Spinal Cord-derived Neural Stem Cells Research Articles

Stem Cell Clinical Trials in Spinal Cord Injury: A Brief Review of Studies in the United States

Background: Although many therapeutic approaches have been attempted to treat spinal cord injury, cellular transplantation offers the greatest promise in reconstituting the architecture of the damaged cord. Methods: A literature review was conducted to search for clinical trials investigating stem cells as treatment for spinal cord injury in the United States. Results: Overall, eight studies met inclusion criteria. Of the included studies, four were identified as being terminated, suspended, or not yet recruiting. Two studies were identified as currently recruiting, including one phase one trial evaluating stereotactic injections of human spinal cord-derived neural stem cells in patients with chronic spinal cord injuries, and one trial of transplantation of autologous bone marrow derived stem cells via paraspinal injections, intravenous injections, and intranasal placement. One study was identified as an active study, a phase one trial of intrathecal injection of 100 million autologous, ex-vivo expanded, adipose-derived mesenchymal stem cells. One trial that was listed as completed is a phase 1/2a, dose escalation study, investigating stereotactic injection of human embryonic stem cell derived oligodendrocyte progenitor cells. Conclusions: Although few significant publications have emerged to this point, current trial results are promising.

Comparison of Regenerative Effects of Transplanting Three-Dimensional Longitudinal Scaffold Loaded-Human Mesenchymal Stem Cells and Human Neural Stem Cells on Spinal Cord Completely Transected Rats.

Stem cell-based therapy has been considered as a potential treatment to restore spinal cord injury (SCI) through reconstructing neural networks and providing a favorable microenvironment for neuronal survival, differentiation, and axonal outgrowth. Biomaterial scaffolds can promote cell attachment and survival, neuronal differentiation, and axonal outgrowth; therefore, they were used to combine with stem cells for implantation in SCI treatment. In addition, a longitudinal scaffold can guide regenerated axons with orientated growth and axial extension. Both human umbilical cord-derived mesenchymal stem cells (hMSCs) and human fetal spinal cord-derived neural stem cells (hNSCs) have been applied in clinical trials worldwide. To our knowledge, a parallel comparison of the therapeutic effects of hMSC and hNSC implantations has not been conducted. Hence, in this study, we grafted hMSCs or hNSCs seeded on longitudinal collagen sponge scaffolds into rats with completely transected SCI to examine differences in SCI repair. Both hMSCs and hNSCs had equivalent effects on reducing glial scar formation around the lesion gap. More neuronal class III β-tubulin-positive neurons and neurofilament-positive nerve fibers were found in the lesion cavity after hNSC implantation. In addition, hNSCs had better capabilities to improve motor function, attenuate inflammation, and promote cell survival than hMSCs. These encouraging results provide a clinical basis for future stem cell-based SCI therapies.

Aligned collagen scaffold combination with human spinal cord-derived neural stem cells to improve spinal cord injury repair.

Neural stem/progenitor cell (NSPC)-based spinal cord injury (SCI) therapy is expected to bridge the lesion site by transplanting exogenous NSPCs for replacement of lost cells. The transplanted NSPCs produce a microenvironment conducive to neuronal regeneration, and ultimately, functional recovery. Although both human fetal brain- and spinal cord- derived NSPCs (hbNSPCs and hscNSPCs, respectively) have been used for SCI repair, it remains unclear whether hscNSPCs are a more appropriate stem cell source for transplantation than hbNSPCs. Therefore, in this study, we transplanted hbNSPCs or hscNSPCs into rats with complete transection SCI to monitor their differences in SCI treatment. An aligned collagen sponge scaffold (ACSS) was used here for cell retention. Aligned biomaterial scaffolds provide a support platform and favorable morphology for cell growth and differentiation, and guide axial axonal extension. The ACSS fabricated by our group has been previously reported to improve spinal cord repair by promoting neuronal regeneration and remyelination. Compared with the hbNSPC-ACSS, the hscNSPC-ACSS effectively promoted long-term cell survival and neuronal differentiation and improved the SCI microenvironment by reducing inflammation and glial scar formation. Furthermore, the transplanted hscNSPC-ACSS improved recovery of locomotor functions. Therefore, hscNSPCs appear to be a superior cell source to hbNSPCs for SCI cell therapy with greater potential clinical applications.

