Transplantation of Schwann cells and/or olfactory ensheathing glia into the contused spinal cord: Survival, migration, axon association, and functional recovery

Schwann cells (SCs) and olfactory ensheathing glia (OEG) have shown promise for spinal cord injury repair. We sought their in vivo identification following transplantation into the contused adult rat spinal cord at 1 week post-injury by: (i) DNA in situ hybridization (ISH) with a Y-chromosome specific probe to identify male transplants in female rats and (ii) lentiviral vector-mediated expression of EGFP. Survival, migration, and axon-glia association were quantified from 3 days to 9 weeks post-transplantation. At 3 weeks after transplantation into the lesion, a 60-90% loss of grafted cells was observed. OEG-only grafts survived very poorly within the lesion (<5%); injection outside the lesion led to a 60% survival rate, implying that the injury milieu was hostile to transplanted cells and or prevented their proliferation. At later times post-grafting, p75(+)/EGFP(-) cells in the lesion outnumbered EGFP(+) cells in all paradigms, evidence of significant host SC infiltration. SCs and OEG injected into the injury failed to migrate from the lesion. Injection of OEG outside of the injury resulted in their migration into the SC-injected injury site, not via normal-appearing host tissue but along the pia or via the central canal. In all paradigms, host axons were seen in association with or ensheathed by transplanted glia. Numerous myelinated axons were found within regions of grafted SCs but not OEG. The current study details the temporal survival, migration, axon association of SCs and OEG, and functional recovery after grafting into the contused spinal cord, research previously complicated due to a lack of quality, long-term markers for cell tracking in vivo.     

Promoting survival, migration, and integration of transplanted Schwann cells by over-expressing polysialic acid

The poor survival and migration of transplanted Schwann cells (SCs) are major drawbacks for their clinical application in cell therapy for neurotrauma. To overcome such drawbacks we genetically modified SCs to over-express polysialic acid (PSA) by lentiviral delivery of polysialyltransferase (PST) to study whether over-expression of PSA could enhance their survival, migration, and integration when transplanted into the spinal cord. It was found that more PSA-expressing SCs (PST/SCs) survived than GFP-expressing SCs (GFP/SCs) after transplantation, although cell loss was still quite significant. PSA expression did not enhance the motility of transplanted SCs in uninjured spinal cord. However, in a spinal cord crush injury model PST/SCs transplanted caudal to the lesion showed that increased number of PST/SCs migrated to the injury site compared with that of GFP/SCs. Induced expression of PSA in spinal cord can further facilitate the infiltration of PST/SCs into the lesion site. PST/SCs were also shown to intermingle well with host spinal cells while GFP/SCs formed boundaries with host tissue. This was confirmed by an in vitro confrontation assay showing that more PST/SCs crossed over to astrocyte territory than GFP/SCs. Furthermore, PST/SCs induced much less expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycan in the surrounding tissues than GFP/SCs, indicating that expression of PSA on SCs do not cause significant stress response of astrocytes. These results demonstrate that expression of PSA on SCs significantly changes their biological properties and makes them more feasible for neural repair after neurotrauma.     

BD PuraMatrix peptide hydrogel as a culture system for human fetal Schwann cells in spinal cord regeneration

BD PuraMatrix peptide hydrogel, a three‐dimensional cell culture model of nanofiber scaffold derived from the self‐assembling peptide RADA16, has been applied to regenerative tissue repair in order to develop novel nanomedicine systems. In this study with PuraMatrix, self‐assembling nanofiber scaffold (SAPNS) and Schwann cells (SCs) were isolated from human fetal sciatic nerves, cultured within SAPNS, and then transplanted into the spinal cord after injury (SCI) in rats. First, the peptide nanofiber scaffold was evaluated via scanning electron microscopy and atomic force microscopy. With phase‐contrast microscopy, the appearance of representative human fetal SCs encapsulated in PuraMatrix on days 3, 5, and 7 in 12‐well plates was revealed. The Schwann cells in PuraMatrix were cultured for 2 days, and the SCs had active proliferative potential. Spinal cord injury was induced by placing a 35‐g weight on the dura of T9–T10 segments for 15 min, followed by in vivo treatment with SAPNS and human fetal SCs (100,000 cells/10 μl/injection) grafted into spinal cord 7 days after SCI. After treatment, the recovery of motor function was assessed periodically using the Basso, Beattie, and Bresnahan scoring system. Eight weeks after grafting, animals were perfusion fixed, and the survival of implanted cells was analyzed with antibody recognizing SCs. Immunohistochemical analysis of grafted lumber segments at 8 weeks after grafting revealed reduced asterogliosis and considerably increased infiltration of endogenous S100⁺ cells into the injury site, suggesting that PuraMatrix may play an important role in the repair observed after SAPNS and human fetal SC transplantation. © 2012 Wiley Periodicals, Inc.     

