脐血干细胞移植治疗中间型脊髓性肌萎缩症1 例并文献复习

目的:探讨脐血干细胞移植治疗中间型脊髓性肌萎缩症的临床治疗可行性及效果。方法:已确诊的中间型脊髓性肌萎缩症患儿,采用脐血干细胞移植治疗,4次为一疗程,移植途径采用静脉输注(1次)加蛛网膜下腔注射(3次)的方法,治疗前和治疗后半年均需完善神经系统体检、实验室检查、FIM评分、肌电图等。结果:移植治疗后患儿神经系统症状明显改善,FIM评分提高,实验室检查肌酶下降,肌电图提示重收缩每10.0ms所检肌运动单位较前增加。随访10月患儿未出现副反应。结论:应用脐血干细胞移植治疗中间型脊髓性肌萎缩症是有效安全的,可以改善患儿神经功能。     

脐血干细胞移植治疗中间型脊髓性肌萎缩症1例并文献复习

目的：探讨脐血干细胞移植治疗中间型脊髓性肌萎缩症的临床治疗可行性及效果。方法：已确诊的中间型脊髓性肌萎缩症患儿,采用脐血干细胞移植治疗,4次为一疗程,移植途径采用静脉输注（1次）加蛛网膜下腔注射（3次）的方法,治疗前和治疗后半年均需完善神经系统体检、实验室检查、FIM评分、肌电图等。结果：移植治疗后患儿神经系统症状明显改善,FIM评分提高,实验室检查肌酶下降,肌电图提示重收缩每10.0ms所检肌运动单位较前增加。随访10月患儿未出现副反应。结论：应用脐血干细胞移植治疗中间型脊髓性肌萎缩症是有效安全的,可以改善患儿神经功能。     

Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation

Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia.Together, these findings reveal that multipotent self-renewing NSPC cultured and passaged from adult human spinal cords of organ transplant donors, respond to exogenous factors that promote selective differentiation, and survive and differentiate after transplantation into the injured spinal cord.     

神经移植的新途径--移植的中枢神经元从蛛网膜下腔迁入脊髓和大脑皮层

我们在神经移植的天空过程中观察到被移植的中枢神经元能从蛛网膜下腔迁入脊髓的大脑皮层。这一新观察为脊髓和脑浅层大范围神经元缺损时的无损伤神经元引入和大范围去神经区域的神经再支配提供了一种颇具吸引力的河能性。实验动物选用Ｗｉｓｔａｒ和Ｓ．Ｄ．大鼠,将含有胚胎中枢单胺或精氨酸血管加压素（ＡＶＰ）能神经元的细胞悬浮液或组织块移植到被横断的脊髓或未被脊髓和脑的蛛网膜下腔内。动物分别在移植的同时切断脊髓;在移     

Comparison of mesenchymal stromal cells from human bone marrow and adipose tissue for the treatment of spinal cord injury

Background aimsBone marrow and subcutaneous adipose tissue are both considered prospective sources of mesenchymal stromal cells (MSCs), which can be used in cell therapy for spinal cord injury (SCI). The present study investigated whether human adipose tissue-derived mesenchymal stromal cells (hADSCs) transplanted into a rat model of SCI would lead to similar or improved neurologic effects compared with human bone marrow-derived mesenchymal stromal cells (hBMSCs).MethodshADSCs and hBMSCs were isolated from five adult donors. These MSCs were characterized using flow cytometry, immunocytochemistry, real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Immediately after SCI, 2 × 10⁵ hBMSCs or hADSCs were injected into the injured spinal cord. Locomotor function, cell survival and differentiation, spinal cord tissue morphology and brain-derived neurotrophic factor (BDNF) expression were compared between groups.ResultshADSCs and hBMSCs showed similar surface protein expression, and hADSCs showed higher proliferative activity with higher expression of vascular endothelial cell growth factor, hepatocyte growth factor and BDNF than hBMSCs. After transplant, both hADSCs and hBMSCs migrated within the injured spinal cord without differentiating into glial or neuronal elements. Administration of hADSCs was associated with marked changes in the SCI environment, with significant increases in BDNF levels. This was simultaneously associated with increased angiogenesis, preserved axons, decreased numbers of ED1-positive macrophages and reduced lesion cavity formation. These changes were accompanied by improved functional recovery.ConclusionsThe present results suggest that hADSCs would be more appropriate for transplant to treat SCI than hBMSCs.     