Transplantation of spinal cord-derived neural stem cells for ALS: Analysis of phase 1 and phase 2 trials

Transplantation of spinal cord-derived neural stem cells for ALS: Analysis of phase 1 and phase 2 trials

Wnt3a is critical for endothelial progenitor cell-mediated neural stem cell proliferation and differentiation.

The present study aimed to determine whether co-culture with bone marrow-derived endothelial progenitor cells (EPCs) affects the proliferation and differentiation of spinal cord-derived neural stem cells (NSCs), and to investigate the underlying mechanism. The proliferation and differentiation of the NSCs were evaluated by an MTT cell proliferation and cytotoxicity assay, and immunofluorescence, respectively. The number of neurospheres and the number of β-tubulin III-positive cells were detected by microscopy. The wingless-type MMTV integration site family, member 3a (Wnt3a)/β-catenin signaling pathway was analyzed by western blot analysis and reverse transcription-quantitative polymerase chain reaction to elucidate the possible mechanisms of EPC-mediated NSC proliferation and differentiation. The results revealed that co-culture with EPCs significantly induced NSC proliferation and differentiation. In addition, co-culture with EPCs markedly induced the expression levels of Wnt3a and β-catenin and inhibited the phosphorylation of glycogen synthase kinase 3β (GSK-3β). By contrast, Wnt3a knockdown using a short hairpin RNA plasmid in the EPCs reduced EPC-mediated NSC proliferation and differentiation, accompanied by inhibition of the EPC-mediated expression of β-catenin, and its phosphorylation and activation of GSK-3β. Taken together, the findings of the present study demonstrated that Wnt3a was critical for EPC-mediated NSC proliferation and differentiation.

(285) Spinal cord-derived neural stem cells restore hyperalgesic responses previously diminished by chemical ablation of NK-1 expressing neurons in the rat spinal cord

(285) Spinal cord-derived neural stem cells restore hyperalgesic responses previously diminished by chemical ablation of NK-1 expressing neurons in the rat spinal cord

Analysis of graft survival in a trial of stem cell transplant in ALS.

ObjectiveThe first US Food and Drug Administration–approved clinical trial to treat amyotrophic lateral sclerosis (ALS) with neural stem cell–based therapy is in progress. The goal of the current study was to identify and assess the survival of human spinal cord–derived neural stem cells (HSSCs) transplanted into the spinal cord in patients with ALS.MethodsSpinal cords transplanted with HSSCs were examined from six autopsy cases. Homogenized tissues were interrogated for the presence of donor versus recipient DNA using real-time PCR methods (qPCR). Fluorescence in situ hybridization (FISH) was performed using DNA probes for XY chromosomes to identify male donor HSSCs in one female case, and immunohistochemistry (IHC) was used to characterize the identified donor cells.ResultsGenomic DNA from donor HSSCs was identified in all cases, comprising 0.67–5.4% of total tissue DNA in patients surviving 196 to 921 days after transplantation. In the one female patient a “nest” of cells identified on H&E staining were XY-positive by FISH, confirming donor origin. A subset of XY-positive cells labeled for the neuronal marker NeuN and stem cell marker SOX2.InterpretationThis is the first study to identify human neural stem cells transplanted into a human spinal cord. Transplanted HSSCs survived up to 2.5 years posttransplant. Some cells differentiated into neurons, while others maintained their stem cell phenotype. This work is a proof of concept of the survival and differentiation of human stems cell transplanted into the spinal cord of ALS patients.

FGL-functionalized self-assembling nanofiber hydrogel as a scaffold for spinal cord-derived neural stem cells.