Human Schwann cells exhibit long‐term cell survival,are not tumorigenic and promote repair when transplanted into the contused spinal cord

The transplantation of rodent Schwann cells (SCs) provides anatomical and functional restitution in a variety of spinal cord injury (SCI) models, supporting the recent translation of SCs to phase 1 clinical trials for human SCI. Whereas human (Hu)SCs have been examined experimentally in a complete SCI transection paradigm, to date the reported behavior of SCs when transplanted after a clinically relevant contusive SCI has been restricted to the use of rodent SCs. Here, in a xenotransplant, contusive SCI paradigm, the survival, biodistribution, proliferation and tumorgenicity as well as host responses to HuSCs, cultured according to a protocol analogous to that developed for clinical application, were investigated. HuSCs persisted within the contused nude rat spinal cord through 6 months after transplantation (longest time examined), exhibited low cell proliferation, displayed no evidence of tumorigenicity and showed a restricted biodistribution to the lesion. Neuropathological examination of the CNS revealed no adverse effects of HuSCs. Animals exhibiting higher numbers of surviving HuSCs within the lesion showed greater volumes of preserved white matter and host rat SC and astrocyte ingress as well as axon ingrowth and myelination. These results demonstrate the safety of HuSCs when employed in a clinically relevant experimental SCI paradigm. Further, signs of a potentially positive influence of HuSC transplants on host tissue pathology were observed. These findings show that HuSCs exhibit a favorable toxicity profile for up to 6 months after transplantation into the contused rat spinal cord, an important outcome for FDA consideration of their use in human clinical trials.     

Olfactory ensheathing cells exhibit unique migratory, phagocytic, and myelinating properties in the X-irradiated spinal cord not shared by Schwann cells

Although several studies have shown that Schwann cells (SCs) and olfactory ensheathing cells (OECs) interact differently with central nervous system (CNS) cells in vitro, all classes of adult myelin-forming cells show poor survival and migration after transplantation into normal CNS. X-irradiation of the spinal cord, however, selectively facilitates migration of oligodendrocyte progenitor cells (OPCs), but not SCs, revealing differences in in vivo migratory capabilities that are not apparent in intact tissue. To compare the in vivo migratory properties of OECs and SCs and evaluate the potential of migrating cells to participate in subsequent repair, we first transplanted freshly isolated GFP-expressing adult rat olfactory bulb-derived OECs and SCs into normal and X-irradiated spinal cords. Both OECs and SCs showed limited survival and migration in normal spinal cord at 3 weeks. However, OECs, unlike SCs, migrated extensively in both grey and white matter of the X-irradiated spinal cord, and exhibited a phagocytic phenotype with OX-42 staining on their processes. If a X-irradiated and OEC transplanted spinal cord was then subjected to a focal demyelinating lesion 3 weeks after transplantation, OECs moved into the delayed demyelinated lesion and remyelinated host axons with a peripheral-like pattern of myelin. These results revealed a clear difference between the migratory properties of OECs and SCs in the X-irradiated spinal cord and demonstrated that engrafted OECs can participate in repair of subsequent lesions.     

Proteomic evaluation reveals that olfactory ensheathing cells but not Schwann cells express calponin

