Transplantation of D15A-Expressing Glial-Restricted-Precursor-Derived Astrocytes Improves Anatomical and Locomotor Recovery after Spinal Cord Injury

The transplantation of neural stem/progenitor cells is a promising therapeutic strategy for spinal cord injury (SCI). In this study, we tested whether combination of neurotrophic factors and transplantation of glial-restricted precursor (GRPs)-derived astrocytes (GDAs) could decrease the injury and promote functional recovery after SCI. We developed a protocol to quickly produce a sufficiently large, homogenous population of young astrocytes from GRPs, the earliest arising progenitor cell population restricted to the generation of glia. GDAs expressed the axonal regeneration promoting substrates, laminin and fibronectin, but not the inhibitory chondroitin sulfate proteoglycans (CSPGs). Importantly, GDAs or its conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons in vitro. GDAs were infected with retroviruses expressing EGFP or multi-neurotrophin D15A and transplanted into the contused adult thoracic spinal cord at 8 days post-injury. Eight weeks after transplantation, the grafted GDAs survived and integrated into the injured spinal cord. Grafted GDAs expressed GFAP, suggesting they remained astrocyte lineage in the injured spinal cord. But it did not express CSPG. Robust axonal regeneration along the grafted GDAs was observed. Furthermore, transplantation of D15A-GDAs significantly increased the spared white matter and decreased the injury size compared to other control groups. More importantly, transplantation of D15A-GDAs significantly improved the locomotion function recovery shown by BBB locomotion scores and Tredscan footprint analyses. However, this combinatorial strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. These results demonstrate that transplantation of D15A-expressing GDAs promotes anatomical and locomotion recovery after SCI, suggesting it may be an effective therapeutic approach for SCI.     

Improvement of contusive spinal cord injury in rats by co-transplantation of gamma-aminobutyric acid-ergic cells and bone marrow stromal cells

Background aimsCell therapy is considered a promising option for treatment of spinal cord injury (SCI). The purpose of this study is to use combined therapy of bone marrow stromal cells (BMSCs) and BMSC-derived gamma-aminobutyric acid (GABA)ergic inhibitory neurotransmitter cells (BDGCs) for the contusion model of SCI in rats.MethodsBDGCs were prepared from BMSCs by pre-inducing them with β-mercaptoethanol followed by retinoic acid and then inducing them by creatine. They were immunostained with BMSC, proneuronal, neural and GABA markers. The BDGCs were intraspinally transplanted into the contused rats, whereas the BMSCs were delivered intravenously. The animals were sacrificed after 12 weeks.ResultsThe Basso, Beattie and Bresnahan test showed improvement in the animals with the combined therapy compared with the untreated animals, the animals treated with GABAergic cells only and the animals that received BMSCs. The immunohistochemistry analysis of the tissue sections prepared from the animals receiving the combined therapy showed that the transplanted cells were engrafted and integrated into the injured spinal cord; in addition, a significant reduction was seen in the cavitation.ConclusionsThe study shows that the combination of GABAergic cells with BMSCs can improve SCI.     

Adult stem cell transplants for spinal cord injury repair: current state in preclinical research

Spinal cord injury (SCI) is a traumatic disorder resulting in a functional deficit that usually leads to severe and permanent paralysis. After the initial insult to the spinal cord, additional structure and function are lost through an active and complex secondary process. Since there is not effective treatment for SCI, several strategies including cellular, pharmacological and rehabilitation therapies have been approached in animal models. Some of them have been proved in clinical trials. In this review we focus on the current state of cell therapies, particularly on cells from adult origin, assayed in preclinical research. Cell types used in SCI therapy include Schwann cells, olfactory ensheathing cells and adult stem cells, such as neural stem cells, umbilical cord blood derived cells, mesenchymal stem cells or induced pluripotent stem cells. There are not yet conclusive evidences on which types of glial or adult stem cells are most effective in SCI treatment. Their ability to incorporate into the damaged spinal cord, to differentiate into neural lineages, to exert neuroprotective effects, to promote regeneration of damaged axons, and to improve functional deficits are still discussed, before translation towards clinical use, as a single therapy or in combination with other strategies.     

