Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats

Neural progenitor cells, including neural stem cells, are a potential expandable source of graft material for transplantation aimed at repairing the damaged CNS. Here we present the first evidence that in vitro-expanded fetus-derived neurosphere cells were able to generate neurons in vivo and improve motor function upon transplantation into an adult rat spinal-cord-contusion injury model. As the source of graft material, we used a neural stem cell-enriched population that was derived from rat embryonic spinal cord (E14.5) and expanded in vitro by neurosphere formation. Nine days after contusion injury, these neurosphere cells were transplanted into adult rat spinal cord at the injury site. Histological analysis 5 weeks after the transplantation showed that mitotic neurogenesis occurred from the transplanted donor progenitor cells within the adult rat spinal cord, a nonneurogenic region; that these donor-derived neurons extended their processes into the host tissues; and that the neurites formed synaptic structures. Furthermore, analysis of motor behavior using a skilled reaching task indicated that the treated rats showed functional recovery. These results indicate that in vitro-expanded neurosphere cells derived from the fetal spinal cord are a potential source for transplantable material for treatment of spinal cord injury.     

胚胎小鼠脊髓源性与纹状体源性神经干细胞的培养及分化特点

目的 探讨小鼠脊髓源性神经干细胞与纹状体源性神经干细胞的分离培养方法 及增殖特点,比较两种来源的神经干细胞发育时期上的异同,寻找更有利于脊髓损伤修复的种子细胞.方法 利用显微解剖、无血清培养和单细胞克隆技术在孕14 d小鼠的胎鼠的脊髓及纹状体中分离培养具有单细胞克隆能力的细胞,免疫荧光染色检测克隆细胞的神经巢蛋白(nestin)抗原和诱导分化后特异性成熟神经细胞抗原的表达,并比较两种来源的干细胞在培养及分化方向上的异同点.结果从胎鼠的脊髓和纹状体中成功分离出神经干细胞.两种来源的干细胞均具有连续克隆能力可传代培养,表达nestin.脊髓血清诱导分化后脊髓源性神经干细胞β-tubulinⅢ阳性细胞(13.5±0.8)较纹状体源性神经干细胞(17.4±1.1)减少,而nestin、GFAP阳性细胞明显增多(45.7±0.3vs 39.2±1.2;25.2±1.3 vs 18.8±0.9),差异均有统计学意义(P<0.05). 结论 依据细胞增殖特点和分化结果的区别,证实纹状体源性神经干细胞更适合用于移植修复脊髓损伤.     

Epidermal neural crest stem cells and their use in mouse models of spinal cord injury

Epidermal neural crest stem cell (EPI-NCSC) grafts cause a significant improvement in sensory connectivity and touch perception in the contused mouse spinal cord. EPI-NCSC are derived from the embryonic neural crest but reside in a postnatal location, the bulge of hair follicles. Both mouse and human EPI-NCSC are multipotent adult stem cells capable of generating all major neural crest derivatives. EPI-NCSC of mouse and human origin express the neural crest stem cell molecular signature, genes that were initially used to create induced pluripotent stem (iPS) cells, and other neural crest and global stem cell genes. Due to their origin in the neural folds and because they share a higher order stem cell, neural crest cells, and thus EPI-NCSC, are closely related to neural tube stem cells. This close ontological relationship with the spinal cord makes EPI-NCSC attractive candidates for cell-based therapy in spinal cord injury. In two different contusion models of spinal cord injury, we have shown that EPI-NCSC integrate into the murine spinal cord tissue and that subsets differentiate into GABAergic neurons and myelinating oligodendrocytes. Intraspinal EPI-NCSC do not form tumours. In the presence of EPI-NCSC grafts, but not in control animals, there is a 24% improvement of sensory connectivity and a substantial improvement in touch perception. Unilateral transplants leading to bilateral functional improvements suggest that underlying mechanisms include diffusible molecules. EPI-NCSC indeed express genes that encode neurotrophins, other trophic factors, angiogenic factors and metalloproteases. Intraspinal EPI-NCSC thus have multiple effects in the contused spinal cord, the sum of which can explain the observed functional improvements.     

