Treatment of spinal cord injury by transplantation of fetal neural precursor cells engineered to express BMP inhibitor

Spontaneous recovery after spinal cord injury is limited. Transplantation of neural precursor cells (NPCs) into lesioned adult rat spinal cord results in only partial functional recovery, and most transplanted cells tend to differentiate predominantly into astrocytes. In order to improve functional recovery after transplantation, it is important that transplanted neural precursor cells appropriately differentiate into cell lineages required for spinal cord regeneration. In order to modulate the fate of transplanted cells, we advocate transplanting gene-modified neural precursor cells. We demonstrate that gene modification to inhibit bone morphogenetic protein (BMP) signaling by noggin expression promoted differentiation of neural precursor cells into neurons and oligodendrocytes, in addition to astrocytes after transplantation. Furthermore, functional recovery of the recipient mice with spinal cord injury was observed when noggin-expressing neural precursor cells were transplanted. These observations suggest that gene-modified neural precursor cells that express molecules involved in cell fate modulation could improve central nervous system (CNS) regeneration.     

人脐带间充质干细胞移植对大鼠脊髓损伤神经功能恢复的评价

目的 观察人脐带间充质干细胞（human umbilical cordmesenchymal stem cell，hUCMSC）移植对大鼠脊髓损伤神经功能恢复的影响。方法 SD大鼠70只，随机分为3组：脊髓半切＋hUCMSC组（n=30）、脊髓半切＋PBS组（n=30）和假手术组（n=10）。脊髓半切＋hUCMSC组和PBS组又分为头侧注射、尾侧注射和头尾两侧注射三个亚组。移植后1、7、14、21、28d观察大鼠神经功能恢复情况，应用免疫组化检测移植到脊髓的hUCMSC胶质纤维酸性蛋白（GFAP）和神经元特异性烯醇化酶（NSE）表达情况。结果 大鼠脊髓半切损害后，hUCMSC组动物较PBS组有明显的神经功能恢复。植入后28d在宿主脊髓中存活的hUCMSC细胞MABl281（mouse antiuman nuclei monoclonal antibody）染色阳性，免疫组化双标染色显示MABl28l阳性细胞亦分别有NSE或GFAP表达并向损伤部位迁移，hUCMSC来源的GFAP阳性细胞可见明显的树突生长。结论 hUCMSC移植到宿主损伤脊髓后可以存活、向损伤部位迁移，并向神经元样和星形胶质细胞分化，且可促进大鼠脊髓损伤后神经功能恢复。hUCMSC作为一种来源广泛的干细胞用于治疗脊髓损伤可能具有重要的价值。     

神经干细胞移植促进鼠脊髓损伤后髓鞘结构的修复

目的 观察神经干细胞移植治疗对鼠脊髓损伤后髓鞘结构修复的作用并探讨其作用机制。方法 制备鼠T10脊髓损伤模型,体外培养、诱导鼠神经干细胞,定量评价神经干细胞移植对脊髓损伤后髓鞘结构修复的影响。结果 与对照组相比,神经干细胞移植组明显地增强了蛋白前脂蛋白信使核糖核酸(PLP mRNA)的表达,促进了髓鞘碱性蛋白(MBP)性的髓鞘再生和髓鞘结构的修复。结论 神经干细胞移植通过增强髓鞘的再生而促进了脊髓损伤后髓鞘结构的修复,是急性脊髓损伤一种有效的治疗方案。     

甲基强的松龙和神经干细胞移植联合治疗大鼠脊髓损伤

目的：观察甲基强的松龙和神经干细胞移植对大鼠脊髓损伤后神经结构修复和功能恢复的治疗作用并探讨其作用机制。方法：制备大鼠胸10脊髓损伤模型，体外培养、诱导分化大鼠神经干细胞，定量评价甲基强的松龙和神经干细胞移植对脊髓损伤后神经结构修复和功能恢复的影响。结果：与对照组相比，移植组明显地增强了生长相关蛋白（GAP－43）mRNA的表达，促进了乙酰胆碱转移酶（ChAT)阳性脊髓运动神经元的再生、神经结构的修复和下肢运动功能的恢复（P＜0．05）。结论：甲基强的松龙和神经干细胞移植通过增强GAP－43 mRNA的表达、运动神经元的再生而促进了脊髓损伤后神经结构的修复和功能的恢复，是急性脊髓损伤的一种有效的治疗方案。     

Lithium enhances proliferation and neuronal differentiation of neural progenitor cells in vitro and after transplantation into the adult rat spinal cord

