Abstract
This study was designed to investigate functional recovery after the transplantation of mesenchymal stem cells (MSCs) or neurally differentiated MSCs (NMSCs) derived from bone marrow in a rat model of spinal cord injury (SCI). Sprague-Dawley rats were subjected to incomplete SCI using an NYU impactor to create a free drop contusion at the T9 level. The SCI rats were then classified into three groups; MSCs, NMSCs, and phosphate-buffered saline (PBS)-treated groups. The cells or PBS were administrated 1 week after SCI. Basso-Beattie-Bresnahan (BBB) locomotor rating scores were measured at 1-week intervals for 9 weeks. Somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) were also recorded 8 weeks after transplantation. While transplantation of MSCs led to a clear tendency of motor recovery, NMSC-treated rats had significantly improved BBB scores and showed significantly shortened initial latency, N1 latency, and P1 latency of the SSEPs compared to PBS controls. In addition, 5-bromo-2-deoxyuridine (BrdU)-prelabeled MSCs costained for BrdU and glial fibrillary acidic protein (GFAP) or myelin basic protein (MBP) were found rostrally and caudally 5 mm each from the epicenter of the necrotic cavity 4 weeks after transplantation. These results suggest that neurally differentiated cells might be an effective therapeutic source for functional recovery after SCI.
Keywords
Introduction
Recovery from spinal cord injury (SCI) is quite difficult because the central nervous system has a limited ability to regenerate injured cells, replace damaged myelin sheath, and reestablish functional connections. Many patients with SCI have difficulty in walking and accomplishing daily activities during their remaining years. As a therapeutic approach in SCI, stem/progenitor cells or predifferentiated cells may provide a partial solution, although few successful clinical trials have been reported. For example, stem cells or progenitors from adult tissues such as bone marrow (BM) (1,3,12,15,24), umbilical cord blood (UCB) (5,13,20,23,29), and adipose tissue (9,10) can be propagated in large quantities while retaining their multipotent ability to differentiate into different cell types.
Recently, BM-derived mesenchymal stem cells (MSCs) have been shown to be able to differentiate into neural lineage cells under specific in vitro conditions including induction with β-mercaptoethanol, forskolin, or basic fibroblast growth factor (bFGF) (6,17,27). Likewise, adult stem cells from BM are potentially suitable sources for cell-based therapy in SCI. In addition, BM-derived cells can overcome critical ethical issues of the embryonic stem cells and neural stem cells from human fetal tissues, which were well known to be able to differentiate into oligodendrocytes or astrocytes, and regenerate the injured spinal cord (7,18,19).
This study was carried out to investigate whether neurally differentiated MSCs (NMSCs) derived from allogeneic BM after exposure to appropriate environments can serve as an alternative cell source that maintains the capacity with possible potential to develop into neurons, astrocytes, and oligodendrocytes and whether NMSCs may also be able to replace lost neural cells, remyelinate injured axons, and improve functional recovery in a rat model of SCI.
Materials and Methods
Cell Culture and Maintenance
After obtaining BM aspirates of the femurs of adult Sprague-Dawley rats (Orient, Sungnam, Korea) weighing 300–350 g, mononuclear cells (MNCs) (4–6 × 107 cells/ml) were separated using the Ficoll-Hypaque (Histopaque-1077; Sigma-Aldrich, St. Louis, MO, USA) density-gradient method. The BM-derived MNCs were then seeded on T25 or T75 culture flasks in mesenchymal stem cell growth media (MSCGM, Cambrex, Charles City, IA, USA). Cells were incubated in a humidified atmosphere at 37°C with 5% CO2. They were subcultured when they reached 80–90% confluence, and used for transplantation after 4–6 passages. For immunohistological usage, a portion of the cells was incubated with 30 μM 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich) for 3 days to label dividing cells before transplantation.