Human clinical trials have begun worldwide that use olfactory ensheathing cells (OECs) to ameliorate the functional deficits following spinal cord injury. These trials have been initiated largely because numerous studies have reported that OECs transform into Schwann Cell (SC)-like cells that myelinate axons and support new growth in adult rats with spinal injury. This phenomenon is remarkable because OECs do not myelinate olfactory axons in their native environment. Furthermore, these myelinating OECs are morphologically identical to SCs, which can invade the spinal cord after injury. One factor that has contributed to a possible confusion in the identification of these cells is the lack of phenotypic markers to distinguish unequivocally between OECs and SCs. Such markers are required to first assess the degree of SC contamination in OEC cultures before intraspinal implantation, and then to accurately identify grafted OECs and invading SCs in the injured spinal cord. Using two-dimensional gel electrophoresis, we have identified calponin, an actin binding protein, as the first definitive phenotypic marker that distinguishes between OECs and SCs in vitro and in vivo. We have also provided ultrastructural evidence that calponin-immunopositive OECs do not transform into myelinating SC-like cells after intraspinal implantation. Rather, the grafted OECs retain their morphological and neurochemical features. These data yield new insight into the phenotypic characteristics of OECs, which together with invading SCs can enhance regeneration of the injured spinal cord.     

Co-transplantation of Schwann cells and bone marrow stromal cells versus single cell transplantation on repairing hemisected spinal cord injury of rats

BACKGROUND: Bone marrow stromal cells (BMSCs) or Schwann cells (SCs) transplantation alone can treat spinal cord injury. However, the transplantation either cell-type alone has disadvantages. The co-transplantation of both cells may benefit structural reconstruction and functional recovery of spinal nerves.OBJECTIVE: To verify spinal cord repair and related mechanisms after co-transplantation of BMSCs and SCs in a rat model of hemisected spinal cord injury.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Department of Histology and Embryology, Mudanjiang Medical College from January 2008 to May 2009.MATERIALS: Rabbit anti-S-100, glial fibrillary acidic protein, neuron specific enolase and neurofilament-200 monoclonal antibodies were purchased from Sigma, USA.METHODS: A total of 100 Wistar rats were used in a model of hemisected spinal cord injury. The rats were randomly assigned to vehicle control, SCs transplantation, BMSCs transplantation, and co-transplantation groups; 25 rats per group. At 1 week after modeling, SCs or BMSCs cultured in vitro were labeled and injected separately into the hemisected spinal segment of SCs and BMSCs transplantation groups through three injection points [5 μL (1 × 107 cells/mL)] cell suspension in each point). In addition, a 15 μL 1 × 107 cells/mL SCs suspension and a 15 μL 1 × 107 cells/mL BMSC suspension were injected into co-transplantation group by the above method.MAIN OUTCOME MEASURES: The Basso-Beattie-Bresnahan (BBB) locomotor rating scale and somatosensory evoked potential (SEP) tests were used to assess the functional recovery of rat hind limbs following operation. Structural repair of injured nerve tissue was observed by light microscopy, electron microscopy, immunohistochemistry, and magnetic resonance imaging (MRI). In vivo differentiation, survival and migration of BMSCs were evaluated by immunofluorescence.RESULTS: BBB scores were significantly greater in all three transplantation groups compared with vehicle control group 8 weeks after transplantation. In particular, the co-transplantation group displayed the highest scores among the groups (P < 0.05). Moreover, recovery of SEP latency and amplitude was observed in all the transplantation groups, particularly after 8 weeks. Again, the co-transplantation group exhibited the greatest improvement (P < 0.05). In the co-transplantation group, imaging showed a smooth surface and intact inner structure at the injury site, with no scar formation, and a large number of orderly cells at the injured site. Axonal regeneration, new myelination, and a large amount of cell division were detected in the co-transplantation group by electron microscopy. Neuron specific enolase (NSE)- and glial fibrillary acidic protein (GFAP)-positive cells were observed in the spinal cord sections 1 week following co-transplantation by immunofluorescence staining.CONCLUSION: Co-transplantation of SCs and BMSCs effectively promoted functional recovery of injured spinal cord in rats compared with SCs or BMSCs transplantation alone. This repair effect is probably achieved because of neuronal-like cells derived from BMSCs to supplement dead neurons in vivo.     