移植的5—羟色胺能神经元跨软脊膜迁入大鼠脊髓的研究

本文对移植的5-HT神经元从蛛网膜下腔跨软脊膜迁移进入脊髓作了初步研究。将含有5-HT细胞的胚胎中缝核组织小块或神经细胞悬浮液作为移植物,以5-HT免疫组织化学方法跟踪移植细胞,结果如下:(1)在低胸水平横切脊髓,10d后,横断脊髓内的5-HT纤维消失。(2)横切脊髓(方法同上)后,立即将中缝核组织小块移植在胸腰段脊髓的蛛网膜下腔,一月后.在横断脊髓内出现5-HT阳性神经元和纤维。5-HT纤维能在灰白质内延伸。(3)脊髓横断后,若以中缝核的细胞悬浮液代替组织小块,作上述移植,则在移植区附近的灰质内出现大量的5-HT阳性神经元。这些神经元在灰质内的分布范围与神经细胞悬浮液在蛛网膜下腔的移植范围相一致。迁入神经元能在灰质内重新形成5-HT阳性纤维网。(4)经上述移植后,灰质内出现的5-HT阳性纤维随远离细胞体而变得稀疏。白质内的5-HT阳性纤维远比灰质内稀少。本实验结果表明:移植在脊髓蛛网膜下腔的脑干5-HT细胞能跨软脊膜迁移进入脊髓。     

Olfactory ensheathing cells - another miracle cure for spinal cord injury?

Several recent publications describe remarkably promising effects of transplanting olfactory ensheathing cells as a potential future method to repair human spinal cord injuries. But why were cells from the nose transplanted into the spinal cord? What are olfactory ensheathing cells, and how might they produce these beneficial effects? And more generally, what do we mean by spinal cord injury? To what extent can we compare repair in an animal to repair in a human?     

Differential fate of multipotent and lineage-restricted neural precursors following transplantation into the adult CNS

Multiple classes of precursor cells have been isolated and characterized from the developing spinal cord including multipotent neuroepithelial (NEP) stem cells and lineage-restricted precursors for neurons (NRPs) and glia (GRPs). We have compared the survival, differentiation and integration of multipotent NEP cells with lineage-restricted NRPs and GRPs using cells isolated from transgenic rats that express the human placental alkaline phosphatase gene. Our results demonstrate that grafted NEP cells survive poorly, with no cells observed 3 days after transplant in the adult hippocampus, striatum and spinal cord, indicating that most CNS regions are not compatible with transplants of multipotent cells derived from fetal CNS. By contrast, at 3 weeks and 5 weeks post-engraftment, lineage-restricted precursors showed selective migration along white-matter tracts and robust survival in all three CNS regions. The grafted precursors expressed the mature neuronal markers NeuN and MAP2, the astrocytic marker GFAP, the oligodendrocytic markers RIP, NG2 and Sox-10, and the synaptic marker synaptophysin. Similar behavior was observed when these precursors were transplanted into the injured spinal cord. Predifferentiated, multipotent NEP cells also survive and integrate, which indicates that lineage-restricted CNS precursors are well suited for transplantation into the adult CNS and provide a promising cellular replacement candidate.     

Transplantation of human umbilical mesenchymal stem cells from Wharton's jelly after complete transection of the rat spinal cord

Background

Human umbilical mesenchymal stem cells (HUMSCs) isolated from Wharton''s jelly of the umbilical cord can be easily obtained and processed compared with embryonic or bone marrow stem cells. These cells may be a valuable source in the repair of spinal cord injury.

Methodology/Principal Findings

We examine the effects of HUMSC transplantation after complete spinal cord transection in rats. Approximately 5×10⁵ HUMSCs were transplanted into the lesion site. Three groups of rats were implanted with either untreated HUMSCs (referred to as the stem cell group), or HUMSCs treated with neuronal conditioned medium (NCM) for either three days or six days (referred to as NCM-3 and NCM-6 days, respectively). The control group received no HUMSCs in the transected spinal cord. Three weeks after transplantation, significant improvements in locomotion were observed in all the three groups receiving HUMSCs (stem cell, NCM-3 and NCM-6 days groups). This recovery was accompanied by increased numbers of regenerated axons in the corticospinal tract and neurofilament-positive fibers around the lesion site. There were fewer microglia and reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the stem cell group than in the control group. Transplanted HUMSCs survived for 16 weeks and produced large amounts of human neutrophil-activating protein-2, neurotrophin-3, basic fibroblast growth factor, glucocorticoid induced tumor necrosis factor receptor, and vascular endothelial growth factor receptor 3 in the host spinal cord, which may help spinal cord repair.

