Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue.
Citation: Quertainmont R, Cantinieaux D, Botman O, Sid S, Schoenen J, Franzen R (2012) Mesenchymal Stem Cell Graft Improves Recovery after Spinal Cord Injury in Adult Rats through Neurotrophic and Pro-Angiogenic Actions. PLoS ONE 7(6): e39500. https://doi.org/10.1371/journal.pone.0039500
Editor: Pranela Rameshwar, University of Medicine and Dentistry of New Jersey, United States of America
Received: December 16, 2011; Accepted: May 21, 2012; Published: June 20, 2012
Copyright: © 2012 Quertainmont et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant 3.4.563.04 of the National Fund for Scientific Research (Belgium) to JS, the Specific Targeted Research Project ‘RESCUE’ from the 6th Framework Program of the European Community and the “Gunter Verbraeckel Foundation” for scientific research on spinal cord injury (private foundation, Belgium). RQ is a Formation à la Recherche dans l'Industrie et dans l'Agriculture research fellow from the National Fund for Scientific Research (Belgium). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Treatment of spinal cord injury (SCI) faces several problems. First of all, the mechanical damage and axonal disruption in the spinal cord are followed by a progressive cascade of secondary deleterious reactions spreading to the adjacent spared tissue leading to lesion extension and worsening the situation . Secondly, although axonal regeneration is initiated, it is quickly repressed due to the inhibitory environment acting as a chemical and physical barrier for repair . In this context, the self-regeneration and reorganization ability of the central nervous system is insufficient to lead to considerable functional improvements.
Numerous strategies have been managed to improve functional recovery after SCI. These studies focused on neuroprotection or axonal regeneration, by modifying the injured environment to be beneficial for repair, by replacing lost cells, stimulating and guiding axonal growth or boosting remyelination , , , . To act on these events, scientists often exploited the potential of cell therapy using transplantation of various cell types like Schwann cells , , olfactory ensheathing cells , , neural stem cells , , bone marrow stromal cells , , fibroblasts ,  and macrophages . Despite promising results, it ensues from these experiments and from the complexity of SCI that an optimal single focalized strategy to treat it efficiently doesn't exist. It becomes evident that research has to concentrate its effort on multifactorial treatments, playing on the different factors of SCI pathophysiology. In this context, it appears that bone marrow stromal cells, also called mesenchymal stem cells (MSCs) could take an interesting part in these strategies.
Indeed, MSCs are adult stem cells with self-renewing and differentiation abilities . These cells are harvested from bone marrow and easily and quickly expanded in vitro. This accessibility makes them an interesting candidate for autologous transplantation, abolishing immune and ethical problems of embryonic/foetal stem cell grafts . MSCs are also able to modulate the CNS injured environment and to promote repair as they secrete anti-inflammatory, anti-apoptotic molecules and trophic factors able to support axonal growth, immunomodulation, to promote angiogenesis, remyelination and to protect from apoptotic cell death , , , . In addition, some evidence suggests that MSCs may be able to differentiate into neuronal cells in vitro using co-culture ,  or differentiation medium , ,  but also in vivo after transplantation ,  making them a good candidate for neuronal cell replacement strategies.
Different modes of administration have been used for cell transplantation after SCI: intrathecal , intravenous  or intraspinal . For a human application, intravenous injection appears to be the easiest way to bring therapeutics to patient without risking further damage to the spinal cord. Furthermore, MSC intravenous injection is safe, as it has been shown to have no adverse effect like obstruction of blood circulation or tumorigenicity , .
The aim of this study was to graft MSCs by intravenous injection one week after a spinal cord compression injury, in order to assess their effect on functional recovery and find by which mechanisms these cells exert their beneficial effect.
1. Characterisation of rat MSCs in vitro
MSCs were isolated from Wistar adult female rats and cultured in plastic adherent conditions, classically used for MSCs in vitro expansion. They were characterized according to three criteria: adherence to tissue culture plastic dish, specific surface antigen expression and multipotent differentiation ability. MSCs exhibited typical elongated, fibroblast-like morphology or large, flattened shape (figure 1A). After 4–5 passages (P4–P5), all cells express surface antigens CD90 (figure 1B) as well as the neurotrophin co-receptor p75NGFr, also known as CD271 (figure 1C) , while they were negative for CD45 and CD11b (figures 1D and 1E). P4 MSCs are multipotent, as they differentiate into adipocytes and osteocytes according to published protocols (figures 1F and 1G) . After 12 passages, cells were deprived of serum to induce the expression of nestin (figures 1H), a protein expressed by neural stem cells. This strategy was used in order to influence their fate towards a neural phenotype. Indeed, it has been demonstrated that only nestin-positive MSCs were able to differentiate into functional neurons in vitro . The transcriptomic and proteomic comparisons of nestin-positive and nestin-negative MSCs also suggest that the nestin-positive cell population contains a higher percentage of stem cells that would adopt a neural fate . It thus seemed appropriate to use these nestin-positive cells in our transplantation strategy.
(A): Phase contrast microphotography of rat MSCs in culture. Cells exhibit typical elongated, fibroblast-like morphology (arrowheads) or large, flattened shape (asterisks). (B) CD90 and (C) CD271 positive immunofluorescent labelings of P4 MSCs in culture, while CD45 (D) and CD11b (E) immunostainings are negative. (F) Adipogenic differentiation of P4-MSCs revealed with Oil Red O. (G) Alizarin Red staining of P4 MSCs induced to osteocytes reveals mineral deposition. (H) Immunofluorescent visualization of nestin induction in P12-MSCs deprived of serum. Nuclei are labeled with the Vectashield-DAPI mounting medium.
2. BrdU labelling of MSCs
We used in vitro BrdU incorporation to label MSCs before graft. After 72 h of culture in presence of 1.10−6M BrdU, we confirmed by immunocytochemistry that all MSCs were labelled with BrdU (figure 2A). We also assessed the maintenance of the BrdU labelling in MSCs with time to take into account cell population doublings and cell passages. We show that 3 and 7 days after the removal of BrdU from the culture medium, which respectively correspond to 0 and 1 passage, BrdU is still easily detected within the MSCs (figures 2B–C). After 21 days, and 6 passages, the signal is clearly reduced, but remains observable (figure 2D).
(A) BrdU (FITC)-DAPI immunostaining on MSCs cultured with 1.10−6M BrdU for 72 h, demonstrating that cells were all labeled before being transplanted. (B–C–D) BrdU (Rhodamine) maintenance in MSCs 3 days (B), 7 days (C) and 21 days (D) after the removal of BrdU from the culture medium. (E) BrdU immunodetection (FITC) on a longitudinal spinal cord tissue section from a rat that received 3 ip BrdU injections after spinal cord injury. Scale bar: 50 µm (A), 100 µm (B, C, D) and 200 µm (E).
3. Fate of grafted cells
To study the fate of grafted cells, we performed, at 7, 14 and 21 days after the graft, a BrdU immunohistochemistry on the injured transplanted spinal cord, but also on lung, liver, spleen and kidney tissue sections, as MSCs were grafted in the tail vein. Unfortunately, no grafted cell could be found within the lesion site neither in any organs (data not shown). In order to rule out a problem of BrdU detection, we also performed BrdU immunohistochemistry on spinal cord sections from rats that received, during 3 consecutive days following SCI, intraperitoneal BrdU injection (100 mg/kg), and were allowed to survive for 2 more weeks. Figure 2E shows that BrdU is detected within the injured spinal cord, demonstrating that our protocol of detection is efficient.
4. Control of MSC viability and CD profile
To confirm cell viability at the time of iv injection, we placed back into culture the last drop of the MSC suspension from the injection needle and characterized them 24 hours later by immunocytochemistry for nestin and p75NGFr. Transplanted cells were viable at the time of injection as they still could adhere to plastic culture flask and express nestin and p75NGFr (figures 3 A–B). As transplanted cells were P12 MSCs, we checked that their CD profile was still the one that characterizes MSCs, and thus performed CD90, CD271, CD45 and CD11b immunocytochemistry on P12 MSCs (figures 3 C–F). The results confirm that P12 MSCs have conserved their CD profile, as they do still express CD90 and CD271, and are still negative for CD45 and CD11b.
Nestin (A) and p75NGFr (B) immunofluorescent stainings on MSCs: The last drop of MSC suspension from the injection needle was placed back into culture to check their viability at the time of injection. (C–F): CD profile of P12 MSC grafted cells, showing their immunoreactivity for CD90 (C) and CD271 (D), and the absence of CD45 (E) and CD11b (F) expression. Scale bar: 50 µm (A, B), 100 µm (C–F).
5. MSC graft improves functional recovery after spinal cord compression injury
Spinal cord injured animals were submitted to 2 behavioural assessments in order to evaluate the effect of MSC graft on functional recovery. Locomotor skills were assessed by the open field test using the BBB rating scale. Mean scores over time were higher in the MSC grafted group (injured + MSC) compared to control groups (injured + vehicle or injured only). The difference between MSC grafted and control groups was statistically significant (injured + MSC vs injured only p = 0.0029; injured + MSC vs injured + vehicle p<0.0001; figure 4A). Moreover, only MSC grafted animals reached and overtook the score corresponding to weight support stepping (score of 9). Interestingly, locomotor scores from MSC grafted rats begun to be distinguishable from control groups already three days after the graft. The grid navigation test completes this behavioural part of motor recovery assessment. Even if grafted rats did not recover their toe control and the capacity to realise fine movements, their scores were significantly improved with MSC graft (injured + MSC vs injured only p<0.0001; injured + MSC vs injured + vehicle p<0.0001; figure 4B).