Sulfatase‐mediated manipulation of the astrocyte‐Schwann cell interface

Schwann cell (SC) transplantation following spinal cord injury (SCI) may have therapeutic potential. Functional recovery is limited however, due to poor SC interactions with host astrocytes and the induction of astrogliosis. Olfactory ensheathing cells (OECs) are closely related to SCs, but intermix more readily with astrocytes in culture and induce less astrogliosis. We previously demonstrated that OECs express higher levels of sulfatases, enzymes that remove 6‐O‐sulfate groups from heparan sulphate proteoglycans, than SCs and that RNAi knockdown of sulfatase prevented OEC‐astrocyte mixing in vitro. As human OECs are difficult to culture in large numbers we have genetically engineered SCs using lentiviral vectors to express sulfatase 1 and 2 (SC‐S1S2) and assessed their ability to interact with astrocytes. We demonstrate that SC‐S1S2s have increased integrin‐dependent motility in the presence of astrocytes via modulation of NRG and FGF receptor‐linked PI3K/AKT intracellular signaling and do not form boundaries with astrocytes in culture. SC‐astrocyte mixing is dependent on local NRG concentration and we propose that sulfatase enzymes influence the bioavailability of NRG ligand and thus influence SC behavior. We further demonstrate that injection of sulfatase expressing SCs into spinal cord white matter results in less glial reactivity than control SC injections comparable to that of OEC injections. Our data indicate that sulfatase‐mediated modification of the extracellular matrix can influence glial interactions with astrocytes, and that SCs engineered to express sulfatase may be more OEC‐like in character. This approach may be beneficial for cell transplant‐mediated spinal cord repair. GLIA 2016 GLIA 2017;65:19–33     

Early necrosis and apoptosis of Schwann cells transplanted into the injured rat spinal cord

Poor survival of cells transplanted into the CNS is a widespread problem and limits their therapeutic potential. Whereas substantial loss of transplanted cells has been described, the extent of acute cell loss has not been quantified previously. To assess the extent and temporal profile of transplanted cell death, and the contributions of necrosis and apoptosis to this cell death following spinal cord injury, different concentrations of Schwann cells (SCs), lentivirally transduced to express green fluorescent protein (GFP), were transplanted into a 1-week-old moderate contusion of the adult rat thoracic spinal cord. In all cases, transplanted cells were present from 10 min to 28 days. There was a 78% reduction in SC number within the first week, with no significant decrease thereafter. Real-time polymerase chain reaction showed a similar 80% reduction in GFP-DNA within the first week, confirming that the decrease in SC number was due to death rather than decreased GFP transgene expression. Cells undergoing necrosis and apoptosis were identified using antibodies against the calpain-mediated fodrin breakdown product and activated caspase 3, respectively, as well as ultrastructurally. Six times more SCs died during the first week after transplantation by necrosis than apoptosis, with the majority of cell death occurring within the first 24 h. The early death of transplanted SCs indicates that factors present, even 1 week after a moderate contusion, are capable of inducing substantial transplanted cell death. Intervention by strategies that limit necrosis and/or apoptosis should be considered for enhancing acute survival of transplanted cells.     

Schwann cells engineered to express the cell adhesion molecule L1 accelerate myelination and motor recovery after spinal cord injury

Functional recovery after spinal cord lesion remains an important goal. A combination of inhibitory molecules and lack of appropriate permissive factors in the lesioned spinal cord results in failure of fiber tract reconnection and function. Experimental transplantation in rodent and primate models of CNS injuries has led to the idea that Schwann cells (SCs) are promising candidates for autologous transplantation to assist myelination of lesions and to deliver therapeutic agents in the CNS. In this study, we used retroviral transduction to genetically modify SCs from transgenic GFP-mice in order to overexpress the cell adhesion molecule L1, a protein promoting neurite outgrowth and implicated in myelination. SCs transduced to express L1 or its chimeric secreted form L1-Fc were mixed and grafted rostrally to the lesion site of adult mice immediately after spinal cord compression injury. Our results indicate that 3 weeks postoperatively, but not thereafter, mice transplanted with L1/L1-Fc-expressing SCs exhibited faster locomotor recovery as compared to animals which received SCs transduced with a control vector or no cells at all. Morphological analysis indicated that the accelerated functional recovery correlated with earlier and enhanced myelination by both grafted and host SCs. Moreover, increased sprouting of serotonergic fibers into and across the lesion site was observed in the L1/L1-Fc group as compared with controls. Our results suggest that transplantation of L1-overexpressing SCs enhances early events in spinal cord repair after injury and may be considered in combinatorial strategies together with other regeneration-promoting molecules.     

Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 ( 1 ). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 10⁶ cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DβH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.     

