Transplantation of an adipose stem cell cluster in a spinal cord injury

We investigated whether transplantation of a three-dimensional cell mass of adipose-derived stem cells (3DCM-ASCs) improved hind limb functional recovery by the stimulation of angiogenesis and neurogenesis in a spinal cord injury. In in-vitro experiments, we confirmed that 3DCM-ASCs differentiated into CD31-positive endothelial cells. To evaluate the therapeutic effect of 3DCM-ASCs in vivo, PBS, human adipose tissue-derived stem cells, and 3DCM-ASCs were transplanted into a spinal cord injury model. The 3DCM-ASCs transplanted into the injured spinal cord differentiated into CD31-positive endothelial cells and remained differentiated. Transplantation of 3DCM-ASCs into the injured spinal cord significantly elevated the density of vascular formations through angiogenic factors released by the 3DCM-ASCs at the lesion site, and enhanced axonal outgrowth at the lesion site. Consistent with these results, the transplantation of 3DCM-ASCs significantly improved functional recovery compared with both ASC transplantation and PBS treatment. These findings suggest that transplantation of 3DCM-ASCs may be an effective stem cell therapy for the treatment of spinal cord injuries and neural ischemia.     

Hypoxia-specific VEGF-expressing neural stem cells in spinal cord injury model

We established three stable neural stem cell (NSC) lines to explore the possibility of using hypoxia-specific vascular endothelial growth factor (VEGF) expressing NSC lines (EpoSV-VEGF NSCs) to treat spinal cord injury. The application of EpoSV-VEGF NSCs into the injured spinal cord after clip compression injury not only showed therapeutic effects such as extended survival and angiogenesis, but also displayed its safety profile as it did not cause unwanted cell proliferation or angiogenesis in normal spinal cord tissue, as EpoSV-VEGF NSCs consistently showed hypoxia-specific VEGF expression patterns. This suggests that our EpoSV-VEGF NSCs are both safe and therapeutically efficacious for the treatment of spinal cord injury. Furthermore, this hypoxia-inducible gene expression system may represent a safe tool suitable for gene therapy.     

Multipotent embryonic spinal cord stem cells expanded by endothelial factors and Shh/RA promote functional recovery after spinal cord injury

Cell transplantation is a promising way to treat spinal cord injury and neurodegenerative disorders. Neural stem cells taken from the embryonic spinal cord are an appealing source of cells for transplantation because these cells are committed to making spinal cord progeny. However these stem cells are rare and require expansion in tissue culture to generate sufficient cells for transplantation. We have developed a novel method for expanding embryonic mouse spinal cord stem cells using a co-culture system with endothelial cells. This method improves neural stem cell survival and preserves their multipotency, including their ability to make motor neurons. Transplantation of endothelial-expanded neural stem cells that were treated with sonic hedgehog(Shh) and retinoic acid (RA) during the expansion phase, into an adult mouse SCI model resulted in significant recovery of sensory and motor function.     

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.     

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.     

Co-transplantation of schwann cells promotes the survival and differentiation of neural stem cells transplanted into the injured spinal cord

The present study investigates whether Schwann cells (SCs) could promote the survival and differentiation of neural stem cells in the injured spinal cord. Neural stem cells were dissociated and cloned from the hippocampal tissue of newborn rats. SCs were also dissociated and purified simultaneously from the sciatic nerves of 4-day-old rats. The results showed that the number of surviving neural stem cells and differentiated neuron-like cells was significantly increased in the co-grafted (SCs and neural stem cells) group compared with the control group (neural stem cells only). Neuron-like cells that developed axon-like processes were observed more commonly in the co-grafted group. These results demonstrate that SCs can promote the survival and differentiation of transplanted neural stem cells in the injured spinal cord.     