Chondroitinase and growth factors enhance activation and oligodendrocyte differentiation of endogenous neural precursor cells after spinal cord injury

The adult spinal cord harbours a population of multipotent neural precursor cells (NPCs) with the ability to replace oligodendrocytes. However, despite this capacity, proliferation and endogenous remyelination is severely limited after spinal cord injury (SCI). In the post-traumatic microenvironment following SCI, endogenous spinal NPCs mainly differentiate into astrocytes which could contribute to astrogliosis that exacerbate the outcomes of SCI. These findings emphasize a key role for the post-SCI niche in modulating the behaviour of spinal NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation in SCI by reducing the survival, migration and integration of engrafted NPCs within the injured spinal cord. These inhibitory effects were attenuated by administration of chondroitinase (ChABC) prior to NPC transplantation. Here, in a rat model of compressive SCI, we show that perturbing CSPGs by ChABC in combination with sustained infusion of growth factors (EGF, bFGF and PDGF-AA) optimize the activation and oligodendroglial differentiation of spinal NPCs after injury. Four days following SCI, we intrathecally delivered ChABC and/or GFs for seven days. We performed BrdU incorporation to label proliferating cells during the treatment period after SCI. This strategy increased the proliferation of spinal NPCs, reduced the generation of new astrocytes and promoted their differentiation along an oligodendroglial lineage, a prerequisite for remyelination. Furthermore, ChABC and GF treatments enhanced the response of non-neural cells by increasing the generation of new vascular endothelial cells and decreasing the number of proliferating macrophages/microglia after SCI. In conclusions, our data strongly suggest that optimization of the behaviour of endogenous spinal NPCs after SCI is critical not only to promote endogenous oligodendrocyte replacement, but also to reverse the otherwise detrimental effects of their activation into astrocytes which could negatively influence the repair process after SCI.     

Umbilical Cord Blood Stem Cell Mediated Downregulation of Fas Improves Functional Recovery of Rats after Spinal Cord Injury

Human umbilical cord blood stem cells (hUCB), due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, represent a potentially useful source for cell-based therapies after spinal cord injury (SCI). To evaluate their therapeutic potential, hUCB were stereotactically transplanted into the injury epicenter, one week after SCI in rats. Our results show the presence of a substantial number of surviving hUCB in the injured spinal cord up to five weeks after transplantation. Three weeks after SCI, apoptotic cells were found especially in the dorsal white matter and gray matter, which are positive for both neuron and oligodendrocyte markers. Expression of Fas on both neurons and oligodendrocytes was efficiently downregulated by hUCB. This ultimately resulted in downregulation of caspase-3 extrinsic pathway proteins involving increased expression of FLIP, XIAP and inhibition of PARP cleavage. In hUCB-treated rats, the PI3K/Akt pathway was also involved in antiapoptotic actions. Further, structural integrity of the cytoskeletal proteins α-tubulin, MAP2A&2B and NF-200 has been preserved in hUCB treatments. The behavioral scores of hind limbs of hUCB-treated rats improved significantly than those of the injured group, showing functional recovery. Taken together, our results indicate that hUCB-mediated downregulation of Fas and caspases leads to functional recovery of hind limbs of rats after SCI.     

Early Intervention for Spinal Cord Injury with Human Induced Pluripotent Stem Cells Oligodendrocyte Progenitors

Induced pluripotent stem (iPS) cells are at the forefront of research in regenerative medicine and are envisaged as a source for personalized tissue repair and cell replacement therapy. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs) can be derived from iPS cells generated using either an episomal, non-integrating plasmid approach or standard integrating retroviruses that survive and differentiate into mature oligodendrocytes after early transplantation into the injured spinal cord. The efficiency of OP differentiation in all 3 lines tested ranged from 40% to 60% of total cells, comparable to those derived from human embryonic stem cells. iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors and glial progenitors while the episomal plasmid-derived iPS line generated more OPs expressing late markers O1 and RIP. Moreover, we discovered that iPS-derived OPs (iPS-OPs) engrafted 24 hours following a moderate contusive spinal cord injury (SCI) in rats survived for approximately two months and that more than 70% of the transplanted cells differentiated into mature oligodendrocytes that expressed myelin associated proteins. Transplanted OPs resulted in a significant increase in the number of myelinated axons in animals that received a transplantation 24 h after injury. In addition, nearly a 5-fold reduction in cavity size and reduced glial scarring was seen in iPS-treated groups compared to the control group, which was injected with heat-killed iPS-OPs. Although further investigation is needed to understand the mechanisms involved, these results provide evidence that patient-specific, iPS-derived OPs can survive for three months and improve behavioral assessment (BBB) after acute transplantation into SCI. This is significant as determining the time in which stem cells are injected after SCI may influence their survival and differentiation capacity.     