Conclusions/Significance

Transplantation of HUMSCs is beneficial to wound healing after spinal cord injury in rats.     

A Neonatal Mouse Spinal Cord Injury Model for Assessing Post-Injury Adaptive Plasticity and Human Stem Cell Integration

Despite limited regeneration capacity, partial injuries to the adult mammalian spinal cord can elicit variable degrees of functional recovery, mediated at least in part by reorganization of neuronal circuitry. Underlying mechanisms are believed to include synaptic plasticity and collateral sprouting of spared axons. Because plasticity is higher in young animals, we developed a spinal cord compression (SCC) injury model in the neonatal mouse to gain insight into the potential for reorganization during early life. The model provides a platform for high-throughput assessment of functional synaptic connectivity that is also suitable for testing the functional integration of human stem and progenitor cell-derived neurons being considered for clinical cell replacement strategies. SCC was generated at T9–T11 and functional recovery was assessed using an integrated approach including video kinematics, histology, tract tracing, electrophysiology, and high-throughput optical recording of descending inputs to identified spinal neurons. Dramatic degeneration of axons and synaptic contacts was evident within 24 hours of SCC, and loss of neurons in the injured segment was evident for at least a month thereafter. Initial hindlimb paralysis was paralleled by a loss of descending inputs to lumbar motoneurons. Within 4 days of SCC and progressively thereafter, hindlimb motility began to be restored and descending inputs reappeared, but with examples of atypical synaptic connections indicating a reorganization of circuitry. One to two weeks after SCC, hindlimb motility approached sham control levels, and weight-bearing locomotion was virtually indistinguishable in SCC and sham control mice. Genetically labeled human fetal neural progenitor cells injected into the injured spinal cord survived for at least a month, integrated into the host tissue and began to differentiate morphologically. This integrative neonatal mouse model provides opportunities to explore early adaptive plasticity mechanisms underlying functional recovery as well as the capacity for human stem cell-derived neurons to integrate functionally into spinal circuits.     

间充质干细胞-透明质酸-多聚赖氨酸治疗脊髓损伤的实验研究

目的研究间充质干细胞—透明质酸—多聚赖氨酸复合物治疗脊髓损伤的可行性,评价其治疗效果并探讨其可能机制。方法从人骨髓中分离、培养人骨髓间充质干细胞（human bone marrow mesenchymal stem cell,hBMSC）;制作大鼠脊髓半横断模型,按照实验分组分别将hBMSC、透明质酸-多聚赖氨酸（hyaluronic acid-poly-L-lysine,HA-PLL）、hBMSC-HA-PLL复合物注入损伤区域,单纯损伤组作为对照。术后按照不同时间点评价损伤和移植后的大鼠运动功能。8周后杀死大鼠,观察不同移植组体内轴突和血管生长的情况,对不同细胞、材料及复合物移植对大鼠脊髓损伤修复效果进行评估。结果 hBMSC移植组和hBMSC-HA-PLL移植组的大鼠运动功能的改善显著好于单纯损伤及HA-PLL移植组。电镜结果证实复合物移植组可显著促进轴突和血管生长,新生的轴突和血管结构较为完整。结论 hBMSC具有促进神经功能恢复的作用,将其与HA-PLL相结合,可以促进大鼠脊髓损伤修复,其机制可能包括材料框架作用和hBMSC在体内对大鼠神经细胞的营养作用以及促进微血管的生成。     