Transplantation of placenta-derived mesenchymal stem cell-induced neural stem cells to treat spinal cord injury

Because of their strong proliferative capacity and multi-potency, placenta-derived mesenchymal stem cells have gained interest as a cell source in the field of nerve damage repair. In the present study, human placenta-derived mesenchymal stem ceils were induced to differentiate into neural stem cells, which were then transplanted into the spinal cord after local spinal cord injury in rats. The motor functional recovery and pathological changes in the injured spinal cord were observed for 3 successive weeks. The results showed that human placenta-derived mesenchymal stem cells can differentiate into neuron-like cells and that induced neural stem cells contribute to the restoration of injured spinal cord without causing transplant rejection. Thus, these cells promote the recovery of motor and sensory functions in a rat model of spinal cord injury. Therefore, human placenta-derived mesenchymal stem cells may be useful as seed cells during the repair of spinal cord injury.     

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。     

Multimodal treatment for spinal cord injury: a sword of neuroregeneration upon neuromodulation

Spinal cord injury is linked to the interruption of neural pathways,which results in irreversible neural dysfunction.Neural repair and neuroregeneration are critical goals and issues for rehabilitation in spinal cord injury,which require neural stem cell repair and multimodal neuromodulation techniques involving personalized rehabilitation strategies.Besides the involvement of endogenous stem cells in neurogenesis and neural repair,exogenous neural stem cell transplantation is an emerging effective method for repairing and replacing damaged tissues in central nervous system diseases.However,to ensure that endogenous or exogenous neural stem cells truly participate in neural repair following spinal cord injury,appropriate interventional measures(e.g.,neuromodulation)should be adopted.Neuromodulation techniques,such as noninvasive magnetic stimulation and electrical stimulation,have been safely applied in many neuropsychiatric diseases.There is increasing evidence to suggest that neuromagnetic/electrical modulation promotes neuroregeneration and neural repair by affecting signaling in the nervous system;namely,by exciting,inhibiting,or regulating neuronal and neural network activities to improve motor function and motor learning following spinal cord injury.Several studies have indicated that fine motor skill rehabilitation training makes use of residual nerve fibers for collateral growth,encourages the formation of new synaptic connections to promote neural plasticity,and improves motor function recovery in patients with spinal cord injury.With the development of biomaterial technology and biomechanical engineering,several emerging treatments have been developed,such as robots,brain-computer interfaces,and nanomaterials.These treatments have the potential to help millions of patients suffering from motor dysfunction caused by spinal cord injury.However,large-scale clinical trials need to be conducted to validate their efficacy.This review evaluated the efficacy of neural stem cells and magnetic or electrical stimulation combined with rehabilitation training and intelligent therapies for spinal cord injury according to existing evidence,to build up a multimodal treatment strategy of spinal cord injury to enhance nerve repair and regeneration.     

EGF-responsive neural stem cells are a transient population in the developing mouse spinal cord

The adult mouse forebrain, which exhibits substantial ongoing cell genesis, contains self-renewing multipotent neural stem cells that respond to epidermal growth factor (EGF), but the adult spinal cord, which exhibits limited cell genesis, does not. Spinal cord development is a process characterized by defined periods of cell histogenesis. Thus, in the present study we asked whether EGF-responsive neural stem cells are present within the spinal cord during development. At embryonic day (E) 11, subsequent to the onset of neurogenesis, only fibroblast growth factor (FGF) receptors and FGF-2 (requiring heparan sulphate)-responsive stem cells are present in the spinal cord. Between E12 and 14, at the peak of spinal cord neurogenesis and the onset of gliogenesis, EGF receptors appear along with clonally derived highly expandable EGF-responsive neural stem cells. Following the cessation of cell histogenesis, the adult spinal cord is largely devoid of both EGF receptors and EGF-responsive stem cells. On the other hand, the FGF receptor1c subtype and multipotent FGF-2-responsive neural stem cells are present in early development and in the adult. The order of appearance of spinal cord neural stem cells and in vitro lineage analysis suggests that a more primitive FGF-2-responsive stem cell produces the EGF-responsive stem cell. These findings suggest that EGF-responsive neural stem cells appear transiently in the spinal cord, during the peak period of cell histogenesis, but are no longer present in the relatively quiescent adult structure.     

Embryonic mouse spinal cord motor neuron hybrid cells.