Transplants of Human Mesenchymal Stem Cells Improve Functional Recovery After Spinal Cord Injury in the Rat

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell replacement therapy after nervous tissue damage. They can be expanded in culture and reintroduced into patients as autografts or allografts with unique immunologic properties. The aim of the present study was to investigate (i) survival, migration, differentiation properties of hMSCs transplanted into non-immunosuppressed rats after spinal cord injury (SCI) and (ii) impact of hMSC transplantation on functional recovery. Seven days after SCI, rats received i.v. injection of hMSCs (2×10⁶ in 0.5 mL DMEM) isolated from adult healthy donors. Functional recovery was assessed by Basso–Beattie–Bresnahan (BBB) score weekly for 28 days. Our results showed gradual improvement of locomotor function in transplanted rats with statistically significant differences at 21 and 28 days. Immunocytochemical analysis using human nuclei (NUMA) and BrdU antibodies confirmed survival and migration of hMSCs into the injury site. Transplanted cells were found to infiltrate mainly into the ventrolateral white matter tracts, spreading also to adjacent segments located rostro-caudaly to the injury epicenter. In double-stained preparations, hMSCs were found to differentiate into oligodendrocytes (APC), but not into cells expressing neuronal markers (NeuN). Accumulation of GAP-43 regrowing axons within damaged white matter tracts after transplantation was observed. Our findings indicate that hMSCs may facilitate recovery from spinal cord injury by remyelinating spared white matter tracts and/or by enhancing axonal growth. In addition, low immunogenicity of hMSCs was confirmed by survival of donor cells without immunosuppressive treatment.     

Neural progenitors isolated from newborn rat spinal cords differentiate into neurons and astroglia

Permanent functional deficit in patients with spinal cord injury (SCI) is in part due to severe neural cell death. Therefore, cell replacement using stem cells and neural progenitors that give rise to neurons and glia is thought to be a potent strategy to promote tissue repair after SCI. Many studies have shown that stem cells and neural progenitors can be isolated from embryonic, postnatal and adult spinal cords. Recently, we isolated neural progenitors from newborn rat spinal cords. In general, the neural progenitors grew as spheres in culture, and showed immunoreactivity to a neural progenitor cellular marker, nestin. They were found to proliferate and differentiate into glial fibrillary acidic protein-positive astroglia and multiple neuronal populations, including GABAergic and cholinergic neurons. Neurotrophin 3 and neurotrophin 4 enhanced the differentiation of neural progenitors into neurons. Furthermore, the neural progenitors that were transplanted into contusive spinal cords were found to survive and have migrated in the spinal cord rostrally and caudally over 8 mm to the lesion center 7 days after injury. Thus, the neural progenitors isolated from newborn rat spinal cords in combination with neurotrophic factors may provide a tool for cell therapy in SCI patients.     

Increase of NG2-positive cells associated with radial glia following traumatic spinal cord injury in adult rats

In the CSN including the spinal cord, NG2 proteoglycan is a marker of oligodendrocyte progenitors. To elucidate the dynamics of the endogenous neural stem (progenitor) cells in adult rats with spinal cord injury (SCI), we examined an immunohistochemical analysis of NG2, GFAP, and 3CB2, a specific marker of radial glia (RG). SD rats were divided into a SCI group (n = 25) and a sham-operated group (n = 5). In the injury group, laminectomy was performed at Th11–12 and contusive compression injury was created by applying a weight of 30 g for 10 min. Rats were sacrificed at 24 h, and 1, 4, 8 and 12 weeks post-injury. Frozen 20-μ m sections of tissue 5 and 10 mm rostral and caudal to the epicenter of injury were prepared. Immunohistochemistry was performed using antibodies against NG2, GFAP and 3CB2. At 4 weeks after injury, NG2-positive glial cells arose from below the pial surface as bipolar cells with processes extending throughout the entire white matter. NG2 expression peaked at 4 weeks after injury, showing a 7-fold increase compared to the 24 h after injury. The NG2-positive cells with processes which increased in the white matter of the spinal cord were GFAP-positive and also co-localized with 3CB2 antigen. The pattern of NG2 expression of these cells was temporally and spatially different from the pattern of NG2 expression that accumulated around the hemorrhagic and necrotic epicenter. These results suggest that NG2 positive cells which derived from subpial layer, may have some lineage to RG after SCI in adult rodents.     