Neural Induction
To induce neural differentiation, rat MSCs were cultured according to a modified protocol from Woodbury et al. (27). Briefly, MSCs after four passages were preinduced with DMEM (Gibco, Grand Island, NY, USA), 20% FBS (Gibco), and 5 ng/ml bFGF (Sigma-Aldrich) for 24 h. Thereafter, the medium was changed to specific medium containing DMEM with N2 supplement, 200 μM butylated hydroxyanisole, 25 mM KCl, 2 mM valproic acid, and 10 μM forskolin (all from Sigma-Aldrich) to promote neurogenic differentiation. Cells were then incubated in the neural induction medium for 2–16 h at 37°C.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 30 min, and blocked with 1% bovine serum albumin for 1 h at 37°C. This procedure was followed by incubation with a FITC-conjugated hamster anti-rat CD29 mAb (BD Bioscience, San Jose, CA, USA) or RPE-conjugated mouse anti-rat CD90 mAb (BD Bioscience) to label MSCs and PE-CD31 (BD Bioscience), FITC-CD34 (Santa Cruz, Santa Cruz, CA, USA), PE-CD45 (BD Bioscience), FITC-CD54 (BD Bioscience) to label lympho-hematopoietic cells. NMSCs were incubated for one of the following markers: 1) βIII-tubulin using mAb TuJ1 (Covance, NJ, USA), 2) NeuN (Chemicon, Pemecula, CA, USA), 3) glial fibrillary acidic protein (GFAP; Chemicon), 4) S100β (Chemicon), 5) CNPase (DAKO, Hamburg, Germany). In addition, cells were stained with 4′ 6-diamino-2-phenylindole (DAPI; 1 μg/ml, Sigma-Aldrich) to visualize nuclei.
Spinal Cord Injury
Animals were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The Institution Review Board approved the experimental procedures. After all rats were anesthetized with sodium pentobarbital (50 mg/kg, IP), the thoracic back area was shaved and disinfected with povidone iodine, and a laminectomy was then performed at the T9 spinal cord level with the duramater intact. Contused incomplete SCI was induced by dropping a 10-g weight rod from a 25-mm height onto the exposed dorsal surface of the spinal cord using an NYU impactor. During the recovery period, body temperature was maintained at 37°C in a heating chamber. The postoperative care included bladder expression 1–2 times per day until bladder function had recovered. Prophylactic kanamycin (1 mg/kg) was regularly administered for 1 week after surgery. Among a total of 50 animals, nine rats were excluded from this experiment due to asymmetrical spinal cord damage.
Cell Transplantation
One week after SCI, the injured rats were randomly assigned to three groups without bias: MSC-treated (n = 16), NMSC-treated (n = 12), and PBS-treated (n = 13) groups. Rats were fixed in a stereotaxic frame (Stoelting Co., Wood Dale, IL, USA). Using a 25-gauge cannula connecting to 10-μl Hamilton syringe, a total of 10 μl of the cultured cells (2.5 × 105 cells) was transplanted into the epicenter of the injury over a 10-min period (infusion rate, 60 μl/h) after reopening the wound. All rats received cyclosporine A (10 mg/kg, IP) daily from 2 days before the transplantation until the completion of this experiment to suppress immune rejection.
Behavioral Assessment
The Basso, Beattie, and Bresnahan (BBB) locomotor rating scale was used to measure functional recovery of the rats. This scale measures hindlimb movements starting with a score of 0, indicating no observable movement. Increasing scores are given for movement of individual joints, limb coordination, weight-supported plantar stepping, etc., up to a maximum score of 21, which indicates normal movement. Two investigators observed the hindlimb movements in an open field for 5 min after rats were gently adapted to the field. Locomotor functions were scored weekly from day 1 to 9 weeks after SCI. The score was obtained by averaging the value of both limbs. A total of 23 rats (9 MSC-, 7 NMSC-, 7 PBS-treated rats) survived to the end of the experiment.
Electrophysiological Study
Somatosensory evoked potentials (SSEPs) (n = 6 each) and motor evoked potentials (MEPs) (n = 5 each) were measured 8 weeks after transplantation. The rats were randomly assigned to the recording of SSEPs or MEPs without any indication of the extent of the lesion. The animals were anesthetized with urethane (1.25 g/kg, IP). They were also treated with atropine sulfate (0.8 mg/kg, IP) to reduce tracheal secretions, and pancuronium bromide (1.0 mg/kg, IP) to induce muscle relaxation. The rats were then intubated with a tracheostomy and artificially respired using an animal respirator (Harvard Apparatus, South Natik, MA, USA). The left sciatic nerve was exposed and a pair of electrodes was hooked around the nerve. A single square pulse of electrical stimulation was delivered with a stimulus isolator (A365, World Precision Instruments, New Haven, CT, USA), which was driven by a pulse generator (Pulse-master A300, World Precision Instruments, New Haven, CT, USA) with a 0.1-ms pulse duration of stimuli and a 6-mA intensity at 1–4 Hz. For the SSEP recording, a 4 × 4-mm-sized craniectomy was performed in the contralateral frontoparietal area. A recording electrode (NE-120, Rhodes Medical Instruments, Tujunga, CA, USA) was fixed on the sensorimotor cortex at 2 mm posterior to bregma and 2 mm lateral to the sagittal suture after craniectomy. For the MEP recording, a laminectomy was performed at the L1 spinal cord level. Then, an electrode was inserted into the contralateral gray matter of the spinal cord near the motor conduction tracts. The pointed tip of the electrode was used as an active electrode, and the rounded part of the electrode was used as a reference electrode. Each evoked potential consisted of an average of 100 single sweep epochs.