三七总皂苷对脊髓损伤后BDNF 的表达及运动功能的影响

目的:探讨三七总皂苷(total panax notoginseng saponins,tPNS)对脊髓半横断损伤后对脑源性神经营养因子(Brain-derivedneurotrophic factor,BDNF)表达以及运动功能恢复的作用的影响。方法:大鼠随机分为正常组和实验组,实验组大鼠脊髓T10右侧半横断模型,损伤后15min,腹腔注射三七总皂苷,剂量为20mg.kg-1,以后每天给药一次,溶媒对照组注射等量生理盐水。术后进行BBB评分和斜板实验检测;动物分别存活1d、3d、7d、14d、28d后,采用免疫荧光化学方法检测脊髓损伤远侧端BDNF表达的变化。结果:BBB评分及斜板实验结果显示,三七总皂苷能明显促进脊髓损伤后运动功能的恢复,尤其是损伤后7d和14d,三七总皂苷组评分明显高于溶媒对照组。免疫组化结果显示:脊髓半横断损伤后,损伤远侧端损伤侧BDNF的表达强于对侧,损伤侧BDNF的表达呈现出1d,3d逐渐增强,7d达高峰的趋势,14dBDNF的表达逐渐下降,至28d仍略高于正常组。三七总皂苷组和溶媒对照组相比,BDNF表达的时间趋势相同,但相同时间点BDNF的表达强于对照组,尤其是3d、7d。结论:三七总皂苷能增强脊髓半横断损伤后BDNF的表达,这可能是其改善脊髓再生的微环境,促进脊髓损伤后运动功能恢复的机制之一。     

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck & Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.     

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.     

Characterization of the immune response to a secondary encephalitogenic epitope of basic protein in Lewis rats. II. Biased T cell receptor V beta expression predominates in spinal cord infiltrating T cells.

The immune response of Lewis rat lymph node T cells to guinea pig myelin basic protein (GP-BP) in experimental allergic encephalomyelitis is directed primarily against a region of basic protein encompassed by residues 72-89. T cells that respond to this epitope are restricted by the RT1.B class II molecule of the MHC and use V beta 8.2 exclusively in their TCR. A second region of GP-BP, residues 87-99, also induces experimental allergic encephalomyelitis in Lewis rats but this response is restricted primarily by RT1.D. Elsewhere we describe the biologic characteristics of T cell clones responding to the synthetic peptide, s87-99, and to a related peptide, s85-99. We present a detailed analysis of TCR V beta gene expression among these clones, derived from the lymph node and spinal cord of immunized animals, and among spinal cord derived T cell clones reactive to GP-BP 72-89. We find that spinal cord-derived clones, reactive to s85-99 and to s87-99, use V beta 6 predominantly. In contrast, T cell clones derived from lymph nodes and reactive to the same peptides express multiple V beta genes including V beta 6. This difference in heterogeneity of V beta usage at the clonal level is also seen in T cell lines derived from spinal cord and immune lymph node. DNA sequence comparison of the CDR3 regions in V beta 6+ spinal cord clones revealed a conserved amino acid motif also found in the majority of V beta 6 sequences from the spinal cord anti-s85-99 line. Although V beta 6 was expressed in some lymph node-derived clones, only one contained a CDR3 region similar to that seen in spinal cord isolates. All spinal cord-derived T cell clones reactive to GP-BP 72-89 used V beta 8.2 and most (five of six) contained the AspSer residues in CDR3 previously shown to be associated with V beta 8.2 receptors expressed by the majority of lymph node T cells responding to GP-BP 72-89. These data indicate that TCR V beta usage in peripheral T cells responding to an autoantigen does not always predict the V beta usage among T cells at the site of an autoimmune attack. Possible explantations for the relative homogeneity in TCR V beta expression seen in T cell clones derived from the spinal cord are discussed.     

Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5 cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.     

Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5?cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.     

Human neural stem cells and astrocytes,but not neurons,suppress an allogeneic lymphocyte response

Transplantation of human neural stem cells (NSCs) and their derivatives is a promising future treatment for neurodegenerative disease and traumatic nervous system lesions. An important issue is what kind of immunological reaction the cellular transplant and host interaction will result in. Previously, we reported that human NSCs, despite expressing MHC class I and class II molecules, do not trigger an allogeneic T cell response. Here, the immunocompetence of human NSCs, as well as differentiated neural cells, was further studied. Astrocytes expressed both MHC class I and class II molecules to a degree equivalent to that of the NSCs, whereas neurons expressed only MHC class I molecules. Neither the NSCs nor the differentiated cells triggered an allogeneic lymphocyte response. Instead, these potential donor NSCs and astrocytes, but not the neurons, exhibited a suppressive effect on an allogeneic immune response. The suppressive effect mediated by NSCs most likely involves cell–cell interaction. When the immunogenicity of human NSCs was tested in an acute spinal cord injury model in rodent, a xenogeneic rejection response was triggered. Thus, human NSCs and their derived astrocytes do not initiate, but instead suppress, an allogeneic response, while they cannot block a graft rejection in a xenogeneic setting.