Studies of motor neurons are difficult because of limitations in their isolation and culture. One solution is to produce clonal neural hybrid cells that can express motor neuron characteristics; we fused an aminopterin-sensitive and neomycin-resistant mouse neuroblastoma cell line to isolated embryonic mouse spinal cord motor neurons. Several hybrid neuron cell lines expressing high levels of choline acetyltransferase (CHAT) enzyme activity were found. These were cloned and clones with high CHAT activity isolated. The hybrid nature of cloned cells was confirmed by karyotyping and determining glucose phosphate isomerase allozymes. The availability of these embryonic clonal hybrid cells will enable molecular, physiological, and biochemical studies to define motor neuron-specific properties.     

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。     

Therapeutic potential of induced pluripotent stem cells for spinal cord injury

Once the safety issue has been overcome, induced pluripotent stem cells (iPSCs), which do not entail ethical or immunological concerns, may become the preferred cell source for regenerative medicine. Various types of iPSCs have been established by different methods, and each type exhibits different biological properties. Before iPSC-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Recently, we demonstrated the directed neural differentiation of mouse iPSCs and examined their therapeutic potential in a mouse spinal cord injury (SCI) model. Mouse iPSC-derived neural stem/progenitor cells (NS/PCs), which had been pre-evaluated as non-tumorigenic by their transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-scid) mouse brain, were transplanted into the spinal cord 9 days after SCI. Mouse iPSC-derived NS/PCs differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in re-myelination and induced the axonal re-growth, promoting motor functional recovery. Nevertheless, our results constitute only the first step toward clinical application. The safety and effectiveness of human iPSC-derived NS/PCs need to be more intensively investigated in future preclinical studies, for example, using non-human primate SCI models. In particular, human iPSCs established by delivering reprogramming factors using a safer method than retrovirus system, such as an integration-free virus system, virus-free system, or transgene-free system should be evaluated.     

组织工程脊髓移植治疗大鼠脊髓半切块状损伤

目的 研究组织工程脊髓移植治疗大鼠脊髓半切块状损伤的疗效.方法 以聚乳酸-羟基乙酸(PLGA)为细胞支架,多聚赖氨酸为细胞外基质,神经十细胞(NSCs)为种子细胞,体外构建组织工程脊髓.制作大鼠T10脊髓右半切块状损伤模型,随机分成3组:实验组在损伤区移植组织工程脊髓,对照组A移植NSCs,对照组B移植PLGA.移植治疗12周,每周均行BBB评分定量评价肢体运动功能.伤后第12周辣根过氧化物酶(HRP)神经逆行示踪评价脊髓传导束的恢复程度,并取损伤处脊髓组织行免疫组织化学染色,观察移植区的形态结构修复.结果 伤后12周实验组的BBB运动功能评分较对照组明显提高,差异有统计学意义(P＜0.05).HRP神经逆行示踪显示:实验组鼠右侧大脑组织中可见大量的HRP标记阳性神经元,而两对照组仅见有少量HRP阳性神经元;免疫组织化学染色显示:实验组移植区NF阳性神经元和GAP-43阳性神经轴索数量较多,修复了缺损,而对照组极少,仍留下不同程度的缺损.结论 组织工程脊髓移植治疗促进了半切块状损伤脊髓的形态结构修复和功能恢复,疗效明显优于单纯的NSCs移植和PLGA移植.     

Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury

The greatest challenge to successful treatment of spinal cord injury is the limited regenerative capacity of the central nervous system and its inability to replace lost neurons and severed axons following injury. Neural stem cell grafts derived from fetal central nervous system tissue or embryonic stem cells have shown therapeutic promise by differentiation into neurons and glia that have the potential to form functional neuronal relays across injured spinal cord segments. However, implementation of fetal-derived or embryonic stem cell-derived neural stem cell therapies for patients with spinal cord injury raises ethical concerns. Induced pluripotent stem cells can be generated from adult somatic cells and differentiated into neural stem cells suitable for therapeutic use, thereby providing an ethical source of implantable cells that can be made in an autologous fashion to avoid problems of immune rejection. This review discusses the therapeutic potential of human induced pluripotent stem cell-derived neural stem cell transplantation for treatment of spinal cord injury, as well as addressing potential mechanisms, future perspectives and challenges.     

Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pc DNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1–4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the subarachnoid cavity to help repair spinal cord injury and promote the recovery of spinal cord function better than neural stem cell transplantation alone. These findings may lead to significant improvements in the clinical treatment of spinal cord injuries.     