Integration and Long Distance Axonal Regeneration in the Central Nervous System from Transplanted Primitive Neural Stem Cells

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.     

Optimal Location and Time for Neural Stem Cell Transplantation into Transected Rat Spinal Cord

Implanted neural stem cells (NSC) could improve neurological functions following spinal cord injury (SCI), but the optimal conditions for NSC transplantation are largely unknown, especially in transected spinal cord. This study investigated the effect and fate of NSC engrafted into spinal cords at different locations and time points following T₉ spinal cord transection. Engrafted NSC could survive and migrate in host spinal cords. Significant improvement in hindlimb locomotor functions associated with NSC survival was found in rats receiving NSC transplantation in the spinal cords rostral to the transection site at the subacute stage (7 days post operation), compared with those caudal to the transection site at the acute stage (at the time of injury). At 4 weeks post operation, CD68 immunohistochemical staining confirmed that macrophages were less in rostrally transplanted sites and in subacute groups than seen in caudal and acute transplanted rats. The present findings indicated that NSC transplantation into spinal cords rostral to transection site at the subacute stage is an optimal strategy for engrafted NSC survival and host behavioral improvement. It therefore would be available to the usage of NSC for the treatment of SCI in the future clinic trial.     

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.¹ Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5° (animals rotated at 22.5°), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya''s staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.     

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.     

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

Increased expression of nitric oxide synthase interacting protein (NOSIP) following traumatic spinal cord injury in rats

Previous studies indicated that nitric oxide (NO) is involved in secondary damage of spinal cord injury (SCI), which worsens the primary physical injury to the central nervous systems. Recently, nitric oxide synthase interacting protein (NOSIP) has been identified to interact with neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase by inhibiting the NO production. However, its expression and function after a central nervous system injury remains unclear. In this study, we examined the expression and cellular localization of NOSIP in the spinal cord of an adult rat. Western blot analysis indicated that NOSIP protein levels increased at day1 post-injury and peaked at day 14. Double immunofluorescence staining showed that NOSIP was primarily expressed in neurons and glial cells in the intact spinal cord. Interestingly, this study also showed that the expression of NOSIP significantly increased in astrocytes after injury. Furthermore, injury-induced expression of NOSIP was co-expressed with proliferating cell nuclear antigen (PCNA) positive astrocytes after injury. We also showed the NOSIP was co-localized with nNOS in gray matter and white matter after SCI. All these data taken together suggested that NOSIP may play an important roles in astrogliogenesis after a spinal cord injury.     

骨髓间充质干细胞移植对急性脊髓损伤脱髓鞘病变的保护作用

骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)已被广泛应用于治疗脊髓损伤,但目前对其治疗机制了解甚少。BMSCs被移植至脊髓钳夹损伤模型大鼠,以研究其保护作用。通过LFB(Luxol fast blue)染色、锇酸染色、TUNEL(Td T-mediated d UTP nick-end labeling)染色和透射电镜对白质有髓神经纤维进行观察。免疫印迹检测BMSCs移植对脑源性神经营养因子(Brain derived neurotrophic factor,BDNF)和caspase 3蛋白表达的影响。通过脊髓损伤后1、7、14 d三个时间点移植BMSCs并进行后肢运动评分(Basso,beattie and bresnahan;BBB评分)和CNPase(2′,3′-cyclic-nucleotide 3′-phosphodiesterase)、髓鞘碱性蛋白(Myelin basic protein,MBP)、caspase 3蛋白水平的检测。免疫荧光观察BMSCs移植到受损脊髓后分化情况及CNPase-caspase 3~+共表达情况。骨髓间充质干细胞移植7 d后,部分移植的BMSCs可表达神经元和少突胶质细胞标记物,大鼠后肢运动能力和髓鞘超微结构特征均明显改善。骨髓间充质干细胞移植后BDNF蛋白表达水平增加,caspase 3蛋白表达水平则降低。相对于脊髓损伤后1 d和14 d,7 d移植BMSCs后MBP和CNPase蛋白表达水平最高;caspase 3蛋白表达水平则最低。骨髓间充质干细胞移植后CNPase-caspase 3~+细胞散在分布于脊髓白质。结果表明,急性脊髓损伤后,BMSCs移植到受损脊髓有分化为神经元和少突胶质细胞的倾向,并促进BDNF的分泌介导抗少突胶质细胞凋亡而对神经脱髓鞘病变有保护作用,且最佳移植时间为脊髓损伤后7 d。