Transduced Schwann cells promote axon growth and myelination after spinal cord injury

We sought to directly compare growth and myelination of local and supraspinal axons by implanting into the injured spinal cord Schwann cells (SCs) transduced ex vivo with adenoviral (AdV) or lentiviral (LV) vectors encoding a bifunctional neurotrophin molecule (D15A). D15A mimics actions of both neurotrophin-3 and brain-derived neurotrophic factor. Transduced SCs were injected into the injury center 1 week after a moderate thoracic (T8) adult rat spinal cord contusion. D15A expression and bioactivity in vitro; D15A levels in vivo; and graft volume, SC number, implant axon number and cortico-, reticulo-, raphe-, coerulo-spinal and sensory axon growth were determined for both types of vectors employed to transduce SCs. ELISAs revealed that D15A-secreting SC implants contained significantly higher levels of neurotrophin than non-transduced SC and AdV/GFP and LV/GFP SC controls early after implantation. At 6 weeks post-implantation, D15A-secreting SC grafts exhibited 5-fold increases in graft volume, SC number and myelinated axon counts and a 3-fold increase in myelinated to unmyelinated (ensheathed) axon ratios. The total number of axons within grafts of LV/GFP/D15A SCs was estimated to be over 70,000. Also 5-HT, DbetaH, and CGRP axon length was increased up to 5-fold within D15A grafts. In sum, despite qualitative differences using the two vectors, increased neurotrophin secretion by the implanted D15A SCs led to the presence of a significantly increased number of axons in the contusion site. These results demonstrate the therapeutic potential for utilizing neurotrophin-transduced SCs to repair the injured spinal cord.     

神经干细胞与许旺细胞共移植于大鼠损伤脊髓

目的 观察神经干细胞与许旺细胞共移植于大鼠半横断脊髓损伤处神经干细胞的迁移、存活、分化及对损伤脊髓的修复作用.方法 绿色荧光蛋白(GFP)标记脊髓神经下细胞后与许旺细胞共移植于大鼠半横断脊髓损伤处,免疫荧光染色和电镜技术分别观察神经下细胞的迁移、存活、分化及新生的髓鞘.皮层运动诱发电位(CMEPs)及BBB评分分别检测大鼠运动功能的恢复.结果 在神经干细胞与许旺细胞共移植组,损伤脊髓的头端、尾端及对侧町见明显的GFP阳性细胞及GaLC/GFP、GFAP/GFP、NSE/GFP、SYN/GFP舣阳性细胞,电镜下新生的髓鞘最多,CMEPs恢复百分率和振幅明显高于其他两组,但BBB评分与神经干细胞单移植组差异无统计学意义.结论 神经干细胞和许旺细胞体内共移植可促进神经干细胞的辽移、存活、分化及脊髓运动功能的恢复.     

Extensive cell migration, axon regeneration, and improved function with polysialic acid-modified Schwann cells after spinal cord injury

Schwann cell (SC) implantation after spinal cord injury (SCI) promotes axonal regeneration, remyelination repair, and functional recovery. Reparative efficacy, however, may be limited because of the inability of SCs to migrate outward from the lesion-implant site. Altering SC cell surface properties by overexpressing polysialic acid (PSA) has been shown to promote SC migration. In this study, a SCI contusion model was used to evaluate the migration, supraspinal axon growth support, and functional recovery associated with polysialyltransferase (PST)-overexpressing SCs [PST-green fluorescent protein (GFP) SCs] or controls (GFP SCs). Compared with GFP SCs, which remained confined to the injection site at the injury center, PST-GFP SCs migrated across the lesion:host cord interface for distances of up to 4.4 mm within adjacent host tissue. In addition, with PST-GFP SCs, there was extensive serotonergic and corticospinal axon in-growth within the implants that was limited in the GFP SC controls. The enhanced migration of PST-GFP SCs was accompanied by significant growth of these axons caudal to lesion. Animals receiving PST-GFP SCs exhibited improved functional outcome, both in the open-field and on the gridwalk test, beyond the modest improvements provided by GFP SC controls. This study for the first time demonstrates that a lack of migration by SCs may hinder their reparative benefits and that cell surface overexpression of PSA enhances the ability of implanted SCs to associate with and support the growth of corticospinal axons. These results provide further promise that PSA-modified SCs will be a potent reparative approach for SCI. © 2012 Wiley Periodicals, Inc.     