(A) Locomotor scores as assessed by the BBB rating scale. MSC grafted rats reach significantly higher scores compared to both control groups. Only MSC transplanted rats reach the weight-supporting step level (score of 9). Injured + MSC vs injured only p = 0.0029; injured + MSC vs injured + vehicle p<0.0001. (B) Grid test scores assessing deficits in descending fine motor control. MSC grafted rats reach higher scores, significantly different from control rats. Injured + MSC vs injured only p<0.0001; injured + MSC vs injured + vehicle p<0.0001.
6. MSC graft increases NGF level expression within the injured tissue
As behavioural improvements appear already during the first week following MSC graft, we performed cytokine array on proteins extracted from the injured spinal cord of control and MSC-treated rats (3 days post-graft), to assess and compare changes in cytokine/trophic factor expression. We analyzed the expression of Ciliary neurotrophic factor (CNTF), Granulocyte macrophage-colony stimulating factor (GM–CSF), Interleukin-1 alpha (IL–1α), Interleukin−1 beta (IL–1β), Vascular endothelial growth factor (VEGF), Nerve growth factor beta (ßNGF), Interleukin-10 (IL–10), Interferon-gamma (IFN-γ), Monocyte chemo-attractant protein-1 (MCP–1) and Tumor necrosis factor alpha (TNF–α). The expression levels of all these molecules were higher in grafted animals compared to vehicle-treated animals. The sole factor for which we observed a difference statistically significant is ßNGF (p = 0.042) (figure 5A). All other tested molecules showed only a slight increase tendency. As SCI is known to trigger NGF up-regulation by itself, the higher rate of NGF expression obtained 3 days after the graft in the “injured + MSC group” compared to the “injured + vehicle-treated group”, raises the hypothesis that the source of this increased NGF are the grafted MSCs. We thus checked if MSCs produce NGF by performing an ELISA assay on P12-MSC conditioned medium. The analysis of 2 different culture media (from 2 distinct cultures) demonstrates that MSCs secrete the neurotrophin NGF (figure 5B). Immunocytochemistry for NGF on P12 MSCs in culture also reveals that all of them express NGF in vitro (figure 5C). In parallel, ELISA for BDNF was performed on the same culture media, showing that MSC do also secrete BDNF, but in a much lower quantity compared to NGF (figure 5B). This correlates with the weaker BDNF expression obtained by immunocytochemistry on MSCs in vitro (figure 5D).
(A) Cytokine arrays. Spinal cord extracts from injured vehicle-treated (n = 4) and MSC-treated (n = 5) rats. ß-NGF is significantly increased within the lesioned site 3 days after MSC injection compared to controls. * p<0.05 (mean±S.E.). (B) Histogram showing the amounts, in pg/ml, of the neurotrophins NGF and BDNF as quantified by Elisa, within 2 distinct P12 MSC-conditioned media (M1 and M2). (C) NGF and (D) BDNF fluorescent immunocytochemistry on P12 MSCs in vitro. Scale bar : 50 µm.
7. MSC graft promotes tissue sparing after spinal cord compression injury
To further elucidate the mechanisms that could explain the beneficial effects obtained with MSC transplantation, we investigated the possibility of a tissue sparing effect. We thus performed a histological luxol fast blue/eosin staining on serial cross sections from injured spinal cord at 28 days after injury, in MSC grafted and in control “injured-only” rats. Sections were then analyzed and the ratio “injured area/total area” from each section determined. (figures 6A–B). The mean values were reported on figure 6C. This analysis revealed a reduced surface of damaged tissue for the grafted group compared to the control one (white matter and grey matter together). The difference was statistically significant (p = 0.0454). It thus strongly supports a neuroprotective effect of the MSC graft.
MSC transplants favor tissue sparing after SCI. Luxol fast blue/hematoxylin staining on cross sections of MSC-grafted (A) and control injured only (B) rats, 21 days after transplantation. The quantification of spared tissue (C), as assessed by the mean ratio of injured area on total area of the sections, reveals a significant decrease of the lesion extension in MSC treated rats compared to control ones. * p<0.05.
8. MSC graft does not increase axonal regrowth
We analysed the effect of MSC graft on axonal regrowth 21 days after transplantation, by an immunostaining against GAP-43 on transversal spinal cord sections taken at the level of the lesion site (figures 7A–B). The quantification of the proportion of the total surface of the section stained for GAP–43 doesn't show a significant difference between control (2,25%±0,44) and grafted (2,96%±0,35) animals, though these latter exhibit higher values of GAP-43 immunoreactivity (figure 7C; p = 0.23 Student's t-Test).
(A–B) GAP43 immunoreactivity on transversal spinal cord sections, 28 days after SCI, in injured + MSC (A) and injured-only (B) groups. Sections were taken at the lesion site. (C) Image analysis doesn't show a significant difference (Student's t-Test, p = 0,23) in the percentage of total lesioned area immunoreactive for GAP43 between treated (2,96 ± 0,35 %) and control (2,25 ± 0,44 %) groups
9. MSC graft favours vascularisation
To assess a potential effect of MSC graft on tissue vascularisation, we performed a RECA-1 immunostaining, a specific marker for rat endothelial cells, on longitudinal spinal cord sections centred on the lesion, both on MSC-treated and control groups (3 days after MSC or vehicle treatment). A first qualitative analysis revealed a higher number of blood vessels in the injury epicentre of MSC-treated cords compared to controls (figures 8 A–B). Quantification confirmed a significant increase in the number of blood vessels in the injury site of grafted animals compared to controls (figure 8C). Despite the higher surface analysed in the control group (which correspond to the injured tissue: 2.067.378±198.630 µm2 for injured + MSC and 2.207.101± 212?647 µm2 for vehicle-treated), the number of blood vessels is lower in that group compared to injured + MSC group.
(A–B) RECA-1 immunohistological staining on longitudinal sections of MSC-treated (A) and control vehicle-treated (B) spinal cords. Scale bar: 500 µm. (C) Blood vessel quantification within the lesioned site reveals a significant increase in MSC treated rats compared to control-vehicle treated ones. *p<0.05.
Our work confirms that MSC treatment is beneficial after SCI and is in accordance with previous studies reviewed by . We show by two complementary locomotor tests that MSCs have the ability to significantly improve functional recovery. Using the open field test with the BBB rating scale, we were able to precisely study locomotion faculties after SCI. The animals grafted with MSCs do not only reach higher motor scores, but were also able to support their weight and make coordinated steps whereas control animals were not able of weight support and seemed to reach a plateau. MSCs appear to act quickly after iv injection as the difference of recovery of MSC-grafted animals and control groups is already important 3 days after the cell injection (BBB scores of 4.5 and 2.6 respectively). We also show a beneficial effect of MSCs on the grid-walking task. Again, MSC-grafted rats obtained higher scores than controls, reinforcing the conclusion that MSCs do really exert a beneficial effect on recovery after SCI. According to the literature , the evidence of a functional improvement after iv injection of MSCs in SCI is scarcely described. Indeed, our results can be compared to one single study, conducted by Urdzikova et al, who also described locomotor improvement after bone marrow stromal cell iv injection performed one week after a thoracic compression injury . Their behavioral data also demonstrate a rapid effect of the treatment, but no evidence of MSC integration within the host tissue to replace lost cells has been described, even if some grafted MSCs could be found within the lesioned tissue.
In spite of the efficacy and maintenance of the BrdU labeling of MSCs, the use of an effective protocol to detect BrdU in vivo, and the confirmation of the viability of the cells at the time of injection, we were not able to find the grafted MSCs at various delays (7, 14 and 21 days post injection) in the injured spinal cord, neither in highly vascularized organs such as lungs, liver, spleen and kidneys where we thought the cells could have migrate after systemic injection , . These observations are in accordance with Karp and Teo analysis of exogenously infused-MSC homing . Indeed, according to this review, the homing of cultured-expanded iv-injected MSCs is inefficient, and apparently due to (i) a lack of relevant cell adhesion and chemokine receptors and (ii) to the increased size of MSCs in culture, which likely promotes cell entrapment and reduces the number of MSCs that reach the target site. If homing towards the injury site of MSCs delivered by iv injections has nevertheless been reported in SCI , ,  as well as in brain injuries , , these cells reach the host tissue in small numbers and are not sufficient to explain the rapid functional recovery . It thus seems clear that iv delivery of MSCs is not the most effective way to introduce cells after injury (see also , ) but it nevertheless remains the least invasive, making it worth trying. Other delivery methods (intra-thecal, intra-arterial) might have better potential in the perspective of clinical application . Yet, it remains that even if we couldn't find grafted cells in the host tissue, we observed a clear, significant and early beneficial effect of MSC iv transplantation on locomotion, strongly suggesting that MSCs act throughout their secretions. Likewise, several reviews on the paracrine therapeutic effect of MSCs , , ,  reinforce our findings.
