To assess Gd-hMSC migration, animals were imaged 3, 7, 14, and 28 days after intracerebral Gd-hMSC injection using high resolution 3-Tesla MRI (whole-body Sigma EchoSpeed MR scanner, General Electric, Milwaukee, Wis) as described previously.13 T1-weighted fast spin echo sequences were optimized to detect Gd-hMSCs. Acquisition parameters were TE/TR 59.4/600 ms, echo train length 53, and NEX 8. Each image was determined by a consensus of 2 observers blinded to the Gd-hMSC injection.
Proton MR Spectroscopy Assessment
Proton MR spectroscopy (1H-MRS) was performed using the same MRI scanner with a single-voxel technique, and then T2-weighted transverse, coronal, or sagittal images were used to localize the volume of interest as previously described with modification.13 Volume of interest indicates the region measured by the three parameters (N-acetylaspartate [NAA], creatine [Cr], and choline and choline-containing compounds [Cho]) in the MRI computer software, which includes both the core and the penumbra regions of the infarcted brain. The volume of interest (3×3×3 mm3) was localized centrally to the infarcted region using 2 or 3 images (transverse and sagittal/coronal). Spectroscopic acquisition parameters were as follows: water suppression was provided for by CHESS pulses and localization by a standard PRESS-type sequence (TR=2000 ms; TE=68, 136, and 272 ms). Spectra were processed using the NMR1 program (NMR1, Syracuse, NY). Metabolic peaks were fitted by the Lorentzian line shape at the known frequencies of NAA at 2.02 ppm, Cr at 3.03 ppm, and Cho at 3.22 ppm. From this, NAA/Cr and NAA/Cho ratios were calculated. Metabolic ratios are presented as mean±SE.
Histological Evaluation of Brain Tissue
Animals were anesthetized with chloral hydrate (0.4 g/kg, intraperitoneally) and their brains fixed by transcardial perfusion with saline, followed by perfusion and immersion with 4% paraformaldehyde as previously described.13
Laser-Scanning Confocal Microscopy for Immunofluorescence Colocalization Analysis
To demonstrate the differentiation potential of transplanted cells, the expression of cell type-specific markers in bis-benzimide-labeled IhMSCs were identified by immunofluorescence analysis for each brain section as previously described.13 Because bis-benzimide-labeled cells showed spontaneous blue fluorescence in their nuclei, cell-type specific antibodies, such as GFAP (1:400; Sigma), MAP-2 (1:200; BM), von Willebrand factor (1:20; Sigma), Nestin (1:400; Sigma), and Neu-N (1:200; Chemicon) conjugated with Cy-3 (Jackson Immunoresearch), were stained to determine whether they colocalized with bis-benzimide in the same cell. The total number of differentiated cells that colocalized with bis-benzimide-labeled cells was measured as previously described.13
Quantitative Reverse-Transcription Polymerase Chain Reaction of Growth Factors Synthesis
Experimental rats were anesthetized with chloral hydrate (0.4 g/kg, intraperitoneally) at 3, 7, 14, and 28 days after cell or vehicle transplantation. Ischemic cortical and striatal areas were immediately removed on ice. Subsequently, brain tissue samples were homogenized by a plastic homogenizer in 1 g/mL lysis buffer (Promega), and total RNA and cDNA synthesis were performed as previously described.13
Unaltered Cell Viability after Gd-DTPA Labeling
To determine whether there was any detrimental effect on cells labeled with contrast agent, cell viability tests were performed after each labeling procedure. Evaluation of cell viability by trypan blue exclusion tests showed an initial transient reduction in cell numbers. After 24-hour incubation under the same culture conditions, cellular viability was 93±5% for nonlabeled cells and 91±4% for Gd-hMSCs. No signs of apoptosis were detected by DNA fragmentation assays (data not shown).
Improved Labeling Efficiency and Increased Longevity of Gd-DTPA Cell Maintenance
To determine whether Effectene affected the transfection of Gd-DTPA into cells, the level of gadolinium in cells was measured by spectrophotometry. Effectene-mediated labeling efficiency was determined to be 90±3%, which was higher than that obtained (52±4%) by pure endocytosis. Saturation of the system was defined by adding 50 μL 0.5 mol/L Gd-DTPA. Results of the longevity of Gd-DTPA in cells showed that passaged cells remained healthy and retained Gd-DTPA intracellularly for a period of 28 days (data not shown).