Transplantation of neural progenitor cells (NPCs) holds great potential for the treatment of spinal cord injuries. The survival and differential fates of transplanted NPCs in the cord are key factors contributing to the success of the therapy. In this study, we investigate the effects of lithium, a widely used antidepressant drug, on the survival, proliferation and differentiation of spinal cord-derived NPCs in cultures and after transplantation into the spinal cord. Our results show that clinically relevant doses of lithium increase the proliferation of grafted NPCs at 2 weeks post-grafting and neuronal generation by grafted NPCs at 2 weeks and 4 weeks post-grafting. However, lithium does not cause preferential differentiation of NPCs into astrocytes or oligodendrocytes both in vitro and after transplantation. Our results also show that chronic treatment with lithium (up to 4 weeks) reduces microglia and macrophage activation, indicating that lithium treatment can affect the host immune response. The results of the present study provide evidence that lithium may have therapeutic potential in cell replacement strategies for CNS injury due to its ability to promote proliferation and neuronal generation of grafted NPCs and reduce the host immune reaction.     

大鼠骨髓间充质干细胞静脉移植对脊髓损伤的修复作用

目的初步探讨骨髓间充质干细胞（BMSCs）静脉移植对脊髓损伤后神经功能恢复和神经修复的影响。方法体外培养BMSCs，改良Allen法制备大鼠脊髓损伤模型，经尾静脉移植Brdu标记的BMSCs，损伤后24h、移植后1、3、5周评价实验动物的神经功能状况，并检测BMSCs在体内迁移、存活以及分化情况，电子显微镜观察组织形态学变化。结果移植的BMSCs在宿主损伤脊髓中聚集并存活，3～5周后有部分移植细胞表达神经元特异性烯醇化酶（NSE）、神经丝蛋白（NF）、微管相关蛋白（MAP2）；BMSCs静脉移植组大鼠运动功能改善，BBB评分高于对照组（P〈0．05）；5周后组织学观察，与对照组相比移植组损伤区脊髓结构较完整。结论BMSCs经静脉移植后可向脊髓损伤处聚集并存活分化，促进神经修复及神经功能的恢复。     

Effect of intravenous transplantation of bone marrow mesenchymal stem cells on neurotransmitters and synapsins in rats with spinal cord injury

Bone marrow mesenchymal stem cells were isolated, purified and cultured in vitro by Percoll density gradient centrifugation combined with the cell adherence method. Passages 3-5 bone marrow mesenchymal stem cells were transplanted into rats with traumatic spinal cord injury via the caudal vein. Basso-Beattie-Bresnahan scores indicate that neurological function of experimental rats was significantly improved over transplantation time (1-5 weeks). Expressions of choline acetyltransferase, glutamic acid decarboxylase and synapsins in the damaged spinal cord of rats was significantly increased after transplantation, determined by immunofluorescence staining and laser confocal scanning microscopy. Bone marrow mesenchymal stem cells that had migrated into the damaged area of rats in the experimental group began to express choline acetyltransferase, glutamic acid decarboxylase and synapsins, 3 weeks after transplantation. The Basso-Beattie- Bresnahan scores positively correlated with expression of choline acetyltransferase and synapsins. Experimental findings indicate that intravenously transplanted bone marrow mesenchymal stem cells traverse into the damaged spinal cord of rats, promote expression of choline acetyltransferase, glutamic acid decarboxylase and synapsins, and improve nerve function in rats with spinal cord injury.     

骨髓间质干细胞移植对大鼠脊髓损伤神经功能恢复的影响

目的：观察成人骨髓间质干细胞（hBMSCs)移植对大鼠脊髓损伤神经功能恢复的影响.方法：Wistar大鼠90只，随机分为脊髓半切＋hBMSCs组、脊髓半切＋PBS组、单纯脊髓半切组和假手术组。脊髓半切＋hBMSCs组和PBS组又分别分为头侧注射、尾侧注射和头尾两侧注射三个亚组。移植后1、7、14、21、28d观察大鼠神经功能恢复情况，应用免疫组化和免疫荧光技术检测BrdU标记hBMSCs的胶质纤维酸性蛋白（GFAP）和神经元特异性核蛋白（NeuN)表达情况。结果：大鼠脊髓半切损害后，hBMSCs组动物较PBS组死亡率下降并有明显的神经功能恢复。移植的hBMSCs 在宿主脊髓中存活，从第7天开始即有NeuN和GFAP表达并向损伤部位及对侧迁移，第28天hBMSCs来源GFAP阳性细胞可见明显的树突生长。结论：hBMSCs可在宿主损伤脊髓中存活、向损伤部位迁移并向神经元和星形胶质细胞分化，并促进神经功能恢复，降低死亡率，成人骨髓间质干细胞作为一种独特的干细胞来源用于治疗脊髓损伤可能具有非常重要的价值。     