To explore this hypothesis of an early paracrine effect of the MSCs, we performed cytokine array on the injured tissue 3 days post injection. Among the cytokines analyzed, we observed a slight increase in the expression of CNTF, MCP-1 and GM-CSF. CNTF plays a role in neuroprotection  and promotes survival and differentiation of oligodendrocyte precursor cells (OPCs) after SCI . MCP-1 induces monocyte recruitment during inflammation , enhancing myelin debris clearance in CNS injuries . GM-CSF activates macrophages , inhibits apoptosis of neuronal cells and gliosis after SCI ,  and treatment with GM-CSF or GM-CSF with bone marrow stromal cell transplantation promotes axonal regeneration and functional recovery after SCI , . Several authors reviewed the therapeutic properties of MSCs and reported that these cells are able to secrete, among others, CNTF, MCP-1 and GM-CSF , , . We also detected a slight tendency of increased expression of anti-inflammatory cytokines IFN-γ, IL-10, and pro-inflammatory IL-1β after MSC injection but the levels concerned and the differences with the control group are insignificant. All together these cytokines and trophic factors could have a beneficial effect after SCI on the reduction of the inflammatory reaction, cell death and tissue protection, but regarding the low difference we have seen in their expression in injured tissue between MSC-grafted and control rats, they cannot be the only explanation for an important functional recovery.
Concerning nerve growth factor (NGF), we observed a high and statistically significant increase of its expression in MSC-grafted animals compared to controls, which totally agrees with the results obtained after MSC iv transplantation in traumatic brain injury . NGF has been shown to play a role in protecting neuronal injured tissue from toxic events, inducing regrowth, repair and reorganization of neuronal connections , ,  but also stimulating neurogenesis , . NGF is also known to activate monocytes, macrophages and microglia in CNS by increasing their phagocytic activity , , . After CNS injury, NGF is secreted by microglia , but we believe that the higher NGF expression observed in the injured tissue in MSC-grafted spinal cord results from its secretion by MSC themselves. Indeed, we show that they do express NGF in vitro, and that they secrete NGF as demonstrated by our Elisa results. BDNF, to a lesser extent, is also expressed and secreted by MSCs in vitro. These results are in accordance with the literature , , . We however can not rule out the possibility that the elevated expression of NGF found in the grafted tissue could be due to endogenous cells being stimulated by injected MSCs. BDNF is beneficial to SCI repair, both for axonal growth and tissue protection , . If no significant effect on axonal regrowth with GAP-43 staining was obtained in our MSC-treated rats, we however show a neuroprotective effect of our transplanted cells. Actually, one of the most documented effects of MSC treatment after SCI is the reduction of cavity formation or tissue sparing, which is frequently reported without the demonstration of an axonal regrowth-associated effect , , , , . Bearing in mind the large neuroprotective and repair-inducing effect of NGF, its higher expression could thus partially explain the largest amount of spared tissue in MSC grafted rats.
Among the molecules secreted by MSCs, pro-angiogenic factors like VEGF  are interesting to study in SCI as blood supply is essential for repair of damaged tissue. Furthermore, tissue sparing is often associated with stimulation of angiogenesis. VEGF/PDGF-stimulated angiogenesis results in lesion size reduction and in white matter sparing with functional outcome after SCI , . In our study, we only detect a slight tendency of increase in the expression of VEGF after MSC graft, which is probably not sufficient to be responsible of the higher blood vessel density that we found. As it has been previously shown, MSCs have the capacity to provide other trophic support for angiogenesis such as bFGF, BMP-2, angiogenin, MCP-1 or IL-6 , . Matrix metalloproteinases also facilitate angiogenesis by helping the invasion of endothelial cells , . MSCs could also be able to increase endothelial cell survival in an injured environment via other paracrine secretions like anti-apoptotic molecules , , .
Throughout the literature, MSCs show differences in their survival and integration within host tissue as well as in their effects on repair. We explain these discrepancies by the difference between MSCs themselves. Indeed, variations in the morphology, behavior and characteristics have been shown depending on the number of passages, the plating densities and the duration of culture . It has been reported that subpopulations of MSCs with different properties do exist , . Using different markers, some authors were able to select subpopulations of MSCs . Furthermore, it has been shown that MSCs in the bone marrow have at least two developmental origins, one of them being the neural crest , . Also, it has been reported that deprivation of serum ,  or hypoxia culture conditions  modify MSC characteristics. The existence of such subpopulations with distinctive properties is reinforced by differences in MSC properties in culture or in their post-graft effect, even more for human MSC from different donors , . So, we believe the way a heterogeneous population of MSCs is cultured selects some subpopulations with different characteristics and properties.
In conclusion, despite differences in the mechanisms and effects of MSC graft after SCI, their beneficial effect on functional recovery remains constant. We confirm an early significant behavioral improvement in MSC-transplanted rats, which is likely due to their paracrine secretions, as no cells could be found within the host. We also show a tissue sparing effect and a higher blood vessel density in the injured site, which can also play a beneficial role in tissue sparing and functional recovery by improving blood flow in the lesion . These effects are mediated at least partially through the important NGF secretion, but, in regard of the large array of factors MSCs are able to secrete, we cannot exclude the effect of other molecular actors we didn't study here. Additional work is needed to study and further characterize the paracrine effect of MSCs after SCI.
Materials and Methods
1. Mesenchymal stem cell isolation, culture and characterization
MSCs were harvested from Wistar adult female rats. After sacrifice, femurs and tibias were aseptically removed and bone marrow flushed from each piece with approximately 5 ml culture medium using a sterile syringe. Bone marrow was placed in T75 culture flask in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% of foetal bovine serum (Invitrogen). The culture medium was changed once per day for three days in order to remove non-adherent cells. Then the culture medium was changed twice a week and cells were passaged when they reached confluence. The enrichment in MSCs was made simply by several passages due to their preferential adherence to plastic. Cells were characterized by their specific surface antigen expression with immunocytochemistry against CD90 (+), CD271 ( = p75NGFr) (+) , CD11b (−) and CD45 (−). Their multipotency was monitored by in vitro differentiation into adipocytes and osteocytes , . After fixation with 4% paraformaldehyde, the adipogenic differentiation was evaluated by accumulation of lipid vacuoles and staining with Oil Red O (Sigma) and the osteogenic differentiation was visualized by Alizarin Red S staining (Sigma) of matrix mineralization associated with osteoblasts. We considered that the culture contained 95% of MSCs after 4 to 5 passages.
50 adult female Wistar rats from the animal facility of the University of Liege were used for this study (±250 g). All experiments were conducted according to the ethical laws and regulations of Belgian National Fund for Scientific Research. The project research has been approved by the ethical commission for animal use of the University of Liege, Belgium. Animals were subdivided into 3 experimental groups (Table 1):
- Spinal cord injured rats without treatment (n = 10) = Injured only.
- Spinal cord injured rats injected with fresh culture medium without serum (vehicle) (n = 20) = Injured + vehicle-treated.
- Spinal cord injured rats injected with a suspension of MSCs in fresh culture medium without serum (n = 20) = Injured +MSC-treated.
Animals were housed individually with water and food ad libitum. One week before the beginning of the experiments, animals were placed daily in the different surroundings of the behavioural tests, to get accustomed to them.
3. Spinal cord injury procedure and animal care
Animals were anaesthetized by inhalation of Isoflurane (Forene®, Abbott, Queenborough, Kent, England) 2 to 3% in a flow of 1 L/minute of oxygen. The compression injury was performed as described by Vanicky et al. . Briefly, a 2-French Fogarty arterial embolectomy catheter (Edwards Lifesciences LLC, Irvine, CA, USA) was inserted in the epidural space at the T10 level and moved rostrally for 2 metameric levels before being inflated with a distilled water volume of 15 µl and left in place during 5 minutes. The balloon was then deflated and carefully removed. Skin and muscle were carefully closed in two layers. 1mL of NaCl 0.09% was injected subcutaneously to prevent dehydration during the time animals recover from anaesthesia. Antibiotic (amoxicilline-clavulanic acid) was administrated by intraperitoneal (ip) injection after the surgery to prevent urinary tract infection. Animals had their bladder manually emptied twice daily until recovery of micturition and were carefully inspected for weight loss, dehydration and autophagia.
4. Mesenchymal stem cell injection procedure
72 hours before injection, MSCs were cultured in D-MEM (Invitrogen) without serum to induce the expression of nestin, a protein expressed by neural stem cells. This was performed in order to favour their fate towards neural stem cells , which could further support their integration within the host tissue. In order to label MSCs, 1.10−6M of 5-Bromo-2′-deoxyuridine (BrdU) was added to the culture medium during this period. The culture medium was changed every 24 hours. MSCs were injected 7 days after injury. We used passage 12 MSCs. After reaching confluence, cells were detached and collected in D-MEM without serum. The number of cells per mL was calculated and cells were diluted in order to obtain 1.106 cells in an injection volume of 500 µL. Animals were anaesthetized by inhalation of Isoflurane (Forene®, Abbott, Queenborough, Kent, England) 2 to 3% in a flow of 1 L/minute of oxygen. Intravenous 24 GA catheter (Becton Dickinson) was inserted in one of the caudal vein and the cell suspension was injected with the UltraMicroPump (UMP-3) and SYS-Micro4 controller (World Precision Instruments) in a flow rate of 50 µL/min. The catheter was removed 2 min after the end of injection to avoid cell loss during its withdrawal. The last drop of cell suspension collected from the catheter was put back in culture on polyornithine-coated glass coverslips to assess cell viability at the time of the injection procedure.