Gd-hMSC Migration: In Vivo Tracing With MRI
To examine whether Gd-hMSCs could migrate throughout the ischemic brain, Gd-hMSCs were injected intracerebrally into 3 holes and tracked with MRI at 3, 7, 14, and 28 days without the use of immunosuppressive agents (n=6 at each time point).9 Within 2 to 3 days, 3 strong spots of increased signal intensity became visible, showing a white tract through the cerebral cortex from the anterior to posterior portion of the rat brain under MRI (Figure 1B through 1E). Controls (IhMSCs without labeling, n=6) lacked spots of increased signal intensity (Figure 1A). The area of one spot of increased signal intensity over each plane was measured at ≈0.5×0.5 mm2 from an MRI coronal view (Figure 1F through 1I). Sequentially from days 3 to 28 after immortalized hMSC transplantation, the confluent (versus dispersed) area of increased signal intensity (Figure 2A) rapidly spread from the striatum to the ipsilateral corpus callosum and hippocampus toward the peri-infarcted cortical area (Figure 2B through 2C), and even migrated across the midline to the contralateral corpus callosum and hippocampus (Figure 2D). Fluorescent histological examination confirmed that this developing MRI signal was caused by transplanted Gd-hMSCs (Figure 2A through 2J). MRI showed that many Gd-hMSCs gathered near the subventricular zone, giving an area of increased signal intensity (Figure 2G). Generally, in animals treated with Gd-hMSCs, this cell accumulation extended over time to line the lateral ventricular wall and the peri-infarcted area (Figure 2H through 2J). Furthermore, Gd-hMSCs were observed on the choroid plexus of the lateral ventricle in the ischemic hemisphere (Figure 2G).
IhMSC Treatment Increases Neurochemical Activity
To evaluate any improvement in neuronal metabolism after transplantation, experimental rats were studied using 1H-MRS to assess the neurochemical activity of ischemic rats. Seven days after cell transplantation, 1H-MRS showed a significant decrease in the metabolic ratio of NAA/Cho and NAA/Cr (1.61±0.03 and 1.79±0.04, respectively) (n=6) in IhMSC-treated rats (Figure 3C) and 1.52±0.03 and 1.63±0.03, respectively, in untreated rats (n=5) compared with the prestroke stage (2.6±0.11 and 2.1±0.08, respectively) (Figure 3D). Consistent with the recovery of neurological behavior test scores (data not shown), significant improvement in neurochemical activity under 1H-MRS was observed specifically with regard to NAA/Cho and NAA/Cr (1.72±0.04 and 1.90±0.06, respectively) at 14 days (n=6) and NAA/Cho and NAA/Cr (1.84±0.08 and 2.08±0.13, respectively) at 28 days (n=6) in the treated group (Figure 3F and 3H) in comparison to NAA/Cho and NAA/Cr (1.59±0.06 and 1.71±0.14, respectively) at 14 days (n=6) and NAA/Cho and NAA/Cr (1.66±0.07 and 1.80±0.13, respectively) at 28 days (n=6) in the control group (Figure 3E and 3G). Measurement data for NAA/Cho and NAA/Cr are displayed graphically for the IhMSC-treated and control groups (Figure 3I).
Neuroplasticity After Intracerebral Transplantation of Gd-hMSCs After Cerebral Ischemia
To study whether the Effectene-labeling procedure impeded the potential of stem cells to differentiate, fluorescent immunohistochemistry was used to analyze the colocalization between cell-type specific markers and bis-benzimide-labeled cell nuclei. The results showed that some bis-benzimide-labeled cells colocalized with antibodies for GFAP and MAP-2 (Figure 4A and 4B), and Neu-N and Nestin (Figure 4C and 4D). Some bis-benzimide-labeled cells showing vascular phenotypes of von Willebrand factor (Figure 4E) were also found around the perivascular and endothelial regions in the ischemic hemispheres of Gd-hMSCs-treated rats. The transplanted cells did not induce any inflammatory cell infiltration (immune reaction) or tumor formation in the host brain.