Reconstruction of the contused cat spinal cord by the delayed nerve graft technique and cultured peripheral non-neuronal cells

Summary Previously, surgical reconstruction of the transected dog spinal cord by the delayed nerve graft technique has been shown to result in reinnervation of the nerve graft by axons. In the present study, we compared the results of surgical reconstruction of the severely contused cat spinal cord by the delayed nerve graft technique alone to those after reconstruction with a similar nerve graft plus cultured peripheral nonneuronal cells implanted between the grafted nerve and the spinal cord stumps. The spinal cord-nerve graft junction was examined by light and electron microscopy. The cultured cells were prelabelled with tritiated thymidine and their location after implantation determined by autoradiography. By 3 days after spinal cord reconstruction, the pelabelled cells were present at the junction and had migrated into the nerve graft and also into the spinal cord stumps where they were observed near axons. By 7 days, physical connections were observed bridging the junction between the spinal cord and nerve graft and axons ensheathed by Schwann cells had already penetrated at least 1 mm into the nerve graft. Wound healing took at least a week longer in animals repaired with a nerve graft alone. At one year or later after reconstructive surgery, in both groups of animals, the grafted nerve was reinnervated with myelinated and unmyelinated axons. Thus, the severely contused cat spinal cord could be reconstructed with the delayed nerve graft technique alone but the use of the cultured cells appeared to enhance wound healing and decrease the time required for axon elongation into the nerve graft.Supported by grants from the Veterans Administration and the National Institutes of Health (NS-14413)     

Migration and differentiation of nuclear fluorescence‐labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice

There is increasing evidence that bone marrow stromal cells (BMSC) have the potential to migrate into the injured neural tissue and to differentiate into the CNS cells, indicating the possibility of autograft transplantation therapy. The present study was aimed to clarify whether the mouse BMSC can migrate into the lesion and differentiate into the CNS cells when transplanted into the mice subjected to focal cerebral infarct or spinal cord injury. The BMSC were harvested from mice and characterized by flow cytometry. Then, the BMSC were labeled by bis‐benzimide, a nuclear fluorescence dye, over 24 h, and were stereotactically transplanted into the brain or spinal cord of the mice. The cultured BMSC expressed low levels of CD45 and high levels of CD90 and Sca‐1 on flow cytometry. A large number of grafted cells survived in the normal brain 4 weeks after transplantation, many of which were located close to the transplanted sites. They expressed the neuronal marker including NeuN, MAP2, and doublecortin on fluorescent immunohistochemistry. However, when the BMSC were transplanted into the ipsilateral striatum of the mice subjected to middle cerebral artery occlusion, many of the grafted cells migrated into the corpus callosum and injured cortex, and also expressed the neuronal markers 4 weeks after transplantation. In particular, NeuN was very useful to validate the differentiation of the grafted cells, because the marker was expressed in the nuclei and was overlapped with bis‐benzimide. Similar results were obtained in the mice subjected to spinal cord injury. However, many of the transplanted BMSC expressed GFAP, an astrocytic protein, in injured spinal cord. The present results indicate that the mouse BMSC can migrate into the CNS lesion and differentiate into the neurons or astrocytes, and that bis‐benzimide is a simple and useful marker to label the donor cells and to evaluate their migration and differentiation in the host neural tissues over a long period.     

胚胎脊髓移植在恢复损伤脊髓传导功能中的作用

目的：探讨胚胎脊髓移植在恢复损伤脊髓传导功能中的作用。方法：将Ｅ１４胚胎脊髓植入成鼠损伤脊髓后３０、４５、６０天时，用单位放电记录技术观察了正常脊髓神经元和移植物神经元的自发放电活动，及其对刺激坐骨神经、红核和同时刺激的反应。结果：正常脊髓神经元的自发单位放电多是一个低频的单发脉冲活动。无论选择那种刺激方式，都可见兴奋、抑制和无反应三种反应。术后３０天时，胚胎神经元的自发单位放电以高频电脉冲活动为主，簇状放电所占比例较大，对刺激有反应的放电单位数也较少；随着动物存活时间的延长，这些单位放电的情况逐渐向着低频、单脉冲以及高反应率的方向发展。至术后６０天时，胚胎神经元单位放电的频率、形式以及对刺激的反应情况都变得和正常神经元的相似。结论：胚胎神经元移植后经历了一个逐渐发育分化过程，在这个过程中它们有可能逐渐和宿主神经元形成了功能性突触连接。     

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.     