Schwann cells differentiated from spheroid-forming cells of rat subcutaneous fat tissue myelinate axons in the spinal cord injury

In the present study, we found that nestin-expressing spheroid cells derived from multipotent adipose stem cells of subcutaneous fat tissue could efficiently differentiate into Schwann cells (SCs) in vitro based on expression of SC markers such as A2B5, GFAP, O4, p75, S100, Sox10, Krox-20, and L1. The induced SC is engrafted to spinal cord injury lesions and formed a peripheral nervous system (PNS)-type myelin sheath on central nervous system (CNS) axons. PNS-type myelin sheath formation in repaired tissue was confirmed by transplantation of both induced PKH26-labeled SC and induced EGFP-expressing SC generated from EGFP transgenic rats. In addition to direct participation as myelin sheath-forming SC in repaired tissue, the induced SC also expressed several neurotrophic factors, as did native SC, which may suggest an additional role for induced SC in stimulation of endogenous healing responses. Thus, spheroid-forming cells from subcutaneous fat tissue demonstrated rapid and efficient induction into SC, and such cells show therapeutic promise for repair of damage to the CNS and PNS.     

Acute transplantation of olfactory ensheathing cells or Schwann cells promotes recovery after spinal cord injury in the rat

We compared the neurological and electrophysiological outcome, glial reactivity, and spared spinal cord connectivity promoted by acute transplantation of olfactory ensheathing cells (group OEC) or Schwann cells (group SC) after a mild injury to the rat spinal cord. Animals were subjected to a photochemical injury of 2.5 min irradiation at the T8 spinal cord segment. After lesion, a suspension containing 180,000 OECs or SCs was injected. A control group (group DM) received the vehicle alone. During 3 months postsurgery, behavioral skills were assessed with open field-BBB scale, inclined plane, and thermal algesimetry tests. Motor (MEPs) and somatosensory evoked potentials (SSEPs) were performed to evaluate the integrity of spinal cord pathways, whereas lumbar spinal reflexes were evaluated by the H reflex responses. Glial fibrillary acidic protein and proteoglycan expressions were quantified immunohistochemically at the injured spinal segments, and the preservation of corticospinal and raphespinal tracts caudal to the lesion was evaluated. Both OEC- and SC-transplanted groups showed significantly better results in all the behavioral tests than the DM group. Furthermore, the OEC group had higher MEP amplitudes and lower H responses than the other two groups. At the injury site, the area of spared parenchyma was greater in transplanted than in control injured rats. OEC-transplanted animals had reduced astrocytic reactivity and proteoglycan expression in comparison with SC-transplanted and DM rats. Taken together, these results indicate that transplantation of both OEC and SC has potential for restoration of injured spinal cords. OEC grafts showed superior ability to reduce glial reactivity and to improve functional recovery.     

Olfactory ensheathing glia and Schwann cells exhibit a distinct interaction behavior with meningeal cells

Schwann cells (SCs) and olfactory ensheathing glia (OEG) have both been used as cellular transplants to promote spinal cord repair. Both cell types support axonal regeneration and have beneficial effects on functional recovery. A significant difference between SCs and OEG is the effect of these cell types on astrocytes (ACs) present in the neural scar. In contrast to OEG, which associate and intermingle with ACs, SCs and ACs form separate cellular territories. Here, we show that OEG and SCs also interact differently with meningeal cells (MCs), another major cellular component of the neural scar. Whereas OEG intermingle with MCs in cocultures, SCs aggregate into well‐defined cell clusters. Our data suggest that (a) soluble factor(s) as well as direct cellular contact are involved in the MC‐induced SC clustering. Furthermore, the cluster formation of SCs in coculture with MCs is different from the previously reported segregation of SCs and ACs in coculture. The present results help to understand the differential behavior of both cell types after transplantation in the injured spinal cord and will be important to either determine which cell has optimal capacities to render the scar more permissive for regeneration, or to exploit the transplantation of both cell types in combination. © 2009 Wiley‐Liss, Inc.     