A class of designed self-assembling peptide nanofiber scaffolds has been shown to be a good biomimetic material in tissue engineering. Here, we specifically made a new peptide hydrogel scaffold FGLmx by mixing the pure RADA16 and designer functional peptide RADA16-FGL solution, and we analyzed the physiochemical properties of each peptide with atomic force microscopy (AFM) and circular dichroism (CD). In addition, we examined the biocompatibility and bioactivity of FGLmx as well as RADA16 scaffold on spinal cord-derived neural stem cells (SC-NSCs) isolated from neonatal rats. Our results showed that RADA16-FGL displayed a weaker β-sheet structure and FGLmx could self-assemble into nanofibrous morphology. Moreover, we found that FGLmx was not only noncytotoxic to SC-NSCs but also promoted SC-NSC proliferation and migration into the three-dimensional (3-D) scaffold, meanwhile, the adhesion and lineage differentiation of SC-NSCs on FGLmx were similar to that on RADA16. Our results indicated that the FGL-functionalized peptide scaffold might be very beneficial for tissue engineering and suggested its further application for spinal cord injury (SCI) repair.

ERK1/2 pathway-mediated differentiation of IGF-1-transfected spinal cord-derived neural stem cells into oligodendrocytes.

Spinal cord injury (SCI) is a devastating event that causes substantial morbidity and mortality, for which no fully restorative treatments are available. Stem cells transplantation offers some promise in the restoration of neurological function but with limitations. Insulin-like growth factor 1 (IGF-1) is a well-appreciated neuroprotective factor that is involved with various aspects of neural cells. Herein, the IGF-1 gene was introduced into spinal cord-derived neural stem cells (NSCs) and expressed steadily. The IGF-1-transfected NSCs exhibited higher viability and were promoted to differentiate into oligodendrocytes. Moreover, the most possible underlying mechanism, through which IGF-1 exerted its neuroprotective effects, was investigated. The result revealed that the differentiation was mediated by the IGF-1 activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) and its downstream pathway. These findings provide the evidence for revealing the therapeutic merits of IGF-1-modified NSCs for SCI.

Collagen nanofibers facilitated presynaptic maturation in differentiated neurons from spinal-cord-derived neural stem cells through MAPK/ERK1/2-Synapsin I signaling pathway.

Neural stem cells (NSCs) are deemed to be a potential cell therapy for brain and spinal cord reconstruction and regeneration following injury. In this study, we investigated the role of nanofibrous scaffolds on NSCs-derived neurons in the formation of neural networks. Miniature excitatory postsynaptic currents (mEPSCs) were recorded using the whole-cell patch clamp recording method after the spinal cord-derived NSCs were differentiated into neurons and cultured in vitro for 10-14 days. It was observed that the frequency of mEPSCs in the differentiated neurons cultured on both randomly oriented and aligned collagen nanofibrous scaffolds was higher than that on the collagen-coated control and can be inhibited by an ERK inhibitor (PD98059), indicating that the collagen nanofibers affected the maturation of the synapses from presynaptic sites via the MAPK/ERK1/2 pathway. In addition, both of the collagen nanofibers increased the phosphorylation of Synapsin I and facilitated the interaction of p-ERK1/2 and p-Synapsin I. All these results suggested that the collagen nanofibrous scaffolds contributed to the presynaptic maturation via the ERK1/2-Synapsin I signaling pathway.