嗅鞘细胞静脉移植治疗脊髓损伤

背景：嗅鞘细胞移植治疗脊髓损伤在众多疗法中效果较佳,成为最有前景的治疗方法之一。目前移植方法为局部移植,存在操作复杂、创伤大、重复移植治疗困难等缺点。寻找一种简单易行且疗效好的移植方法成为各国学者研究的热点。 目的：分析嗅鞘细胞静脉移植治疗脊髓损伤的可行性和疗效。 方法：制备Wistar大鼠脊髓半切模型,随机分4组：嗅鞘细胞髓内局部移植组、嗅鞘细胞静脉移植组、D/F12静脉移植组和空白对照组。定期行CBS功能评分及组织学检查,评价脊髓修复情况。 结果与结论：嗅鞘细胞髓内局部移植组、嗅鞘细胞静脉移植组的功能恢复和组织学改变优于D/F12静脉移植组和空白对照组,嗅鞘细胞髓内局部移植组、嗅鞘细胞静脉移植组间无显著差别。说明嗅鞘细胞静脉移植可向脊髓损伤部位迁移并修复脊髓损伤,其疗效与嗅鞘细胞髓内局部移植相当。     

不同来源成体神经干细胞促进大鼠脊髓损伤功能修复的比较研究

目的 评价大脑、骨髓和脂肪组织3种不同来源的神经干细胞对大鼠脊髓挫伤的治疗效果.方法 选取来源于同一大鼠成体中大脑、骨髓和脂肪的3个部位的组织,分离、诱导分化为不同来源的神经干细胞;应用自由落体损伤模型装置造成大鼠脊髓挫伤.将不同来源的神经干细胞分别移植入大鼠脊髓损伤部位,通过BBB评分比较修复脊髓损伤功能的效果,应用免疫荧光染色检测不同移植细胞在损伤脊髓中的存活、分布、迁移的情况.另设假手术对照组和生理盐水对照组.结果 与假手术对照组和生理盐水对照组比较,3个细胞处理组BBB评分在2～8周开始增加,9周以后更加明显,差异开始有统计学意义(P<0.05).在移植后1周和4周,细胞移植组中脑源性神经干细胞(SVZ-NSs)组Brdu/nestin+>神经元存活的数目明显高于其他2组.但差异没有统计学意义(P>0.05);到了第8周,3组均仅有少量Brdu/nestin+>细胞存活,相互之间比较差异无统计学意义(P>0.05).结论 植入来源于大脑、骨髓和脂肪组织的神经干细胞都可以在一定程度上提高脊髓损伤后运动功能恢复,但SVZ-NSs组的脊髓损伤大鼠运动功能恢复要比脂肪来源的神经干细胞(AD-NSs)组及骨髓来源的神经干细胞(BM-NSs)组更好.AD-NSs由于来源广泛和强有力的增殖能力,相比其他来源的神经干细胞,可能是更好的选择.     

Transplantation of embryonic noradrenergic neurons in two models of adult rat spinal cord injury: ultrastructural immunocytochemical study

The synaptic connections established by grafted noradrenergic (NA) neurons into the lesioned adult rat spinal cord were analysed using immunocytochemistry at the electron microscopic level. An embryonic cell suspension of the locus coeruleus region from E-13 rat embryos was transplanted into the spinal cord following either: (1) spinal cord transection or (2), partial selective denervation by 6-hydroxy dopamine (6-OH DA). One month after grafting, the NA-neurons established, in the two models, an innervation pattern similar to that found in the intact spinal cord. In both models, the transplanted NA-immunoreactive neurons formed extensive synaptic contacts with dendrites, spines and perikarya. The proportion of axodendritic and axospinous contacts was inverse in the two models. The first model thus reproduced more closely the normal synaptic pattern prefering dendritic targets, which could correspond to a better integration of the graft. In the second model, a partially NA-denervated spinal cord, there existed a competition between residual intrinsic and grafted neuron-derived fibres, which presumably affects synaptogenesis. In conclusion, the present study illustrate the complexity of cell interations conducting to the formation of a specific circuitry. Recognition phenomenon are likely modulated by space constraints, which ultimately shape-up the geometry of synaptic contacts.     