Intracerebral Transplantation of IhMSCs Modulates Neurotrophic Factor Expression in the Ischemic Hemisphere
To identify molecular mechanisms for improvement of neurological dysfunction after cerebral ischemia in IhMSC-treated animals, we examined the expression of neurotrophic factors known to neuroprotect the ischemic cortical area (n=4). The results revealed significantly increased expression levels of SDF-1 and BDNF in the ischemic rats treated with stem cells in comparison to vehicle controls (Figure 5A and 5B). The ratio of SDF-1 and BDNF to GAPDH peaked at ≈2-fold increase in comparison to the control 14 days after transplantation of stem cells (Figure 5C and 5D).
In molecular biological experiments, standardized transfection protocols with either liposomes or viral vectors are widely used to transport extracellular DNA or other substances into targeted cells.14 Here we used Effectene to transfect the MRI contrast agent Gd-DTPA into stem cells, because it is relatively more efficient and less toxic than liposome, calcium phosphate, or viral vectors for transfection into primary cells.15–17 There was a possibility that phagocytosed or dead transplanted cells with Gd-DTPA were detected by the MRI. However, a previous pharmacokinetic study reported that Gd-DTPA in dead cells or interstitial spaces was washed out within 24 hours.18 Furthermore, we did not find any phagocytosed implanted cells or fusion cells19 in our Gd-hMSC-treated rat brain. In addition, our experimental results showed improvement in neurological dysfunction, increased neurotrophic factor synthesis, and enhanced neuronal activity after stem cell transplantation. On the basis of these pieces of evidence, we conclude that the possibility of having detected dead or phagocytosed transplanted cells in the ischemic brain was very low.
Many experiments have used other paramagnetic substances to track cell movement under MRI, including iron-containing agents (eg, Feridex) and a new generation of contrast agents magnetodendrimers and particles conjugated with Tat peptides.20 However, magneto dendrimers and particles conjugated with Tat peptides require complicated methods of manufacture. Furthermore, iron-containing agent-labeled stem cells transplanted into brain are visualized as signal void images (black signal) using these paramagnetic substances. This can create problems because enlarged “false” black signaling effects of iron-labeled cells can exceed true stem cell mass.20 In addition, iron can be toxic at high concentrations,21 with its accumulation in tissue also catalyzing the Fenton reaction and potentiating oxygen toxicity through the generation of a wide range of free radical species. Furthermore, leakage of iron or the death of labeled cells may cause the release of iron oxide crystals into tissue, which can result in a potentially toxic uptake in surrounding healthy cells.22 In contrast, the pharmacological properties of Gd-DTPA have been extensively investigated17 and clinically applied. Under MRI, Gd-DTPA-labeled cells in the brain show increased signal intensity (white signal) rather than void signals. Although the MR detection thresholds in stem cell labeling were lower in the iron-containing particle (2.5×105 cells) than that of Gd-DTPA (5×105 cells), it is highly desirable for clinical applications to have the choice of identifying labeled stem cells with high signal intensity on MRI such as Gd-DTPA.17 In this study, the increased signal intensity seen in the MRI of ischemic rat brains correlated well with the true size of engrafted stem cells and corresponding anatomical brain structures. However, one major advantage of using iron-based contrast agents rather than Gd-DTPA would be their much lower detection threshold.
In summary, the present study demonstrates a strategy to detect implanted stem cells using an imaging system in an intact animal. We speculate that MRI tracking of grafted cells might become a powerful tool for understanding the molecular mechanisms that are ultimately responsible for the successful migration and expansion of neurotransplanted cells.
We thank Dr Junya Toguchida for providing the human immortalized MSC cell line.
Sources of Funding
This work was supported in part by research grants from Chen-Han Foundation for Education, Mackay Memorial Hospital (MMH-E-94001), Academia Sinica (94M003), and National Science Council (NSC94-2314-B-303-009).
W.-C.S. and C.-P.C. contributed equally to this article.
- Received April 7, 2006.
- Revision received August 16, 2006.
- Accepted September 10, 2006.
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