5. Behavioural analyses
Following SCI, locomotion and sensorimotor skills were monitored as follows, twice a week, during 3 weeks:
Open field locomotor test.
The Basso, Beattie and Bresnahan (BBB) locomotor rating scale  was used. Rats were observed walking freely in an open field and scores were attributed by two blinded observers according to the recovery of hind limb movements.
Grid navigation test.
This test was used to assess the deficit in descending motor control. The animals had to cross 5 times a 1 m long grid with round metal bars separated from 4 cm and a score was attributed by two blinded observers according to the recovery of hind limb movements: 0. Hindlimb drag, no foot placement; 1. Consistent foot faults, foot miss and fall through wire mesh; 2. Consistent foot faults, foot slip off grid after accurate step and fall through wire mesh; 3. Accurate foot placement on grid but toes do not grip; 4. Normal course over grid with gripping toes.
For statistical analysis, we used a GLMM model (generalized linear mixed models) and p<0.05 was considered statistically significant.
6. Histological analyses
Animals were deeply anaesthetized with pentobarbital (200 mg/kg) ip injection (Nembutal, CEVA Santé Animale, Libourne, France) and perfused with 250 mL of NaCl 0.09% (4°C) followed by 500 mL of 4% paraformaldehyde (4°C) in 0.1 M phosphate buffer saline (PBS, pH 7.4). The spinal cords were rapidly dissected out and post-fixed in the same fixative for 24 h at 4°C and then kept for 48 h in 30% sucrose for cryoprotection. Two tissue blocks of 0.75 cm length were taken from both sides of the lesion epicentre. Tissue blocks were then either transversely, either longitudinally, cryo-sectioned in 20 µm serial sections, mounted on superfrost+ glass slides and stored at −20°C until used.
To analyze the lesion extension, we performed Eosin – Luxol fast blue histological staining. We calculated the ratio between the measured injured area and the entire area of the spinal cord slice on serial transversal sections, covering a tissue bloc of 1,5 cm centered on the lesion epicenter. We used ANOVA followed by the Scheffé's post-hoc test. p<0.05 was considered statistically significant.
Sections were fixed with 4% PFA for 5 min. After 1 TBS (Tris buffered saline) rinse, tissue sections were first permeabilized with 0.3% Triton in TBS for 15 min at RT. They were then rinsed 3×5 min in TBS at 37°C, and further incubated in HCl 2N for 30 min at 37°C followed by Na Tetraborate 0.1 M pH 8.5 for 10 min at 37°C. After 3 TBS rinses, they were incubated in a blocking buffer (1% BSA and 10% goat normal serum in 0.3% Triton X-100-TBS) for 1h, and thereafter overnight at 4°C in the BrdU antibody diluted in the blocking buffer (containing 3% of normal goat serum instead of 10%). After 3 TBS−0.05% Tween20 and one TBS rinses, the sections were incubated for 1 h at RT with Alexa Fluor®488 goat anti-rat (1/1000, Invitrogen), rinsed 3 times in TBS−0.05%Tween20, and once in TBS. Sections were finally coverslipped with Vectashield® with DAPI mounting medium (Labconsult). Negative controls were obtained by omission of the primary antibody.
In order to check the maintenance of BrdU within MSCs throughout their divisions, we performed a BrdU immunocytochemistry on P12 MSCs as follows: P12-MSCs were cultured in D-MEM +10% FBS until 60% confluence. Cells were then rinsed once with PBS and cultured in D-MEM without FBS + 1.10−6 M BrdU for 3 days (as it was done for the cells used for in vivo transplantation). The medium was renewed every day. Then, MSCs were placed again in D-MEM +10% FBS for 3, 7 and 21 days. During this period, the culture was maintained by medium changes and passages (0, 1 and 6 passages, respectively). At each delay, cells were detached and placed on glass coverslips during a few hours in order to perform a BrdU immunostaining.
Cells were cultured on polyornithine (Sigma-Aldrich, Schnelldorf, Germany) coated glass coverslips and fixed during 5 minutes in 4% PFA or in 4% PFA buffered in 50 mM sodium borate at pH 9.5 for Nestin immunostaining. For BrdU staining, cells were fixed with 4% PFA for 5 min, incubated in HCl 2N at 37°C for 30 min and neutralized in sodium tetraborate 0,1 M for 10 min. After 3 PBS rinses, cells were incubated in a blocking buffer (1% BSA and 10% of appropriate normal serum in PBS +0.3% Triton X-100) for 1 h, and thereafter overnight at RT in the primary antibody diluted in the blocking buffer (1% BSA and 3% appropriate normal serum in PBS +0.3% Triton X-100). After 3 PBS−0.05% Tween20 and one PBS rinses, cells were incubated for 1 h at RT with the secondary antibody diluted in PBS +1% of appropriate normal serum, rinsed 3 times in PBS +0.05% Tween20, and once in PBS. Coverslips were finally turned over on glass slide using Vectashield® with DAPI mounting medium (Labconsult). Negative controls were obtained by omission of the primary antibody.
Tissues were fixed with 4% PFA for 5 min, then incubated in a 0.3% H2O2, 0.1% Na azide solution in PBS for 20 min at RT to reduce endogenous peroxydase activity. Nonspecific binding was prevented by 1h incubation in 3% normal serum and 1% bovine serum albumin solutions in PBS +0.2% triton. Sections were then incubated overnight at RT with the primary antibody. After 3 PBS rinses, they were incubated with their respective secondary biotinylated antibodies (Vector Laboratories) diluted and centrifuged in PBS with 1% BSA and 2% of rat serum, for 1 h at RT. Then sections were incubated 1 h with the avidin-biotin-peroxydase complex (Vector Laboratories), diluted 1/1000 in PBS and the immunostaining revealed with 3,3′-diaminobenzidine.
The primary antibodies used for immunostaining were the following: mouse anti-CD90 (1/100, AbCam), rabbit anti-CD45 (1/100, AbCam), mouse anti-CD11b (1/200, AbD Serotec), mouse anti-Nestin (1/200, Chemicon), rat anti-BrdU (1/100, AbD serotec), rabbit anti-NGF (1/100, AbCam), rabbit anti-p75NGFr (1/500, AbCam), mouse anti-RECA-1 (1/200, AbCam), chicken anti-BDNF (1/250, R&D Systems). Secondary antibodies were rhodamine- or FITC-coupled donkey anti-mouse, rhodamine- or FITC- donkey anti-rabbit, rhodamine donkey anti-chicken (1/500, Jackson Immunoresearch), Alexa Fluor®488 goat anti-rat (1/1000, Invitrogen) or Alexa Fluor®488 donkey anti-mouse and anti-rabbit (1/500, Invitrogen) for immunofluorescence, and biotinylated goat anti-rabbit and horse anti-mouse (1/500, Vector Laboratories) for DAB immunostainings.
Immunoreactivity and tissue stainings were examined and captured using an Olympus AX70 microscope equipped with an Olympus DP50 digital camera and an Olympus FSX100 microscope. Images were analysed by AnalySIS software (Soft Imaging System, Olympus) for spared tissue quantification. For GAP-43 immunostaining quantification, bright field images of transversal sections of the “lesion-caudal” tissue block were taken and pictures were converted to gray scale. A threshold intensity of gray-colored staining was fixed for each slice. Then, a surface covering the entire lesion area was selected ( = total lesion area). Thereafter, immunostaining was quantified within non-overlapping successive fields covering the total lesion area ( = total stained area) using Analysis software (Olympus). Data were expressed as the percentage of total stained area (pixel) per total lesioned area (pixel). The results represent averages from 5 “injured only” rats and 6 “injured + MSC” rats. The statistical significances between injured-grafted and injured only groups were assessed using a Student t-test.
Blood vessel quantification.
We performed RECA-1 immunostainings on longitudinal sections of spinal cords 10 days after injury (3 days after MSC graft for treated animals). The epicentre zone was encircled and the number of blood vessels counted within the zone. The statistical significances between injured-grafted and injured only groups were assessed using a Student t-test.