骨髓基质细胞移植治疗脊髓全横断损伤超微结构观察

目的观察骨髓基质细胞（MSCs）移植治疗脊髓全横断损伤（SCI）超微结构,探讨内源性细胞与再生轴突关系。方法通过全骨髓法培养、纯化MSCs,SCI9d后移植MSCs,通过免疫荧光组化观察细胞移植后损伤区轴突再生情况,免疫荧光双标、免疫电镜观察再生轴突与内源性细胞关系。结果移植8W后实验组脊髓损伤区可见大量神经微丝蛋白200（NF200）阳性纤维,对照组脊髓损伤区未见明显的NF200阳性纤维。免疫荧光双标结果显示损伤区NF200阳性纤维和2,3＇-环核苷酸磷酸而酯酶（CNP）阳性细胞之间存在密切的空间关系,免疫电镜显示CNP阳性细胞通过伸长丝状伪足形成再生轴突支架,内源性施万细胞参与再生轴突髓鞘形成。结论MSCs移植可促进损伤区轴突再生,宿主自身CNP阳性细胞和施万细胞参与损伤轴突的再生和髓鞘形成。     

Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury

Emerging studies of treating spinal cord injury （SCI） with adult stem cells led us to evaluate the effects of transplantation of hair follicle stem cells in rats with a compression-induced spinal cord lesion. Here, we proposed a hypothesis that rat hair follicle stem cell transplantation can promote the recovery of injured spinal cord. Compression-induced spinal cord injury was induced in Wistar rats in this study. The bulge area of the rat vibdssa follicles was isolated, cultivated and characterized with nestin as a stem cell marker. 5-Bromo-2＇-deoxyuridine （BrdU） labeled bulge stem cells were transplanted into rats with spinal cord injury. Immunohistochemical staining results showed that some of the grafted cells could survive and differentiate into oligodendrocytes （receptor-interacting protein positive cells） and neuronal-like cells （~lll-tubulin positive cells） at 3 weeks after transplantation. In addition, recovery of hind limb locomotor function in spinal cord injury rats at 8 weeks following cell transplantation was assessed using the Basso, Beattie and Bresnahan （BBB） locomotor rating scale. The results demon- strate that the grafted hair follicle stem cells can survive for a long time period in vivo and differentiate into neuronal- and glial-like cells. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury.     

Transplantation of nasal olfactory tissue promotes partial recovery in paraplegic adult rats

Recent reports have highlighted the potential therapeutic role of olfactory ensheathing cells for repair of spinal cord injuries. Previously ensheathing cells collected from the olfactory bulbs within the skull were used. In humans a source of these cells for autologous therapy lies in the nasal mucosa where they accompany the axons of the olfactory neurons. The aim of the present study was to test the therapeutic potential of nasal olfactory ensheathing cells for spinal cord repair. Olfactory ensheathing cells cultured from the olfactory lamina propria or pieces of lamina propria from the olfactory mucosa were transplanted into the transected spinal cord. Three to ten weeks later these animals partially recovered movement of their hind limbs and joints which was abolished by a second spinal cord transection. Control rats, receiving collagen matrix, respiratory lamina propria or culture medium, did not recover hind limb movement. Recovery of movement was associated with recovery of spinal reflex circuitry, assessed using the rate-sensitive depression of the H-reflex from an interosseous muscle. Histological analysis of spinal cords grafted with olfactory tissue demonstrated nerve fibres passing through the transection site, serotonin-positive fibres in the spinal cord distal to the transection site, and retrograde labelling of brainstem raphe and gigantocellularis neurons from injections into the distal cord, indicating regeneration of descending pathways. Thus, olfactory lamina propria transplantation promoted partial restoration of function after relatively short recovery periods. This study is particularly significance because it suggests an accessible source of tissue for autologous grafting in human paraplegia.     

Olfactory ensheathing cells from the nose: Clinical application in human spinal cord injuries

Olfactory mucosa, the sense organ of smell, is an adult tissue that is regenerated and repaired throughout life to maintain the integrity of the sense of smell. When the sensory neurons of the olfactory epithelium die they are replaced by proliferation of stem cells and their axons grow from the nose to brain assisted by olfactory ensheathing cells located in the lamina propria beneath the sensory epithelium. When transplanted into the site of traumatic spinal cord injury in rat, olfactory lamina propria or purified olfactory ensheathing cells promote behavioural recovery and assist regrowth of some nerves in the spinal cord. A Phase I clinical trial demonstrated that autologous olfactory ensheathing cell transplantation is safe, with no adverse outcomes recorded for three years following transplantation. Autologous olfactory mucosa transplantation is also being investigated in traumatic spinal cord injury although this whole tissue contains many cells in addition to olfactory ensheathing cells, including stem cells. If olfactory ensheathing cells are proven therapeutic for human spinal cord injury there are several important practical issues that will need to be solved before they reach general clinical application. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.