The developing human spinal cord contains distinct populations of neural precursors

It is becoming apparent that neural stem cells display some differences in their behaviour depending on the region of the CNS they originate from and on whether they are derived from embryonic or adult tissue. Whereas much work has focused on brain neural stem cells, less attention has been paid to spinal cord neural precursors, particularly in the developing human embryo. We briefly review here some of our work which points at some similarities between neural precursors in developing human spinal cords and in animals which can regenerate their spinal cord (e.g. tailed amphibians), and at differences in the properties of human neural precursors with spinal cord development. Altogether these studies suggest the existence of dynamic neural stem cell populations within the developing spinal cord. They also support the notion that thorough characterization of neural stem cells under different culture conditions and analysis of how these may affect their differentiation in vivo after grafting into different injury models is imperative if we are to develop effective cell therapy strategies for spinal cord injury and diseases.     

Neural stem cell transplantation in a double-layer collagen membrane with unequal pore sizes for spinal cord injur y repair

A novel double-layer collagen membrane with unequal pore sizes in each layer was designed and tested in this study.The inner,loose layer has about 100-μm-diameter pores,while the outer,compact layer has about 10-μm-diameter pores.In a rat model of incomplete spinal cord injury,a large number of neural stem cells were seeded into the loose layer,which was then adhered to the injured side,and the compact layer was placed against the lateral side.The results showed that the transplantation of neural stem cells in a double-layer collagen membrane with unequal pore sizes promoted the differentiation of neural stem cells,attenuated the pathological lesion,and significantly improved the motor function of the rats with incomplete spinal cord injuries.These experimental findings suggest that the transplantation of neural stem cells in a double-layer collagen membrane with unequal pore sizes is an effective therapeutic strategy to repair an injured spinal cord.     

Stem cell transplantation for treating spinal cord injury A literature comparison between studies of stem cells obtained from various sources

OBJECTIVE:To identify global research trends of stem cell transplantation for treating spinal cord injury using a bibliometric analysis of the Web of Science.DATA RETRIEVAL:We performed a bibliometric analysis of data retrievals for stem cell transplantation for treating spinal cord injury from 2002 to 2011 using the Web of Science.SELECTION CRITERIA:Inclusion criteria:(a) peer-reviewed articles on stem cell transplantation for treating spinal cord injury that were published and indexed in the Web of Science;(b) type of articles:original research articles,reviews,meeting abstracts,proceedings papers,book chapters,editorial material,and news items;and(c) year of publication:2002-2011.Exclusion criteria:(a) articles that required manual searching or telephone access;(b) documents that were not published in the public domain;and(c) a number of corrected papers from the total number of articles.MAIN OUTCOME MEASURES:(1) Annual publication output;(2) distribution according to country;(3) distribution according to institution;(4) distribution according to journals;(5) distribution according to funding agencies;and(6) top cited articles over the last 10 years.RESULTS:Bone marrow mesenchymal stem cells and embryonic stem cells have been widely used for treating spinal cord injury.In total,191 studies of bone marrow mesenchymal stem cell transplantation and 236 studies of embryonic stem cell transplantation for treating spinal cord injury appeared in the Web of Science from 2002 to 2011,and almost half of which were derived from American or Japanese authors and institutes.The number of studies of stem cell transplantation for treating spinal cord injury has gradually increased over the past 10 years.Most papers on stem cell transplantation for treating spinal cord injury appeared in journals with a particular focus on stem cell research,such as Stem Cells and Cell Transplantation.Although umbilical cord blood stem cells and adipose-derived stem cells have been studied for treating spinal cord injury,the number of published papers was much smaller,with only 21 and 17 records,respectively,in the Web of Science.CONCLUSION:Based on our analysis of the literature and research trends,we found that stem cells transplantation obtained from various sources have been studied for treating spinal cord injury;however,it is difficult for researchers to reach a consensus on this theme.     