7. Cytokine array
We used a custom Rat Cytokine Antibody Array kit (RayBiotech, Norcross, Georgia, USA). Animals were deeply anaesthetized with pentobarbital (200 mg/kg) ip injection and perfused with 250 mL NaCl 0,09% at 4°C. Tissue block of 1 cm length and centred on the injury epicentre was dissected out and crushed into lysis buffer (RayBiotech) containing protease inhibitors (complete mini, Roche) on ice. After protein dosage (BCA protein assay reagent kit, Pierce), 200 µg of total proteins were used per sample. Analysis was performed as recommended in the protocol supplied with the kit. The cytokine array membranes were scanned with a Las-4000 luminescent image analyser (Fujifilm, Tokyo, Japan). The intensity of the signal was measured with the Quantity One 1-D Analysis software (Bio-RAD Laboratories, Hercules, CA). The results were expressed as the mean relative signal intensity, i.e. the ratio of sample signal intensity/positive control signal intensity. Each signal intensity of positive controls and samples were normalized by background substraction. The statistical significances between injured-grafted and injured only groups were tested using a Student t-test.
8. NGF – BDNF Quantification in the MSC-conditioned medium
NGF and BDNF levels were measured by enzyme-linked immunosorbent assay according to the manufacturer's protocol (ChemiKine Nerve Growth Factor Sandwich ELISA kit CYT304 and ChemiKine Brain Derived Neurotrophic Factor, Sandwich ELISA kit CYT306, Chemicon). The conditioned media from 2 distinct cultures of rat MSCs at passage 12 were harvested and concentrated using Amicon Ultra-15 centrifugal filter units (Millipore). 100 µl of these two concentrated media (M1 and M2) were used for the assay.
The authors are grateful to Ms. Mosen, Ms. Ernst, Ms Pieltain and Ms. Wouters for their technical assistance to this work. Ms. Seidel, from the Department of Statistics of the University of Liege, performed all statistical analysis.
Conceived and designed the experiments: RF JS. Performed the experiments: RQ DC OB SS. Analyzed the data: RQ DC. Contributed reagents/materials/analysis tools: RQ DC . Wrote the paper: RQ RF.
- 1. McDonald JW, Sadowsky C (2002) Spinal-cord injury. The Lancet 359: 417–425.JW McDonaldC. Sadowsky2002Spinal-cord injury.The Lancet359417425
- 2. Nishio T (2009) Axonal regeneration and neural network reconstruction in mammalian CNS. Journal of Neurology 256: 306–309.T. Nishio2009Axonal regeneration and neural network reconstruction in mammalian CNS.Journal of Neurology256306309
- 3. Webb AA, Ngan S, Fowler JD (2010) Spinal cord injury I: A synopsis of the basic science. Canadian Veterinary Journal 51: 485–492.AA WebbS. NganJD Fowler2010Spinal cord injury I: A synopsis of the basic science.Canadian Veterinary Journal51485492
- 4. Wright KT, Masri WE, Osman A, Chowdhury J, Johnson WEB (2011) Concise Review: Bone Marrow for the Treatment of Spinal Cord Injury: Mechanisms and Clinical Applications. Stem Cells 29: 169–178.KT WrightWE MasriA. OsmanJ. ChowdhuryWEB Johnson2011Concise Review: Bone Marrow for the Treatment of Spinal Cord Injury: Mechanisms and Clinical Applications.Stem Cells29169178
- 5. Coutts M, Keirstead HS (2008) Stem cells for the treatment of spinal cord injury. Exp Neurol 209: 368–377.M. CouttsHS Keirstead2008Stem cells for the treatment of spinal cord injury.Exp Neurol209368377
- 6. Reier PJ (2004) Cellular transplantation strategies for spinal cord injury and translational neurobiology. NeuroRx 1: 424–451.PJ Reier2004Cellular transplantation strategies for spinal cord injury and translational neurobiology.NeuroRx1424451
- 7. Honmou O, Felts PA, Waxman SG, Kocsis JD (1996) Restoration of normal conduction properties in demyelinated spinal cord axons in the adult rat by transplantation of exogenous Schwann cells. Journal of Neuroscience 16: 3199–3208.O. HonmouPA FeltsSG WaxmanJD Kocsis1996Restoration of normal conduction properties in demyelinated spinal cord axons in the adult rat by transplantation of exogenous Schwann cells.Journal of Neuroscience1631993208
- 8. Martin D, Robe P, Franzen R, Delree P, Schoenen J, et al. (1996) Effects of Schwann cell transplantation in a contusion model of rat spinal cord injury. Journal of Neuroscience Research 45: 588–597.D. MartinP. RobeR. FranzenP. DelreeJ. Schoenen1996Effects of Schwann cell transplantation in a contusion model of rat spinal cord injury.Journal of Neuroscience Research45588597
- 9. Li BC, Li Y, Chen LF, Chang JY, Duan ZX (2011) Olfactory ensheathing cells can reduce the tissue loss but not the cavity formation in contused spinal cord of rats. Journal of the Neurological Sciences 303: 67–74.BC LiY. LiLF ChenJY ChangZX Duan2011Olfactory ensheathing cells can reduce the tissue loss but not the cavity formation in contused spinal cord of rats.Journal of the Neurological Sciences3036774
- 10. Raisman G (2007) Repair of spinal cord injury by transplantation of olfactory ensheathing cells. C R Biol 330: 557–560.G. Raisman2007Repair of spinal cord injury by transplantation of olfactory ensheathing cells.C R Biol330557560
- 11. Schultz SS (2005) Adult stem cell application in spinal cord injury. Curr Drug Targets 6: 63–73.SS Schultz2005Adult stem cell application in spinal cord injury.Curr Drug Targets66373
- 12. Tarasenko YI, Gao J, Nie L, Johnson KM, Grady JJ, et al. (2007) Human fetal neural stem cells grafted into contusion-injured rat spinal cords improve behavior. Journal of Neuroscience Research 85: 47–57.YI TarasenkoJ. GaoL. NieKM JohnsonJJ Grady2007Human fetal neural stem cells grafted into contusion-injured rat spinal cords improve behavior.Journal of Neuroscience Research854757
- 13. Osaka M, Honmou O, Murakami T, Nonaka T, Houkin K, et al. (2010) Intravenous administration of mesenchymal stem cells derived from bone marrow after contusive spinal cord injury improves functional outcome. Brain Research 1343: 226–235.M. OsakaO. HonmouT. MurakamiT. NonakaK. Houkin2010Intravenous administration of mesenchymal stem cells derived from bone marrow after contusive spinal cord injury improves functional outcome.Brain Research1343226235
- 14. Chopp M, Zhang XH, Li Y, Wang L, Chen J, et al. (2000) Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation. Neuroreport 11: 3001–3005.M. ChoppXH ZhangY. LiL. WangJ. Chen2000Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation.Neuroreport1130013005
- 15. Franzen R, Martin D, Daloze A, Moonen G, Schoenen J (1999) Grafts of meningeal fibroblasts in adult rat spinal cord lesion promote axonal regrowth. Neuroreport 10: 1551–1556.R. FranzenD. MartinA. DalozeG. MoonenJ. Schoenen1999Grafts of meningeal fibroblasts in adult rat spinal cord lesion promote axonal regrowth.Neuroreport1015511556
- 16. Tobias CA, Shumsky JS, Shibata M, Tuszynski MH, Fischer I, et al. (2003) Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration. Experimental Neurology 184: 97–113.CA TobiasJS ShumskyM. ShibataMH TuszynskiI. Fischer2003Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration.Experimental Neurology18497113
- 17. Franzen R, Schoenen J, Leprince P, Joosten E, Moonen G, et al. (1998) Effects of macrophage transplantation in the injured adult rat spinal cord: A combined immunocytochemical and biochemical study. Journal of Neuroscience Research 51: 316–327.R. FranzenJ. SchoenenP. LeprinceE. JoostenG. Moonen1998Effects of macrophage transplantation in the injured adult rat spinal cord: A combined immunocytochemical and biochemical study.Journal of Neuroscience Research51316327
- 18. Barry FP, Murphy JM (2004) Mesenchymal stem cells: clinical applications and biological characterization. The International Journal of Biochemistry & Cell Biology 36: 568–584.FP BarryJM Murphy2004Mesenchymal stem cells: clinical applications and biological characterization.The International Journal of Biochemistry & Cell Biology36568584
- 19. Augello A, Kurth TB, De Bari C (2010) Mesenchymal stem cells: a perspective from in vitro cultures to in vivo migration and niches. Eur Cell Mater 20: 121–133.A. AugelloTB KurthC. De Bari2010Mesenchymal stem cells: a perspective from in vitro cultures to in vivo migration and niches.Eur Cell Mater20121133
- 20. Caplan AI, Dennis JE (2006) Mesenchymal stem cells as trophic mediators. Journal of Cellular Biochemistry 98: 1076–1084.AI CaplanJE Dennis2006Mesenchymal stem cells as trophic mediators.Journal of Cellular Biochemistry9810761084