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury

Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair 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。     

New strategies for repairing the injured spinal cord: the role of stem cells

Thanks to advances in the stem cell biology of the central nervous system, the previously unconceivable regeneration of the damaged spinal cord is approaching reality. A number of potential strategies aim to optimize functional recovery after spinal cord injury. They include minimizing the progression of secondary injury, manipulating the inhibitory environment of the spinal cord, replacing lost tissue with transplanted cells or peripheral nerve grafts, remyelinating denuded axons and maximizing the intrinsic regenerative potential of endogenous progenitor cells. We review the application of stem cell transplantation to the spinal cord, emphasizing the use of embryonic stem cells for remyelinating damaged axons. Recent advancements in neural injury and repair, and the progress towards development of neuroprotective and regenerative interventions are discussed.     

组织工程支架在犬急性脊髓损伤修复中应用的初步研究

目的探讨携带神经干细胞的聚碳酸亚丙酯[poly（propylene carbonate），PPC]可降解支架移植在犬脊髓急性损伤后修复中的作用。方法制作犬T13脊髓左侧半切损伤模型。将实验动物随机分为3组：细胞支架组在损伤后1周时将填充神经干细胞的PPC可降解支架植入损伤区．支架组只植入支架，对照组不作移植。8周后观察支架的组织反应、降解情况及神经干细胞的迁移分化和脊髓轴突再生情况。结果支架部分降解，管腔内未见瘢痕侵入。神经干细胞向支架邻近部位广泛迁移、扩散，并分化为3种神经细胞表型。神经丝蛋白（NF）及髓鞘碱性磷酯蛋白（MBP）免疫组化显示细胞支架组脊髓损伤区邻近部位的继发损害较其他组轻。结论携带神经干细胞的PPC可降解支架在犬脊髓组织中无明显组织反应．能够抵御瘢痕侵入：其携带的神经干细胞能够整合入邻近脊髓组织并起到一定保护作用。     

Tryptophan 2,3-dioxygenase is a key modulator of physiological neurogenesis and anxiety-related behavior in mice

Background

Transplantation of neural stem/progenitor cells is a promising approach toward functional restoration of the damaged neural tissue, but the injured spinal cord has been shown to be an adverse environment for the survival, migration, and differentiation of the donor cells. To improve the efficiency of cell replacement therapy, cell autonomous factors in the donor cells should be optimized. In light of recent ffindings that Rho family GTPases regulate stem cell functions, genetic manipulation of Rho GTPases can potentially control phenotypes of transplanted cells. Therefore we expressed mutant forms of Rho GTPases, Rac, Rho, and Cdc42, in the neural stem/progenitor cells and examined their survival and migration after transplantation.

Results

Manipulation of the individual Rho GTPases showed differential effects on survival, with little variation in their migratory route and predominant differentiation into the oligodendroglial lineage. Combined suppression of both Rac and Rho activity had a prominent effect on promoting survival, consistent with its highly protective effect on drug-induced apoptosis in culture.

Conclusion

Manipulation of Rac and Rho activities fully rescued suppression of cell survival induced by the spinal cord injury. Our results indicate that precise regulation of cell autonomous factors within the donor cells can ameliorate the detrimental environment created by the injury.     

Human fetal neural stem cells grafted into contusion-injured rat spinal cords improve behavior

Grafted human neural stem cells (hNSCs) may help to alleviate functional deficits resulting from spinal cord injury by bridging gaps, replacing lost neurons or oligodendrocytes, and providing neurotrophic factors. Previously, we showed that primed hNSCs differentiated into cholinergic neurons in an intact spinal cord. In this study, we tested the fate of hNSCs transplanted into a spinal cord T10 contusion injury model. When grafted into injured spinal cords of adult male rats on either the same day or 3 or 9 days after a moderate contusion injury, both primed and unprimed hNSCs survived for 3 months postengraftment only in animals that received grafts at 9 days postinjury. Histological analyses revealed that primed hNSCs tended to survive better and differentiated at higher rates into neurons and oligodendrocytes than did unprimed counterparts. Furthermore, only primed cells gave rise to cholinergic neurons. Animals receiving primed hNSC grafts on the ninth day postcontusion improved trunk stability, as determined by rearing activity measurements 3 months after grafting. This study indicates that human neural stem cell fate determination in vivo is influenced by the predifferentiation stage of stem cells prior to grafting. Furthermore, stem cell-mediated facilitation of functional improvement depends on the timing of transplantation after injury, the grafting sites, and the survival of newly differentiated neurons and oligodendrocytes.     