Transplanted hematopoietic stem cells from bone marrow differentiate into neural lineage cells and promote functional recovery after spinal cord injury in mice

Recovery in central nervous system disorders is hindered by the limited ability of the vertebrate central nervous system to regenerate lost cells, replace damaged myelin, and re-establish functional neural connections. Cell transplantation to repair central nervous system disorders is an active area of research, with the goal of reducing functional deficits. Recent animal studies showed that cells of the hematopoietic stem cell (HSC) fraction of bone marrow transdifferentiated into various nonhematopoietic cell lineages. We employed a mouse model of spinal cord injury and directly transplanted HSCs into the spinal cord 1 week after injury. We evaluated functional recovery using the hindlimb motor function score weekly for 5 weeks after transplantation. The data demonstrated a significant improvement in the functional outcome of mice transplanted with hematopoietic stem cells compared with control mice in which only medium was injected. Fluorescent in situ hybridization for the Y chromosome and double immunohistochemistry showed that transplanted cells survived 5 weeks after transplantation and expressed specific markers for astrocytes, oligodendrocytes, and neural precursors, but not for neurons. These results suggest that transplantation of HSCs from bone marrow is an effective strategy for the treatment of spinal cord injury.     

BDNF-GFP转染神经干细胞修复大鼠脊髓损伤：发挥干细胞与神经因子的双重效应？

摘要 背景：神经干细胞移植入大鼠脊髓损伤模型可以促进功能恢复,基因治疗已被广泛用于治疗脊髓损伤。 目的：确定BDNF-GFP转染后神经干细胞移植对大鼠脊髓损伤的修复效果。 设计,时间和背景：本实验是在中国医科大学基础医学院发育生物学实验室与2009年5月至2010年1月完成。 材料：10只新生Wistar大鼠和88只2-3个月大,雌雄不限的Wistar大鼠。 方法：以携带BDNF-GFP基因的腺病毒转染神经干细胞。88只Wistar大鼠中假手术组8只, 80只大鼠制成T9左侧横断模型,并随机分成四组：BDNF和GFP修饰的神经干细胞移植组,GFP修饰的神经干细胞移植组;单纯神经干细胞移植组和模型组。在各神经干细胞移植组,脊髓损伤后向横断处显微注射等体积细胞,模型组在相同的部位注射等体积的PBS。 主要观察指标： BBB评分检测脊髓损伤模型运动功能恢复情况;制备脊髓损伤模型2周后取材,免疫组化评估BDNF-GFP转染的神经干细胞移植后的细胞学特点;制备脊髓损伤模型2、4、6、8周Real-time PCR检测脊髓横断处BDNF表达情况。 结果： BDNF-GFP转染后神经干细胞在脊髓半切模型中存活并表达BDNF和GFP,移植该细胞后的大鼠体内高表达具有生物活性的BDNF,且脊髓损伤动物运动功能较对照组明显恢复。 结论：移植BDNF-GFP转染后神经干细胞可能是一种修复脊髓损伤的有效的方法。 关键词：神经干细胞,脑源性神经营养因子;绿色荧光蛋白;脊髓损伤;移植。     

SHH方案诱导胚胎干细胞分化为神经干细胞移植促进小鼠损伤脊髓结构与功能的恢复

目的比较分析体外诱导小鼠胚胎干细胞分化为神经干细胞的方法并观察神经干细胞移植后对脊髓损伤小鼠的治疗作用。方法采用维甲酸(retinoic acid, RA)方案及音猬因子(sonihedgehog,SHH)方案进行体外诱导培养,然后用抗Nestin、NeuN、GFAP、O1抗体进行免疫细胞化学染色鉴定。脂质体转染法将LacZ基因导入小鼠胚胎干细胞,继而在SHH方案诱导后移植进入脊髓横断小鼠体内,分别于移植后1个月、2个月冰冻切片进行X-gal染色追踪ES细胞并对X-gal阳性细胞行免疫荧光组织化学染色鉴定细胞类型;BBB评分分析了小鼠后肢运动功能的恢复。结果RA与SHH方案得到的Nestin阳性细胞的比例分别为32.54%和68.51%。细胞移植后1个月、2个月均可在脊髓横断小鼠体内检测到大量的X-gal阳性细胞,它们与宿主的脊髓组织相整合。免疫荧光组织化学染色显示X-gal阳性细胞主要表达ChAT和MBP。细胞移植组BBB评分高于对照组。结论与RA相比,SHH是一种较为高效的诱导剂,经SHH诱导得到的神经干细胞移植到脊髓横断的小鼠体内至少可存活2个月,表达成熟用胆碱能神经元的标志及髓鞘碱性蛋白,并且可以促进脊髓损伤小鼠运动功能的恢复。