- 21. da Silva Meirelles L, Fontes AM, Covas DT, Caplan AI (2009) Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine & Growth Factor Reviews 20: 419–427.L. da Silva MeirellesAM FontesDT CovasAI Caplan2009Mechanisms involved in the therapeutic properties of mesenchymal stem cells.Cytokine & Growth Factor Reviews20419427
- 22. Uccelli A, Benvenuto F, Laroni A, Giunti D (2011) Neuroprotective features of mesenchymal stem cells. Best Practice & Research Clinical Haematology 24: 59–64.A. UccelliF. BenvenutoA. LaroniD. Giunti2011Neuroprotective features of mesenchymal stem cells.Best Practice & Research Clinical Haematology245964
- 23. Poll D, Parekkadan B, Borel Rinkes IHM, Tilles AW, Yarmush ML (2008) Mesenchymal Stem Cell Therapy for Protection and Repair of Injured Vital Organs. Cellular and Molecular Bioengineering 1: 42–50.D. PollB. ParekkadanIHM Borel RinkesAW TillesML Yarmush2008Mesenchymal Stem Cell Therapy for Protection and Repair of Injured Vital Organs.Cellular and Molecular Bioengineering14250
- 24. Wislet-Gendebien S, Hans G, Leprince P, Rigo J-M, Moonen G, et al. (2005) Plasticity of Cultured Mesenchymal Stem Cells: Switch from Nestin-Positive to Excitable Neuron-Like Phenotype. Stem Cells 23: 392–402.S. Wislet-GendebienG. HansP. LeprinceJ-M RigoG. Moonen2005Plasticity of Cultured Mesenchymal Stem Cells: Switch from Nestin-Positive to Excitable Neuron-Like Phenotype.Stem Cells23392402
- 25. Krampera M, Marconi S, Pasini A, Galiè M, Rigotti G, et al. (2007) Induction of neural-like differentiation in human mesenchymal stem cells derived from bone marrow, fat, spleen and thymus. Bone 40: 382–390.M. KramperaS. MarconiA. PasiniM. GalièG. Rigotti2007Induction of neural-like differentiation in human mesenchymal stem cells derived from bone marrow, fat, spleen and thymus.Bone40382390
- 26. Jin K, Mao XO, Batteur S, Sun Y, Greenberg DA (2003) Induction of neuronal markers in bone marrow cells: differential effects of growth factors and patterns of intracellular expression. Exp Neurol 184: 78–89.K. JinXO MaoS. BatteurY. SunDA Greenberg2003Induction of neuronal markers in bone marrow cells: differential effects of growth factors and patterns of intracellular expression.Exp Neurol1847889
- 27. Greco SJ, Zhou C, Ye JH, Rameshwar P (2008) A method to generate human mesenchymal stem cell-derived neurons which express and are excited by multiple neurotransmitters. Biol Proced Online 10: 90–101.SJ GrecoC. ZhouJH YeP. Rameshwar2008A method to generate human mesenchymal stem cell-derived neurons which express and are excited by multiple neurotransmitters.Biol Proced Online1090101
- 28. Wang TT, Tio M, Lee W, Beerheide W, Udolph G (2007) Neural differentiation of mesenchymal-like stem cells from cord blood is mediated by PKA. Biochem Biophys Res Commun 357: 1021–1027.TT WangM. TioW. LeeW. BeerheideG. Udolph2007Neural differentiation of mesenchymal-like stem cells from cord blood is mediated by PKA.Biochem Biophys Res Commun35710211027
- 29. Xu H, Miki K, Ishibashi S, Inoue J, Sun L, et al. (2010) Transplantation of neuronal cells induced from human mesenchymal stem cells improves neurological functions after stroke without cell fusion. J Neurosci Res 88: 3598–3609.H. XuK. MikiS. IshibashiJ. InoueL. Sun2010Transplantation of neuronal cells induced from human mesenchymal stem cells improves neurological functions after stroke without cell fusion.J Neurosci Res8835983609
- 30. Kishk NA, Gabr H, Hamdy S, Afifi L, Abokresha N, et al. (2010) Case Control Series of Intrathecal Autologous Bone Marrow Mesenchymal Stem Cell Therapy for Chronic Spinal Cord Injury. Neurorehabilitation and Neural Repair 24: 702–708.NA KishkH. GabrS. HamdyL. AfifiN. Abokresha2010Case Control Series of Intrathecal Autologous Bone Marrow Mesenchymal Stem Cell Therapy for Chronic Spinal Cord Injury.Neurorehabilitation and Neural Repair24702708
- 31. Roussos I, Rodriguez M, Villán D, Ariza A, Rodriguez L, et al. (2005) Development of a Rat Model of Spinal Cord Injury and Cellular Transplantation. Transplantation Proceedings 37: 4127–4130.I. RoussosM. RodriguezD. VillánA. ArizaL. Rodriguez2005Development of a Rat Model of Spinal Cord Injury and Cellular Transplantation.Transplantation Proceedings3741274130
- 32. Ra JC, Shin IS, Kim SH, Kang SK, Kang BC, et al. (2011) Safety of Intravenous Infusion of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Animals and Humans. Stem Cells and Development. 110317082130071 p.JC RaIS ShinSH KimSK KangBC Kang2011Safety of Intravenous Infusion of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Animals and Humans.Stem Cells and Development110317082130071
- 33. Karussis D, Karageorgiou C, Vaknin-Dembinsky A, Gowda-Kurkalli B, Gomori JM, et al. (2010) Safety and Immunological Effects of Mesenchymal Stem Cell Transplantation in Patients With Multiple Sclerosis and Amyotrophic Lateral Sclerosis. Archives of Neurology 67: 1187–1194.D. KarussisC. KarageorgiouA. Vaknin-DembinskyB. Gowda-KurkalliJM Gomori2010Safety and Immunological Effects of Mesenchymal Stem Cell Transplantation in Patients With Multiple Sclerosis and Amyotrophic Lateral Sclerosis.Archives of Neurology6711871194
- 34. Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C, et al. (2002) Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 30: 783–791.N. QuiriciD. SoligoP. BossolascoF. ServidaC. Lumini2002Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies.Exp Hematol30783791
- 35. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147.MF PittengerAM MackaySC BeckRK JaiswalR. Douglas1999Multilineage potential of adult human mesenchymal stem cells.Science284143147
- 36. Wislet-Gendebien S, Laudet E, Neirinckx V, Alix P, Leprince P, et al. (2012) Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences. Cell Mol Life Sciences. S. Wislet-GendebienE. LaudetV. NeirinckxP. AlixP. Leprince2012Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences.Cell Mol Life Sciences(DOI: 10.1007/s00018-012-0937-1). (DOI: 10.1007/s00018-012-0937-1).
- 37. Tetzlaff W, Okon EB, Karimi-Abdolrezaee S, Hill CE, Sparling JS, et al. (2011) A systematic review of cellular transplantation therapies for spinal cord injury. J Neurotrauma 28: 1611–1682.W. TetzlaffEB OkonS. Karimi-AbdolrezaeeCE HillJS Sparling2011A systematic review of cellular transplantation therapies for spinal cord injury.J Neurotrauma2816111682
- 38. Urdzikova L, Jendelova P, Glogarova K, Burian M, Hajek M, et al. (2006) Transplantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats. J Neurotrauma 23: 1379–1391.L. UrdzikovaP. JendelovaK. GlogarovaM. BurianM. Hajek2006Transplantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats.J Neurotrauma2313791391
- 39. Li ZH, Liao W, Cui XL, Zhao Q, Liu M, et al. (2011) Intravenous transplantation of allogeneic bone marrow mesenchymal stem cells and its directional migration to the necrotic femoral head. Int J Med Sci 8: 74–83.ZH LiW. LiaoXL CuiQ. ZhaoM. Liu2011Intravenous transplantation of allogeneic bone marrow mesenchymal stem cells and its directional migration to the necrotic femoral head.Int J Med Sci87483
- 40. Lu D, Mahmood A, Wang L, Li Y, Lu M, et al. (2001) Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport 12: 559–563.D. LuA. MahmoodL. WangY. LiM. Lu2001Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome.Neuroreport12559563
- 41. Karp JM, Teo GSL (2009) Mesenchymal stem cell homing: the devil is in the details. Cell stem cell 4: 206–216.JM KarpGSL Teo2009Mesenchymal stem cell homing: the devil is in the details.Cell stem cell4206216
- 42. Takeuchi H, Natsume A, Wakabayashi T, Aoshima C, Shimato S, et al. (2007) Intravenously transplanted human neural stem cells migrate to the injured spinal cord in adult mice in an SDF-1- and HGF-dependent manner. Neurosci Lett 426: 69–74.H. TakeuchiA. NatsumeT. WakabayashiC. AoshimaS. Shimato2007Intravenously transplanted human neural stem cells migrate to the injured spinal cord in adult mice in an SDF-1- and HGF-dependent manner.Neurosci Lett4266974
- 43. Iihoshi S, Honmou O, Houkin K, Hashi K, Kocsis JD (2004) A therapeutic window for intravenous administration of autologous bone marrow after cerebral ischemia in adult rats. Brain Res 1007: 1–9.S. IihoshiO. HonmouK. HoukinK. HashiJD Kocsis2004A therapeutic window for intravenous administration of autologous bone marrow after cerebral ischemia in adult rats.Brain Res100719
- 44. Parr AM, Tator CH, Keating A (2007) Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplantation 40: 609–619.AM ParrCH TatorA. Keating2007Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury.Bone Marrow Transplantation40609619