Consequences of noggin expression by neural stem, glial, and neuronal precursor cells engrafted into the injured spinal cord

Bone morphogenetic proteins (BMPs) are a large class of secreted factors, which serve as modulators of development in multiple organ systems, including the CNS. Studies investigating the potential of stem cell transplantation for restoration of function and cellular replacement following traumatic spinal cord injury (SCI) have demonstrated that the injured adult spinal cord is not conducive to neurogenesis or oligodendrogenesis of engrafted CNS precursors. In light of recent findings that BMP expression is modulated by SCI, we hypothesized that they may play a role in lineage restriction of multipotent grafts. To test this hypothesis, neural stem or precursor cells were engineered to express noggin, an endogenous antagonist of BMP action, prior to transplantation or in vitro challenge with recombinant BMPs. Adult rats were subjected to both contusion and focal ischemic SCI. One week following injury, the animals were transplanted with either EGFP- or noggin-expressing neural stem or precursor cells. Results demonstrate that noggin expression does not antagonize terminal astroglial differentiation in the engrafted stem cells. Furthermore, neutralizing endogenous BMP in the injured spinal cord significantly increased both the lesion volume and the number of infiltrating macrophages in injured spinal cords receiving noggin-expressing stem cell grafts compared with EGFP controls. These data strongly suggest that endogenous factors in the injured spinal microenvironment other than the BMPs restrict the differentiation of engrafted pluripotent neural stem cells as well as suggest other roles for BMPs in tissue protection in the injured CNS.     

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.     

神经干细胞移植治疗脊髓损伤的研究与现状

近年来,神经干细胞的发现及其相关研究为脊髓损伤的治疗开辟了一条新途径,神经干细胞因与脊髓损伤区域的细胞同源而具有独特的治疗优势。随着研究的不断深入,胚胎源神经干细胞,永生化神经干细胞,胚胎和成体神经干细胞作为移植的供体细胞以及基因治疗的载体用于临床治疗。文章就脊髓损伤的机制、神经干细胞的基本特性、增殖和定向诱导分化机制及其在脊髓损伤修复中的应用问题进行系统的分析和综述,并对神经干细胞移植治疗脊髓损伤的应用及存在问题进行了展望。     

Engineering angiogenesis following spinal cord injury: a coculture of neural progenitor and endothelial cells in a degradable polymer implant leads to an increase in vessel density and formation of the blood–spinal cord barrier

Angiogenesis precedes recovery following spinal cord injury and its extent correlates with neural regeneration, suggesting that angiogenesis may play a role in repair. An important precondition for studying the role of angiogenesis is the ability to induce it in a controlled manner. Previously, we showed that a coculture of endothelial cells (ECs) and neural progenitor cells (NPCs) promoted the formation of stable tubes in vitro and stable, functional vascular networks in vivo in a subcutaneous model. We sought to test whether a similar coculture would lead to the formation of stable functional vessels in the spinal cord following injury. We created microvascular networks in a biodegradable two-component implant system and tested the ability of the coculture or controls (lesion control, implant alone, implant + ECs or implant + NPCs) to promote angiogenesis in a rat hemisection model of spinal cord injury. The coculture implant led to a fourfold increase in functional vessels compared with the lesion control, implant alone or implant + NPCs groups and a twofold increase in functional vessels over the implant + ECs group. Furthermore, half of the vessels in the coculture implant exhibited positive staining for the endothelial barrier antigen, a marker for the formation of the blood–spinal cord barrier. No other groups have shown positive staining for the blood–spinal cord barrier in the injury epicenter. This work provides a novel method to induce angiogenesis following spinal cord injury and a foundation for studying its role in repair.     

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.     

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.     

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.     

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.