Immunohistochemistry
Among the subjects, nine rats (n = 3 each) were randomly assigned to be sacrificed 4 weeks after transplantation. Nine rats (n = 3 each) were also sacrificed 8 weeks after transplantation. Animals were perfusion fixed transcardially with ice-cold PBS and 4% paraformaldehyde, pH 7.4, to evaluate the characteristic phenotypes of the transplanted cells in vivo. Spinal cords were immediately removed, 20-mm-sized transverse segments of the injured region of the thoraco-lumbar spinal cord were dissected and stored in the same fixative overnight, and tissues were serially immersed in 6%, 15%, and 30% sucrose until the tissues sank down. The tissues were frozen and cryosectioned longitudinally at 12 μm thick using a cryomicrotome (Microm/HM500V, Walldorf, Germany). Sections were then immunostained for BrdU and various markers of neural cells. Individual sections were incubated for one of the following markers overnight: 1) βIII-tubulin (Covance) at 1:400, 2) NeuN (Chemicon), 3) GFAP (Chemicon), 4) myelin basic protein (MBP) (Chemicon). The sections were incubated with Alexa 488 or 563 secondary antibodies at 1: 400 for 1 h, then washed, mounted on glass slides with fluorescent mounting medium (Vectorshield, Vector), and examined under a fluorescence microscope (BX51, Olympus, Tokyo, Japan) or an argon and krypton laser scanning confocal imaging system (LSM 510, Zeiss, Gottingen, Germany) to confirm double-labeled cells by visualizing the colocalization for BrdU and the cell type-specific markers at a magnification of 600x.
Statistical Analysis
Statistical comparison of the BBB scores were performed using a repeated ANOVA followed by post hoc Bonferroni comparison through SPSS version 13.0. The latencies and amplitudes of SSEPs or MEPs among groups were also analyzed using a one-way ANOVA with post hoc comparison. Values of p < 0.05 were accepted statistically significant.
Results
Characterization of MSCs Derived From Allogeneic BM
Rat MSCs from allogeneic BM grew in a monolayer of large, flat cells at low plating densities. As the cells approached confluency, they assumed a more spindle-shaped, fibroblastic morphology. Using flow cytometry analysis, undifferentiated rat MSCs were demonstrated as negative for CD31, CD34, CD45, and CD54, which surface markers associated with lympho-hematopoietic cells (Fig. 1a–d). Using both flow cytometry analysis and immunocytochemistry, cells were positive for CD29 and CD90, which are reported to be surface markers of MSCs (Fig. 1e–i).
Characterization of rat MSCs by flow cytometry analysis and immunocytochemistry. Fluorescent intensity of each cell surface marker of undifferentiated MSCs (open peak) was compared to that of isotype control (shaded peak). Numbers of cells analyzed (event) was plotted on the y-axis and intensity of staining was plotted on the x-axis. Distribution of cells stained with antibodies to CD 31 (a), CD34 (b), CD 45 (c), and CD 54 (d) (opened), cell surface markers associated with lympho-hematopoietic cells, did not differ from those of isotype controls (shaded). However, fluorescence intensity of CD90 (e) and CD29 (f) (opened) as surface markers of the rat MSCs was greater (shifted to the right) than those of isotype (shaded). Immunocytochemistry showed the cells expressed CD 29 (g), CD 90 (h), and both of them (i) simultaneously, which could confirm the results of flow cytometry analysis. Scale bars: 10 μm. MSCs, mesenchymal stem cells.
In Vitro Differentiation of MSCs Into Neural Lineage Cells
NMSC was defined as MSC predifferentiated into neural lineage cells after neural induction medium. After exposure to the specific medium, undifferentiated rat MSCs exhibited very rapid morphological changes. Most cells retracted their cytoplasm and formed a spherical cell body, extended cellular processes, and completely stopped proliferating, compared to MSCs in basal conditions (Fig. 2a, b). After neurogenic differentiation, the cells showed intense expression of βIII-tubulin, NeuN, GFAP, S100β, and CNPase (Fig. 2c–g).
In vitro differentiation of MSCs into neural-lineage cells. After exposure to neural induction medium, undifferentiated rat MSCs (a) exhibited very rapid morphological changes (b); most cells retracted their cytoplasm, forming spherical cell body, emitted cellular protrusions, and completely stopped proliferating, compared to MSCs in basal conditions. After neurogenic differentiation, the cells showed intense expression of βIII-tubulin (c), NeuN (d), GFAP (e), S100β (f), CNPase (g), suggesting that rat MSCs had successfully differentiated into neural lineage cells, which consisted of progenitors of neurons, astrocytes, and oligodendrocytes. Cells were stained with DAPI to visualize nuclei. Scale bars: 10 μm. MSCs, mesenchymal stem cells.