- 45. Paul C, Samdani AF, Betz RR, Fischer I, Neuhuber B (2009) Grafting of human bone marrow stromal cells into spinal cord injury: a comparison of delivery methods. Spine (Phila Pa 1976) 34: 328–334.C. PaulAF SamdaniRR BetzI. FischerB. Neuhuber2009Grafting of human bone marrow stromal cells into spinal cord injury: a comparison of delivery methods.Spine (Phila Pa 1976)34328334
- 46. Takahashi Y, Tsuji O, Kumagai G, Hara CM, Okano HJ, et al. (2010) Comparative study of methods for administering neural stem/progenitor cells to treat spinal cord injury in mice. Cell Transplantation. Y. TakahashiO. TsujiG. KumagaiCM HaraHJ Okano2010Comparative study of methods for administering neural stem/progenitor cells to treat spinal cord injury in mice.Cell Transplantation
- 47. Sykova E, Homola A, Mazanec R, Lachmann H, Konradova SL, et al. (2006) Autologous bone marrow transplantation in patients with subacute and chronic spinal cord injury. Cell Transplant. 15: 675–687.E. SykovaA. HomolaR. MazanecH. LachmannSL Konradova2006Autologous bone marrow transplantation in patients with subacute and chronic spinal cord injury. Cell Transplant.15675687
- 48. Kordower JH, Isacson O, Emerich DF (1999) Cellular Delivery of Trophic Factors for the Treatment of Huntington's Disease: Is Neuroprotection Possible? Experimental Neurology 159: 4–20.JH KordowerO. IsacsonDF Emerich1999Cellular Delivery of Trophic Factors for the Treatment of Huntington's Disease: Is Neuroprotection Possible?Experimental Neurology159420
- 49. Cao Q, He Q, Wang Y, Cheng X, Howard RM, et al. (2010) Transplantation of ciliary neurotrophic factor-expressing adult oligodendrocyte precursor cells promotes remyelination and functional recovery after spinal cord injury. Journal of Neuroscience 30: 2989–3001.Q. CaoQ. HeY. WangX. ChengRM Howard2010Transplantation of ciliary neurotrophic factor-expressing adult oligodendrocyte precursor cells promotes remyelination and functional recovery after spinal cord injury.Journal of Neuroscience3029893001
- 50. Deshmane SL, Kremlev S, Amini S, Sawaya BE (2009) Monocyte chemoattractant protein-1 (MCP-1): an overview. Journal of Interferon and Cytokine Research 29: 313–326.SL DeshmaneS. KremlevS. AminiBE Sawaya2009Monocyte chemoattractant protein-1 (MCP-1): an overview.Journal of Interferon and Cytokine Research29313326
- 51. Ousman SS, David S (2001) MIP-1alpha, MCP-1, GM-CSF, and TNF-alpha control the immune cell response that mediates rapid phagocytosis of myelin from the adult mouse spinal cord. Journal of Neuroscience 21: 4649–4656.SS OusmanS. David2001MIP-1alpha, MCP-1, GM-CSF, and TNF-alpha control the immune cell response that mediates rapid phagocytosis of myelin from the adult mouse spinal cord.Journal of Neuroscience2146494656
- 52. Huang X, Choi J-K, Park SR, Ha Y, Park H, et al. (2007) GM-CSF inhibits apoptosis of neural cells via regulating the expression of apoptosis-related proteins. Neuroscience Research 58: 50–57.X. HuangJ-K ChoiSR ParkY. HaH. Park2007GM-CSF inhibits apoptosis of neural cells via regulating the expression of apoptosis-related proteins.Neuroscience Research585057
- 53. Huang X, Kim J-M, Kong TH, Park SR, Ha Y, et al. (2009) GM-CSF inhibits glial scar formation and shows long-term protective effect after spinal cord injury. Journal of the Neurological Sciences 277: 87–97.X. HuangJ-M KimTH KongSR ParkY. Ha2009GM-CSF inhibits glial scar formation and shows long-term protective effect after spinal cord injury.Journal of the Neurological Sciences2778797
- 54. Bouhy D, Malgrange B, Multon S, Poirrier AL, Scholtes F, et al. (2006) Delayed GM-CSF treatment stimulates axonal regeneration and functional recovery in paraplegic rats via an increased BDNF expression by endogenous macrophages. FASEB Journal 20: 1239–1241.D. BouhyB. MalgrangeS. MultonAL PoirrierF. Scholtes2006Delayed GM-CSF treatment stimulates axonal regeneration and functional recovery in paraplegic rats via an increased BDNF expression by endogenous macrophages.FASEB Journal2012391241
- 55. Luo J, Zhang HT, Jiang XD, Xue S, Ke YQ (2009) Combination of bone marrow stromal cell transplantation with mobilization by granulocyte-colony stimulating factor promotes functional recovery after spinal cord transection. Acta Neurochir (Wien) 151: 1483–1492.J. LuoHT ZhangXD JiangS. XueYQ Ke2009Combination of bone marrow stromal cell transplantation with mobilization by granulocyte-colony stimulating factor promotes functional recovery after spinal cord transection.Acta Neurochir (Wien)15114831492
- 56. Mahmood A, Lu D, Chopp M (2004) Intravenous administration of marrow stromal cells (MSCs) increases the expression of growth factors in rat brain after traumatic brain injury. J Neurotrauma 21: 33–39.A. MahmoodD. LuM. Chopp2004Intravenous administration of marrow stromal cells (MSCs) increases the expression of growth factors in rat brain after traumatic brain injury.J Neurotrauma213339
- 57. Sofroniew MV, Howe CL, Mobley WC (2001) Nerve growth factor signaling, neuroprotection, and neural repair. Annual Review of Neuroscience 24: 1217–1281.MV SofroniewCL HoweWC Mobley2001Nerve growth factor signaling, neuroprotection, and neural repair.Annual Review of Neuroscience2412171281
- 58. Pettigrew DB, Li YQ, Kuntz Ct, Crutcher KA (2007) Global expression of NGF promotes sympathetic axonal growth in CNS white matter but does not alter its parallel orientation. Experimental Neurology 203: 95–109.DB PettigrewYQ LiCt KuntzKA Crutcher2007Global expression of NGF promotes sympathetic axonal growth in CNS white matter but does not alter its parallel orientation.Experimental Neurology20395109
- 59. Nguyen TL, Kim CK, Cho JH, Lee KH, Ahn JY (2010) Neuroprotection signaling pathway of nerve growth factor and brain-derived neurotrophic factor against staurosporine induced apoptosis in hippocampal H19-7/IGF-IR. Experimental and Molecular Medicine 42: 583–595.TL NguyenCK KimJH ChoKH LeeJY Ahn2010Neuroprotection signaling pathway of nerve growth factor and brain-derived neurotrophic factor against staurosporine induced apoptosis in hippocampal H19-7/IGF-IR.Experimental and Molecular Medicine42583595
- 60. Munoz JR, Stoutenger BR, Robinson AP, Spees JL, Prockop DJ (2005) Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proceedings of the National Academy of Sciences of the United States of America 102: 18171–18176.JR MunozBR StoutengerAP RobinsonJL SpeesDJ Prockop2005Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice.Proceedings of the National Academy of Sciences of the United States of America1021817118176
- 61. Susaki Y, Shimizu S, Katakura K, Watanabe N, Kawamoto K, et al. (1996) Functional properties of murine macrophages promoted by nerve growth factor. Blood 88: 4630–4637.Y. SusakiS. ShimizuK. KatakuraN. WatanabeK. Kawamoto1996Functional properties of murine macrophages promoted by nerve growth factor.Blood8846304637
- 62. Liuzzo JP, Petanceska SS, Devi LA (1999) Neurotrophic factors regulate cathepsin S in macrophages and microglia: A role in the degradation of myelin basic protein and amyloid beta peptide. Molecular Medicine 5: 334–343.JP LiuzzoSS PetanceskaLA Devi1999Neurotrophic factors regulate cathepsin S in macrophages and microglia: A role in the degradation of myelin basic protein and amyloid beta peptide.Molecular Medicine5334343
- 63. Rabchevsky AG, Streit WJ (1998) Role of microglia in postinjury repair and regeneration of the CNS. Mental Retardation and Developmental Disabilities Research Reviews 4: 187–192.AG RabchevskyWJ Streit1998Role of microglia in postinjury repair and regeneration of the CNS.Mental Retardation and Developmental Disabilities Research Reviews4187192
- 64. Crigler L, Robey RC, Asawachaicharn A, Gaupp D, Phinney DG (2006) Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis. Experimental Neurology 198: 54–64.L. CriglerRC RobeyA. AsawachaicharnD. GauppDG Phinney2006Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis.Experimental Neurology1985464
- 65. Kurozumi K, Nakamura K, Tamiya T, Kawano Y, Ishii K, et al. (2005) Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model. Mol Ther 11: 96–104.K. KurozumiK. NakamuraT. TamiyaY. KawanoK. Ishii2005Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model.Mol Ther1196104
- 66. Sasaki M, Radtke C, Tan AM, Zhao P, Hamada H, et al. (2009) BDNF-hypersecreting human mesenchymal stem cells promote functional recovery, axonal sprouting, and protection of corticospinal neurons after spinal cord injury. Journal of Neuroscience 29: 14932–14941.M. SasakiC. RadtkeAM TanP. ZhaoH. Hamada2009BDNF-hypersecreting human mesenchymal stem cells promote functional recovery, axonal sprouting, and protection of corticospinal neurons after spinal cord injury.Journal of Neuroscience291493214941