Partial Restoration of Cardiovascular Function by Embryonic Neural Stem Cell Grafts after Complete Spinal Cord Transection

High-level spinal cord injury can lead to cardiovascular dysfunction, including disordered hemodynamics at rest and autonomic dysreflexia during noxious stimulation. To restore supraspinal control of sympathetic preganglionic neurons (SPNs), we grafted embryonic brainstem-derived neural stem cells (BS-NSCs) or spinal cord-derived neural stem cells (SC-NSCs) expressing green fluorescent protein into the T4 complete transection site of adult rats. Animals with injury alone served as controls. Implanting of BS-NSCs but not SC-NSCs resulted in recovery of basal cardiovascular parameters, whereas both cell grafts alleviated autonomic dysreflexia. Subsequent spinal cord retransection above the graft abolished the recovery of basal hemodynamics and reflexic response. BS-NSC graft-derived catecholaminergic and serotonergic neurons showed remarkable long-distance axon growth and topographical innervation of caudal SPNs. Anterograde tracing indicated growth of medullar axons into stem cell grafts and formation of synapses. Thus, grafted embryonic brainstem-derived neurons can act as functional relays to restore supraspinal regulation of denervated SPNs, thereby contributing to cardiovascular functional improvement.

Human neural stem cell transplantation in spinal cord injury models: how far from clinical application?

Injuries of the spinal cord trigger local healing but hardly restore normal function. Spinal cord injury (SCI) has been deeply studied to develop strategies for functional recovery. The study by van Gorp and colleagues uses a rat model of acute SCI to characterize the effects of intraspinal grafting of human fetal spinal cord-derived neural stem cells (HSSCs). Among the range of results obtained, several positive outcomes related to a GABA-mediated inhibition were achieved. Their interesting article is a clear step forward in elucidating the neurobiology of SCI. Some issues to consider are related to the efficacy of the cell therapy in chronic spinal lesions and to pain responses, since the latter is a common complaint of people with SCI. Furthermore, the GABA-mediated hypothesis of recovery of function upon HSSC transplant needs to be fully tested by interfering with GABA receptors and analyzing neurobiological mechanisms that may invert the inhibitory role of GABA. The future challenge for SCI work will be the translation of knowledge acquired in basic research into clinically applicable strategies. The study by van Gorp and colleagues is an important contribution to that future.

Novel nanometer scaffolds regulate the biological behaviors of neural stem cells.

Ideal tissue-engineered scaffold materials regulate proliferation, apoptosis and differentiation of cells seeded on them by regulating gene expression. In this study, aligned and randomly oriented collagen nanofiber scaffolds were prepared using electronic spinning technology. Their diameters and appearance reached the standards of tissue-engineered nanometer scaffolds. The nanofiber scaffolds were characterized by a high swelling ratio, high porosity and good mechanical properties. The proliferation of spinal cord-derived neural stem cells on novel nanofiber scaffolds was obviously enhanced. The proportions of cells in the S and G2/M phases noticeably increased. Moreover, the proliferation rate of neural stem cells on the aligned collagen nanofiber scaffolds was high. The expression levels of cyclin D1 and cyclin-dependent kinase 2 were increased. Bcl-2 expression was significantly increased, but Bax and caspase-3 gene expressions were obviously decreased. There was no significant difference in the differentiation of neural stem cells into neurons on aligned and randomly oriented collagen nanofiber scaffolds. These results indicate that novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit apoptosis without inducing differentiation. Nanofiber scaffolds regulate apoptosis and proliferation in neural stem cells by altering gene expression.

Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation

IntroductionIntraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury.MethodsThree-month-old female Sprague–Dawley rats received L3 spinal compression injury. Three days post-injury, animals were randomized and received intraspinal injections of either HSSC, media-only, or no injections. All animals were immunosuppressed with tacrolimus, mycophenolate mofetil, and methylprednisolone acetate from the day of cell grafting and survived for eight weeks. Motor and sensory dysfunction were periodically assessed using open field locomotion scoring, thermal/tactile pain/escape thresholds and myogenic motor evoked potentials. The presence of spasticity was measured by gastrocnemius muscle resistance and electromyography response during computer-controlled ankle rotation. At the end-point, gait (CatWalk), ladder climbing, and single frame analyses were also assessed. Syrinx size, spinal cord dimensions, and extent of scarring were measured by magnetic resonance imaging. Differentiation and integration of grafted cells in the host tissue were validated with immunofluorescence staining using human-specific antibodies.ResultsIntraspinal grafting of HSSC led to a progressive and significant improvement in lower extremity paw placement, amelioration of spasticity, and normalization in thermal and tactile pain/escape thresholds at eight weeks post-grafting. No significant differences were detected in other CatWalk parameters, motor evoked potentials, open field locomotor (Basso, Beattie, and Bresnahan locomotion score (BBB)) score or ladder climbing test. Magnetic resonance imaging volume reconstruction and immunofluorescence analysis of grafted cell survival showed near complete injury-cavity-filling by grafted cells and development of putative GABA-ergic synapses between grafted and host neurons.ConclusionsPeri-acute intraspinal grafting of HSSC can represent an effective therapy which ameliorates motor and sensory deficits after traumatic spinal cord injury.

Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments.

Neural stem (NS) cells are a self-renewing population of symmetrically dividing multipotent radial glia-like stem cells, characterized by homogeneous expansion in monolayer. Here we report that fetal NS cells isolated from different regions of the developing mouse nervous system behave in a similar manner with respect to self-renewal and neuropotency, but exhibit distinct positional identities. For example, NS cells from the neocortex maintain the expression of anterior transcription factors, including Otx2 and Foxg1, while Hoxb4 and Hoxb9 are uniquely found in spinal cord-derived NS cells. This molecular signature was stable for over 20 passages and was strictly linked to the developmental stage of the donor, because only NS cells derived from E14.5 cortex, and not those derived from E12.5 cortex, carried a consistent transcription factor profile. We also showed that traits of this positional code are maintained during neuronal differentiation, leading to the generation of electrophysiologically active neurons, even if they do not acquire a complete neurochemical identity.

Transplantation of neurotrophin-3 modified neural stem cell for spinal cord injury treatment in rats

Objective To investigate the survival and differentiation of enhanced green fluorescent protein (EGFP)-labeled neurotrophin-3 (NT-3) modified fetal spinal cord-derived neural stem cells (NSCs) after transplanted into injured spinal cord.Methods EGFP labeled NT-3 modified NSCs were transplanted into the lesioned site of T9 hemisected rats (n = 10).The survival of grafted cells, expressions of NT-3 and differentiation into oligodendrocytes were detected by immunohistochemical assessment, and compared with the rats in unmodified NSCs transplantation group (n = 10).Results Cell counting presented more surviving cells in NT-3-NSCs group (304 ± 40) than NSCs group (258 ± 41) (P 0.05).The percentage of NT-3 positive cells in NT-3-NSCs group [(74.2 ± 14.1)%] was higher than NSCs group [(22.9 ± 11.1) %] (P < 0.01).More grafted cells in NT-3-NSCs group [(53.8 ± 12.4) %] differentiated into oligedendrocytes than NSCs group [(39.7 ± 13.7) %] (P 0.05).Conclusion NT-3 modification improves the survival of grafted NSCs, and promotes the expression of NT-3 and the differentiation into oligodendrocytes, which can increase the therapeutic effect of NSCs transplantation for spinal cord injury. Key words: Spinal cord injury; Transplantation; Neural stem cells; Neurotrophin3

Superparamagnetic iron oxide labeling of spinal cord neural stem cells genetically modified by nerve growth factor-β

This study established superparamagnetic iron oxide (SPIO)-labeled nerve growth factor-beta (NGF-beta) gene-modified spinal cord-derived neural stem cells (NSCs). The E14 rat embryonic spinal cord-derived NSCs were isolated and cultured. The cells of the third passage were transfected with plasmid pcDNA3-hNGFbeta by using FuGENE HD transfection reagent. The expression of NGF-beta was measured by immunocytochemistry and Western blotting. The positive clones were selected, allowed to proliferate and then labeled with SPIO, which was mediated by FuGENE HD transfection reagent. Prussian blue staining and transmission electron microscopy (TEM) were used to identify the SPIO particles in the cells. The distinctive markers for stem cells (nestin), neuron (beta-III-tubulin), oligodendrocyte (CNPase) and astrocyte (GFAP) were employed to evaluate the differentiation ability of the labeled cells. The immunocytochemistry and western blotting showed that NGF-beta was expressed in spinal cord-derived NSCs. Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells. TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma. The immunocytochemistry demonstrated that the labeled cells were nestin-positive. After differentiation, the cells expressed beta-III-tubulin, CNPase and GFAP. It was concluded that the SPIO-labeled NGF-beta gene-modified spinal cord-derived NSC were successfully established, which are multipotent and capable of self-renewal.