Embryonic and adult stem cells promote raphespinal axon outgrowth and improve functional outcome following spinal hemisection in mice

Spinal cord injury (SCI) often results in permanent neurological deficits below the injury site. Serotonergic raphespinal projections promote functional recovery after SCI, but spontaneous regeneration of most severed axons is limited by the glial cyst and scar that form at the lesion site. Stem cell (SC) transplantation offers a promising approach for inducing regeneration through the damaged area. Here we compare the effects of transplantation of embryonic neural precursors (NPs) or adult mesenchymal SCs, both of which are potential candidates for SC therapy. The spinal cord was hemisected at the L2 neuromer in adult mice. Two weeks post-injury, we transplanted neural precursors or mesenchymal SCs into the cord, caudal to the hemisection. Injured mice without a graft served as controls. Mice were tested for functional recovery on a battery of motor tasks, then killed and analysed for survival of grafted cells, for effects of engraftment on the local cellular environment and for the sprouting of serotonergic axons. Both types of SCs survived and were integrated into the host tissue, but only the NPs expressed neuronal markers. All transplanted animals displayed an increased number of serotonin-positive fibres caudal to the hemisection, compared with untreated mice. And both cell types led to improved motor performance. These results point to a therapeutic potential for such cell grafting.     

A systematic evaluation of Schwann cell injection into acellular cold-preserved nerve grafts

Peripheral nerve regeneration after injury depends on environmental cues and trophic support. Schwann cells (SCs) secrete trophic factors that promote neuronal survival and help guide axons during regeneration. The addition of SCs to acellular nerve grafts is a promising strategy for enhancing peripheral nerve regeneration; however, inconsistencies in seeding parameters have led to varying results. The current work sought to establish a systematic approach to seeding SCs in cold-preserved acellular nerve grafts. Studies were undertaken to (1) determine the needle gauge for optimal cell survival and minimal epineurial disruption during injection, (2) track the seeded SCs using a commercially available dye, and (3) evaluate the seeding efficiency of SCs in nerve grafts. It was determined that seeding with a 27-gauge needle resulted in the highest viability of SCs with the least damage to the epineurium. In addition, Qtracker(?) dye, a commercially available quantum dot nanocrystal, was used to label SCs prior to transplantation, which allowed visualization of the seeded SCs in nerve grafts. Finally, stereological methods were used to evaluate the seeding efficiency of SCs in nerve grafts immediately after injection and following a 1- or 3-day in vitro incubation in SC growth media. Using a systematic approach, the best needle gauge and a suitable dye for SC visualization in acellular nerve grafts were identified. Seeding efficiency in these grafts was also determined. The findings will lead to improvements ability to assess injection of cells (including SCs) for use with acellular nerve grafts to promote nerve regeneration.     

蛛网膜下腔移植自体激活许旺细胞治疗大鼠脊髓损伤***

背景：许旺细胞能够分泌多种神经营养因子,促进脊髓损伤功能的恢复。但异体许旺细胞移植可引发自身免疫反应,且在移植方式上,局部移植无法避免二次损伤,静脉移植虽可以透过血脊髓屏障到达损伤局部,但不能达到有效的治疗浓度。 目的：探讨经蛛网膜下腔移植自体激活许旺细胞对脊髓损伤大鼠功能恢复的影响。 方法：66只大鼠均建立脊髓损伤模型,造模后随机分为3组,自体激活许旺细胞组通过结扎单侧隐神经从而激活许旺细胞,自体未激活许旺细胞组、模型对照组仅在相同部位手术但不结扎神经。切除各组手术远端1 cm神经,采用组织块法进行许旺细胞的体外分离培养及纯化。1周后,自体激活许旺细胞组、自体未激活许旺细胞组分别通过蛛网膜下腔注入经Hoechst33342标记的对应许旺细胞悬液,模型对照组仅注入等量DMEM。对脊髓损伤后肢体功能的恢复进行BBB运动功能评分及脚印分析,通过苏木精-伊红染色和GFAP染色从组织学角度评价脊髓损伤恢复情况。 结果与结论：从术后第4周开始,自体激活许旺细胞组BBB后肢功能评分明显优于另两组(P < 0.05)。移植后2周,可见迁移至大鼠脊髓损伤局部的许旺细胞。与自体未激活许旺细胞组比较,移植后5周自体激活许旺细胞组的前后足中心距离、后肢第3足趾外旋角度均显著减小(P < 0.05),移植后13周损伤区胶质瘢痕面积明显减小(P < 0.05),损伤区空洞面积明显减小(P < 0.05)。证实经蛛网膜下腔移植自体激活许旺细胞可以促进脊髓损伤的恢复。