- 67. Jain A, McKeon RJ, Brady-Kalnay SM, Bellamkonda RV (2011) Sustained delivery of activated Rho GTPases and BDNF promotes axon growth in CSPG-rich regions following spinal cord injury. PLoS One 6: e16135.A. JainRJ McKeonSM Brady-KalnayRV Bellamkonda2011Sustained delivery of activated Rho GTPases and BDNF promotes axon growth in CSPG-rich regions following spinal cord injury.PLoS One6e16135
- 68. Ohta M, Suzuki Y, Noda T, Ejiri Y, Dezawa M, et al. (2004) Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation. Experimental Neurology 187: 266–278.M. OhtaY. SuzukiT. NodaY. EjiriM. Dezawa2004Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation.Experimental Neurology187266278
- 69. Zurita M, Vaquero J (2004) Functional recovery in chronic paraplegia after bone marrow stromal cells transplantation. Neuroreport 15: 1105–1108.M. ZuritaJ. Vaquero2004Functional recovery in chronic paraplegia after bone marrow stromal cells transplantation.Neuroreport1511051108
- 70. Nandoe Tewarie RD, Hurtado A, Ritfeld GJ, Rahiem ST, Wendell DF, et al. (2009) Bone marrow stromal cells elicit tissue sparing after acute but not delayed transplantation into the contused adult rat thoracic spinal cord. Journal of Neurotrauma 26: 2313–2322.RD Nandoe TewarieA. HurtadoGJ RitfeldST RahiemDF Wendell2009Bone marrow stromal cells elicit tissue sparing after acute but not delayed transplantation into the contused adult rat thoracic spinal cord.Journal of Neurotrauma2623132322
- 71. Samdani AF, Paul C, Betz RR, Fischer I, Neuhuber B (2009) Transplantation of human marrow stromal cells and mono-nuclear bone marrow cells into the injured spinal cord: a comparative study. Spine (Phila Pa 1976) 34: 2605–2612.AF SamdaniC. PaulRR BetzI. FischerB. Neuhuber2009Transplantation of human marrow stromal cells and mono-nuclear bone marrow cells into the injured spinal cord: a comparative study.Spine (Phila Pa 1976)3426052612
- 72. Potapova IA, Gaudette GR, Brink PR, Robinson RB, Rosen MR, et al. (2007) Mesenchymal Stem Cells Support Migration, Extracellular Matrix Invasion, Proliferation, and Survival of Endothelial Cells In Vitro. Stem Cells 25: 1761–1768.IA PotapovaGR GaudettePR BrinkRB RobinsonMR Rosen2007Mesenchymal Stem Cells Support Migration, Extracellular Matrix Invasion, Proliferation, and Survival of Endothelial Cells In Vitro.Stem Cells2517611768
- 73. Lutton C, Young YW, Williams R, Meedeniya AC, Mackay-Sim A, et al. (2011) Combined VEGF and PDGF Treatment Reduces Secondary Degeneration after Spinal Cord Injury. J Neurotrauma. C. LuttonYW YoungR. WilliamsAC MeedeniyaA. Mackay-Sim2011Combined VEGF and PDGF Treatment Reduces Secondary Degeneration after Spinal Cord Injury.J Neurotrauma
- 74. Sundberg LM, Herrera JJ, Narayana PA (2011) Effect of vascular endothelial growth factor treatment in experimental traumatic spinal cord injury: in vivo longitudinal assessment. J Neurotrauma 28: 565–578.LM SundbergJJ HerreraPA Narayana2011Effect of vascular endothelial growth factor treatment in experimental traumatic spinal cord injury: in vivo longitudinal assessment.J Neurotrauma28565578
- 75. Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, et al. (2004) Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circulation Research 94: 678–685.T. KinnairdE. StabileMS BurnettCW LeeS. Barr2004Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms.Circulation Research94678685
- 76. Sinescu C, Popa F, Grigorean VT, Onose G, Sandu AM, et al. (2010) Molecular basis of vascular events following spinal cord injury. J Med Life 3: 254–261.C. SinescuF. PopaVT GrigoreanG. OnoseAM Sandu2010Molecular basis of vascular events following spinal cord injury.J Med Life3254261
- 77. Mautes AE, Weinzierl MR, Donovan F, Noble LJ (2000) Vascular events after spinal cord injury: contribution to secondary pathogenesis. Physical Therapy 80: 673–687.AE MautesMR WeinzierlF. DonovanLJ Noble2000Vascular events after spinal cord injury: contribution to secondary pathogenesis.Physical Therapy80673687
- 78. Neuhuber B, Swanger S, Howard L, Mackay A, Fischer I (2008) Effects of plating density and culture time on bone marrow stromal cell characteristics. Experimental Hematology 36: 1176–1185.B. NeuhuberS. SwangerL. HowardA. MackayI. Fischer2008Effects of plating density and culture time on bone marrow stromal cell characteristics.Experimental Hematology3611761185
- 79. Jansen BJ, Gilissen C, Roelofs H, Schaap-Oziemlak A, Veltman JA, et al. (2010) Functional differences between mesenchymal stem cell populations are reflected by their transcriptome. Stem Cells Dev 19: 481–490.BJ JansenC. GilissenH. RoelofsA. Schaap-OziemlakJA Veltman2010Functional differences between mesenchymal stem cell populations are reflected by their transcriptome.Stem Cells Dev19481490
- 80. Bakondi B, Shimada IS, Perry A, Munoz JR, Ylostalo J, et al. (2009) CD133 Identifies a Human Bone Marrow Stem/Progenitor Cell Sub-population With a Repertoire of Secreted Factors That Protect Against Stroke. Mol Ther 17: 1938–1947.B. BakondiIS ShimadaA. PerryJR MunozJ. Ylostalo2009CD133 Identifies a Human Bone Marrow Stem/Progenitor Cell Sub-population With a Repertoire of Secreted Factors That Protect Against Stroke.Mol Ther1719381947
- 81. Morikawa S, Mabuchi Y, Niibe K, Suzuki S, Nagoshi N, et al. (2009) Development of mesenchymal stem cells partially originate from the neural crest. Biochem Biophys Res Commun 379: 1114–1119.S. MorikawaY. MabuchiK. NiibeS. SuzukiN. Nagoshi2009Development of mesenchymal stem cells partially originate from the neural crest.Biochem Biophys Res Commun37911141119
- 82. Nagoshi N, Shibata S, Kubota Y, Nakamura M, Nagai Y, et al. (2008) Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem Cell 2: 392–403.N. NagoshiS. ShibataY. KubotaM. NakamuraY. Nagai2008Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad.Cell Stem Cell2392403
- 83. Sauerzweig S, Munsch T, Lemann V, Reymann KG, Braun H (2009) A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells. Experimental Cell Research 315: 50–66.S. SauerzweigT. MunschV. LemannKG ReymannH. Braun2009A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells.Experimental Cell Research3155066
- 84. Wislet-Gendebien S, Leprince P, Moonen G, Rogister B (2003) Regulation of neural markers nestin and GFAP expression by cultivated bone marrow stromal cells. Journal of Cell Science 116: 3295–3302.S. Wislet-GendebienP. LeprinceG. MoonenB. Rogister2003Regulation of neural markers nestin and GFAP expression by cultivated bone marrow stromal cells.Journal of Cell Science11632953302
- 85. Grayson W, Zhao F, Bunnell B, Ma T (2007) Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells☆. Biochemical and Biophysical Research Communications 358: 948–953.W. GraysonF. ZhaoB. BunnellT. Ma2007Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells☆.Biochemical and Biophysical Research Communications358948953
- 86. Neuhuber B, Timothy Himes B, Shumsky JS, Gallo G, Fischer I (2005) Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations. Brain Research 1035: 73–85.B. NeuhuberB. Timothy HimesJS ShumskyG. GalloI. Fischer2005Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations.Brain Research10357385
- 87. Siddappa R, Licht R, van Blitterswijk C, de Boer J (2007) Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering. Journal of Orthopaedic Research 25: 1029–1041.R. SiddappaR. LichtC. van BlitterswijkJ. de Boer2007Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering.Journal of Orthopaedic Research2510291041
- 88. Tator CH, Fehlings MG (1991) Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. Journal of Neurosurgery 75: 15–26.CH TatorMG Fehlings1991Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms.Journal of Neurosurgery751526
- 89. Vanicky I, Urdzikova L, Saganova K, Cizkova D, Galik J (2001) A simple and reproducible model of spinal cord injury induced by epidural balloon inflation in the rat. J Neurotrauma 18: 1399–1407.I. VanickyL. UrdzikovaK. SaganovaD. CizkovaJ. Galik2001A simple and reproducible model of spinal cord injury induced by epidural balloon inflation in the rat.J Neurotrauma1813991407
- 90. Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. Journal of Neurotrauma 12: 1–21.DM BassoMS BeattieJC Bresnahan1995A sensitive and reliable locomotor rating scale for open field testing in rats.Journal of Neurotrauma12121