The effect of different transplantation time for spinal cord-derived neural stem cells differentiating into neurons: a pilot study in vivo

目的 探讨不同移植时间对胚胎脊髓源性神经干细胞体内分化为神经元影响,为胚胎脊髓源性神经干细胞移植防治骨骼肌失神经萎缩的最佳移植时间点的选择提供实验依据.方法 从孕龄14 d SD大鼠胚胎脊髓组织中分离、获得并鉴定脊髓源性神经干细胞.90只SD大鼠分为9组,分别于胫神经切断后0、1、2、3、4、6、8、12及16周,移植胚胎脊髓源性神经干细胞至切断胫神经远端神经外膜下,移植后4周取胫神经冰冻切片行免疫组化方法 计数神经元数量并进行图像分析.结果 损伤后1周移植时,神经元数量最多,其次为损伤后6周,损伤后16周组无神经元存在.结论 对胚胎脊髓源性神经干细胞体内移植后分化为神经元影响最小的时间点是损伤后1周,因此我们可以推断胚胎脊髓源性神经干细胞移植防治骨骼肌失神经萎缩的最佳移植时间点是损伤后1周。

Spinal cord-derived neural stem cells differentiated into cholinergic neurons induced by NT-3 in rats:a pilot study in vivo and in vitro

目的 观察神经营养因子-3(NT-3)在体内外能否诱导脊髓源性神经干细胞分化为胆碱能神经元.方法 从孕龄15 d的胚胎SD大鼠脊髓组织中分离获得脊髓源性神经干细胞.体外诱导分化研究:实验组在培养脊髓源性神经干细胞时,去除表皮生长因子和碱性成纤维细胞生长因子加入NT-3进行诱导;对照组则单纯培养脊髓源性神经干细胞.体内诱导分化研究:实验组将去除表皮生长因子和碱性成纤维细胞生长因子加入NT-3的脊髓源性神经干细胞混合悬液移植于成年SD大鼠切断胫神经远端,对照组则移植未加NT-3脊髓源性神经干细胞悬液.细胞免疫荧光化学法鉴定分化结果.结果 经NT-3诱导的脊髓源性神经干细胞用细胞免疫荧光化学法鉴定,体内外培养均可检测到胆碱乙酰基转移酶阳性细胞,而对照组则未检测到胆碱乙酰基转移酶阳性细胞.结论 NT-3在体内外均可诱导脊髓源性神经干细胞分化为胆碱能神经元。

In vitro culture and induced neuronal differentiation of rat embryonic spinal cord-derived neural stem cells

目的 分离培养大鼠胚胎脊髓神经干细胞(SNSCs),研究其体外增殖分化的特点,模拟大鼠胚胎时期脊髓发育的微环境诱导SNSCs向神经细胞定向分化.方法显微机械分离孕10～11d胚胎大鼠脊髓神经干细胞,无血清悬浮培养,以复合添加剂N4定向诱导分化,并拍照记录不同时期细胞分化的形态;用不同荧光剂进行细胞免疫荧光染色,并计算阳性细胞的平均分化比例.结果显微机械分离SNSCs方法,所分离的SNSCs具有较高的增殖能力,经复合添加剂N4诱导可以分化为神经元、星型胶质细胞、少突胶质细胞.结论采用显微机械分离SNSCs方法简单、效率高,N4添加剂可以高效诱导SNSCs分化为神经细胞,且分化的细胞中神经元比例高。