Locomotor Function
Behavioral performance was evaluated in all rats that received MSCs, NMSCs, or PBS using the BBB locomotor rating scale at weekly intervals for up to 9 weeks. Prior to transplantation at 1 week after SCI, the BBB testing was performed on all animals at 1 and 7 days post-SCI. A total of nine animals with asymmetrically malfunctioning hindlimbs were excluded for this experiment. Although all rats exhibited a gradual improvement over time, the locomotor function of the NMSCs-treated rats significantly continued to increase to a final score of 11.29 ± 0.57, whereas the function of the PBS-injected animals was only maintained to a score of 7.86 ± 1.09 at 8 weeks posttransplantation (p < 0.05 by a repeated measure ANOVA with post hoc Bonferroni comparison). The former score indicated a gait pattern characterized by frequent weight-supported plantar steps. Some of the animals in the NMSCs-treated group showed occasional forelimb and hindlimb coordination, whereas the latter score of the PBS controls indicated a hindlimb dysfunction characterized by sweeping or plantar placement with no weight support. On the other hand, MSCs-treated rats did not show a statistical improvement with a final score of 10.44 ± 0.50 compared with PBS controls (p = 0.128). Taken together, neurally differentiated cells provided a better outcome in locomotor behavioral testing, even if transplantation of MSCs derived from BM had a clear tendency of functional recovery (Fig. 3).
Locomotor behavioral assessment after transplantation of BM-derived cells. Locomotor performance was evaluated using the BBB locomotor rating scale. Although all rats exhibited a gradual improvement over time, the locomotor function of the rats treated with NMSCs continued to increase to a final score of 11.29 ± 0.57, whereas the function of the PBS-injected animals was only maintained to a score of 7.86 ± 1.09 by 8 weeks after transplantation. Finally, they showed an overall increment of hindlimb locomotor performance compared to PBS controls. Values are mean ± SE. BBB, Basso-Beattie-Bresnahan; NMSCs, neurally differentiated MSCs; PBS, phosphated buffered saline. *p < 0.05 by a repeated measure ANOVA.
Electrophysiological Testing
SSEPs and MEPs were recorded 8 weeks after transplantation. The waves of the SSEPs and MEPs showed representative patterns of negative–positive–negative potentials. The latencies of the evoked potentials were measured from the onset of the first negative deflection (initial latency), from the peak of the first negative deflection (N1 latency), and from the peak of the positive deflection (P1 latency). The amplitudes were also measured from the peak of the first negative deflection to the baseline (negative peak amplitude), from the peak of the first positive deflection to the baseline (positive peak amplitude), and from the peak of the first negative deflection to the peak of the positive deflection (peak to peak amplitude) (Fig. 4a, b).
Electrophysiological studies after transplantation of BM-derived cells. SCI rats transplanted with NMSCs showed significantly shortened initial latency, N1 latency, and P1 latency of SSEPs (a) compared with PBS controls. They also had a clear tendency to show increased amplitude of SSEPs, whereas NMSCs-treated animals exhibited neither a significant improvement in latency nor amplitude of MEPs (b). NMSCs, neurally differentiated MSCs; SSEPs, somatosensory evoked potentials; MEPs, motor evoked potentials.
Somatosensory Evoked Potentials in Rats Treated With Allogeneic BM-Derived Cells 8 Weeks After Transplantation
Values are mean ± SE. SSEP, somatosensory evoked potential; MSC, mesenchymal stem cell; NMSC, neurally differentiated MSC; PBS, phosphate-buffered saline.
p < 0.05 compared with PBS.
Motor Evoked Potentials in Rats Treated With Allogeneic BM-Derived Cells 8 Weeks After Transplantation
Values are mean ± SE. MEP, motor evoked potential; MSC, mesenchymal stem cell; NMSC, neurally differentiated MSC; PBS, phosphate-buffered saline.
Immunohistochemistry
The injured spinal cords exhibited cavitary lesions at contused epicenter (Fig. 5a). Transplanted cells derived from allogeneic BM, which were prelabeled with the cell proliferation marker BrdU, were confirmed by BrdU staining around the cavitary lesion of the injured spinal cord in longitudinal sections. Double immunoreactivity for BrdU and various neural lineage markers showed that transplanted cells differentiated into astrocytes stained for GFAP or oligodendrocytes stained for MBP. They were scattered rostrally and caudally 5 mm each (total 10 mm) from the epicenter of the injured spinal cord 4 weeks after transplantation (Fig. 5b, c). However, definite neurons double-labeled with BrdU and βIII-tubulin or NeuN were not seen. In addition, transplanted cells costained for BrdU and GFAP or MBP were not observed in the spinal cord 8 weeks after transplantation, suggesting that most of the settled cells did not survive to that time point.
Immunohistochemistry findings after transplantation of BM-derived cells. The injured spinal cords exhibited cavitary lesions at contused epicenter (a). Hematoxylin and eosin stain. Scale bar: 500 μm. Transplanted cells were scattered rostrally and caudally 5 mm from the epicenter of the injured spinal cord 4 weeks after transplantation. They were recognized and confirmed by confocal imaging of prelabeled BrdU (green) coexpressed GFAP (b) or MBP (c) (red). Scale bars: 10 μm. BrdU, 5-bromo-2-deoxyuridine;GFAP, glial fibrillary acidic protein; MBP, myelin basic protein.
Discussion
Functional recovery after SCI may be advanced with the use of cell-based therapy promoting strategic mechanisms including cellular replacement for neurons or glial cells lost after SCI and trophic or paracrine support to surviving or replaced cells to increase survival and plasticity. Before clinical trials, sufficient preclinical data regarding the biological mechanisms of neurological improvement are required. Among experimental animal models in the laboratory setting, intralesional or intravenous transplantation of BM-derived cells has been proven to be useful for amelioration of the functional deficit in various neurological diseases such as ischemic brain injury (2,28), demyelinating lesion (1,12), and SCI (22,30). Recently, genetically modified BM cells over-expressing neurotrophic factors such as BDNF and GDNF were used to maximize therapeutic effects (8,14,17,21).
In this study, we investigated neurogenic differentiation from allogeneic BM-derived MSCs with flow cytometry and immunocytochemistry, and showed that almost all of neurally differentiated MSCs strongly expressed βIII-tubulin, NeuN, GFAP, S100β, or CNPase, which indicated that they consisted of progenitors of neurons, astrocytes, and oligodendrocytes in equal proportion. Intraspinal transplantation of the NMSCs promoted functional recovery after SCI. In particular, locomotor performance and latency of SSEPs were significantly improved in NMSC-treated rats, compared with those of PBS controls. Furthermore, transplanted cells prelabeled with BrdU also differentiated into neural lineage cells that expressed specific markers for astrocytes and oligodendrocytes 4 weeks after transplantation, even though the number of integrated cells was not abundant. However, we did not find double-labeled cells with BrdU and βIII-tubulin or NeuN, suggesting that neuronal cells had not settled down in vivo after transplantation, despite the ability of MSCs to differentiate into various neural lineage cells in equal proportion in vitro. Differentiated astrocytes and oligodendrocytes also did not survive longer than 8 weeks posttransplantation, which was similar to what was reported in a previous study (4). In addition, NMSC-transplanted rats showed earlier functional improvement as measured by the BBB locomotor rating scale. Namely, most NMSC-treated rats began exhibiting plantar placement 2 weeks posttransplantation, and showed plantar placement with weight support in their stance 3 weeks posttransplantation. These results suggested that functional recovery due to trophic support or paracrine effect by neurotrophic factors, cytokines, and neuroprotective factors secreted by neurally differentiated cells may be a more prominent therapeutic effect than the integration of the grafted BM cells and establishment of functional connections. It can be explained by reports that neurotrophic factors such as BDNF, GDNF, NT-3, and NGF (11,25) or those secreted from transplanted stem cells (16) enhanced neural regeneration and improved neurological outcome, whereas it was reported that transdifferentiation of hematopoietic cells is extremely rare (26).
Because injured rats showed significant motor recovery at a relatively early stage after transplantation, and only a small number of transplanted cells survived in the injured spinal cord for a limited period, we propose that trophic or paracrine support has a much greater impact on functional improvement. Our electrophysiological study also showed that the latency of the SSEPs was significantly shortened in animals treated with NMSCs, which may be due to the effect of the grafted glial cells that produced a supporting structure around the necrotic tissue and consequent myelination. Therefore, transplantation of neurally differentiated MSCs derived from allogeneic BM might be a promising cell-based therapy for SCI, potentially extending to the use of these cells for the treatment of other neurological diseases, even if further therapeutic mechanism should be discovered.
Footnotes
Acknowledgments
This study was supported by a faculty research grant of Yonsei University College of Medicine for 2006 (6-2006-0066), and a grant (SC-4160) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.