Normal sulfation levels regulate spinal cord neural precursor cell proliferation and differentiation

Background

Sulfated glycosaminoglycan chains are known for their regulatory functions during neural development and regeneration. However, it is still unknown whether the sulfate residues alone influence, for example, neural precursor cell behavior or whether they act in concert with the sugar backbone. Here, we provide evidence that the unique 473HD-epitope, a representative chondroitin sulfate, is expressed by spinal cord neural precursor cells in vivo and in vitro, suggesting a potential function of sulfated glycosaminoglycans for spinal cord development.

Results

Thus, we applied the widely used sulfation inhibitor sodium chlorate to analyze the importance of normal sulfation levels for spinal cord neural precursor cell biology in vitro. Addition of sodium chlorate to spinal cord neural precursor cell cultures affected cell cycle progression accompanied by changed extracellular signal-regulated kinase 1 or 2 activation levels. This resulted in a higher percentage of neurons already under proliferative conditions. In contrast, the relative number of glial cells was largely unaffected. Strikingly, both morphological and electrophysiological characterization of neural precursor cell-derived neurons demonstrated an attenuated neuronal maturation in the presence of sodium chlorate, including a disturbed neuronal polarization.

Conclusions

In summary, our data suggest that sulfation is an important regulator of both neural precursor cell proliferation and maturation of the neural precursor cell progeny in the developing mouse spinal cord.     

神经干细胞的端粒酶活性与其增殖分化的关系

目的探讨体外培养的神经干细胞端粒酶活性与细胞增殖、分化的关系,以及细胞分化后端粒酶逆转录酶的表达情况。方法采用无血清培养法从新生大鼠脑皮质分离培养神经干细胞；通过免疫荧光细胞染色鉴定神经干细胞；细胞计数法检测细胞的增殖情况；TRAP-ELISA法测定神经干细胞的端粒酶活性：RT-PCR法和Western-blot法测定细胞分化前后的端粒酶逆转录酶的表达。结果从新生大鼠脑皮质分离培养的神经干细胞具有端粒酶活性；体外培养12周内,神经干细胞的端粒酶活性未见变化,细胞的增殖速率亦未见明显不同；神经干细胞分化后端粒酶活性丧失,端粒酶逆转录酶的mRNA和蛋白质也均未见表达。结论在体外培养过程中,大鼠脑神经干细胞的端粒酶活性和细胞增殖速率未见变化；神经干细胞分化后端粒酶活性丧失,可能是由于端粒酶逆转录酶停止表达所致。     

Fibroblast growth factor stimulates the proliferation and differentiation of neural precursor cells in vitro

We have developed an in vitro culture system to study the regulation of proliferation and differentiation of neural precursor cells contained within the neuroepithelium of embryonic day 10 mice. A number of soluble growth factors have been tested for their ability to regulate these early events and, of these factors, we have found that the fibroblast growth factors [FGFs] can directly stimulate the proliferation and survival of the neuroepithelial cells. At least 50% of the neuroepithelial cells divide in the presence of FGF whereas in the absence of FGF all of the cells die within 6 days of culture. At higher concentrations of FGF, the cells change from being nonadherent round cells in tight clusters into a more flattened cell type which adheres to the substratum. This morphological change is accompanied by the expression of both neurofilament and GFAP, which are definitive markers of the two major cell types in the central nervous system: neurons and glia. In addition a neuroepithelial cell line, which does not rely on FGF for survival or proliferation, expresses both of these markers in response to FGF. These results indicate that FGF is stimulating the differentiation of the neuroepithelial cells into mature neurons and glia.     

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.     

Erythropoietin and GDNF enhance ventral mesencephalic fiber outgrowth and capillary proliferation following neural transplantation in a rodent model of Parkinson's disease

Low dopaminergic cell survival and suboptimal fiber reinnervation are likely major contributing factors for the limited benefits of neural transplantation in Parkinson's disease (PD) patients. Glial cell lined-derived neurotrophic factor (GDNF) has been shown to enhance dopaminergic cell survival and fiber outgrowth of the graft site as well as promote behavioral recovery in rodent models of PD, while erythropoietin (EPO) can produce dopaminergic neuroprotective effects against 6-hydroxydopamine (6-OHDA) exposure on cultured neurons and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. The aim of this study was to determine if fetal ventral mesencephalic (FVM) tissue exposed to hibernation media containing a combination of GDNF and EPO could enhance dopaminergic graft survival, striatal reinnervation and functional recovery in a 6-OHDA rodent model of PD. FVM tissue was dissected from 14-day-old rat fetuses and placed for 6 days in hibernation media alone, and in hibernation media that received either a daily administration of GDNF, EPO or a combination of GDNF and EPO. Following hibernation, FVM cells were transplanted as a single cell suspension into the striatum of unilateral 6-OHDA-lesioned rats. Rotational behavioral assessment revealed animals that received FVM tissue exposed to GDNF, EPO or the combination of both drugs had accelerated functional recovery. Immunohistochemical and stereological assessment revealed a significant increase in graft fiber density and angiogenesis into the graft when compared with control. These findings suggest that the hibernation of FVM tissue in a combination of GDNF and EPO can enhance graft efficacy and may have important implications for tissue preparation protocols for clinical neural transplantation in PD.     

Neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells

Neudesin encodes a secreted signal with neurotrophic activity in neurons. Most neurotrophic factors are involved in neural cell proliferation and/or differentiation. However, the role of neudesin in neural development remains to be elucidated. We examined the expression of neudesin in mouse embryonic cerebral cortex and cultured mouse neural precursor cells and its roles in neural development. Neudesin was expressed in the embryonic cerebral cortex early in development. Its expression was observed mainly in the preplate, where mostly postmitotic neural cells existed. Because neudesin mRNA was expressed in the neural precursor cells before the appearance of neurons, the roles of neudesin in neural development were examined by using the precursor cells. Neudesin significantly promoted neuronal differentiation and overrode the undifferentiated state of the neural precursor cells sustained by fibroblast growth factor 2 (FGF2). In contrast, it inhibited the differentiation of astrocytes. In addition, neudesin transiently promoted neural cell proliferation early in the developmental process. The effect on cell proliferation was distinct from that of FGF2, a self-renewal-promoting factor for neural precursor cells. The differentiation was mediated though activation of the protein kinase A (PKA) and phosphatidylinositol-3 kinase (PI-3K) pathways. In contrast, the proliferation was mediated through the mitogen-activated protein kinase and PKA pathways. The expression profile and activity indicate that neudesin plays unique roles in neural development. The present findings have revealed new potential roles of neudesin in neural cell proliferation and neuronal differentiation.     

Kainic acid triggers oligodendrocyte precursor cell proliferation and neuronal differentiation from striatal neural stem cells

Glutamate is an excitatory amino acid that serves important functions in mammalian brain development through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/ kainate receptor stimulation. Neural stem cells with self-renewal and multilineage potential are a useful tool to study the signals involved in the regulation of brain development. We have investigated the role played by AMPA/kainate receptors during the differentiation of neural stem cells derived from fetal rat striatum. The application of 1 and 10 microM kainic acid increased significantly the phosphorylation of the cyclic AMP response element binding protein (CREB), raised bromodeoxyuridine incorporation in O4-positive oligodendrocyte precursors, and increased the number of O1-positive cells in the cultures. Increased CREB phosphorylation and proliferation were prevented by the AMPA receptor antagonist 4-4(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine (SYM 2206) and by protein kinase A and protein kinase C inhibitors. Cultures treated with 100 microM kainic acid showed decreased proliferation, a lower proportion of O1-positive cells, and apoptosis of O4-positive cells. None of these effects were prevented by SYM 2206, suggesting that kainate receptors take part in these events. We conclude that AMPA receptor stimulation by kainic acid promotes the proliferation of oligodendrocyte precursors derived from neural stem cells through a mechanism that requires the activation of CREB by protein kinase A and C. In the neurons derived from these cells, either AMPA or kainate receptor stimulation produces neuritic growth and larger cell bodies.     

Long-term proliferation and dopaminergic differentiation of human mesencephalic neural precursor cells

We report on generation of dopamine neurons from long-term cultures of human fetal mesencephalic precursor cells. These CNS precursor cells were successfully expanded in vitro using the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Incubation of these cultures in 3% atmospheric oxygen resulted in higher cellular yields than room air. Following incubation in differentiation media containing interleukin (IL)-1b (IL-1b), IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF), up to 1% of the precursor cells converted into cells immunoreactive for tyrosine hydroxylase (TH), a marker for dopamine neurons. The TH immunoreactive cells exhibited morphological and functional properties characteristic of dopamine neurons in culture. These precursor cells might serve as a useful source of human dopamine neurons for studying the development and degeneration of human dopamine neurons and may further serve as a continuous, on-demand source of cells for therapeutic transplantation in patients with Parkinson's disease.     

成年大鼠脑创伤后神经前体细胞的增殖及迁移

目的 研究液压冲击性脑损伤后成年大鼠神经前体细胞的增殖及迁移。方法 制作液压冲击性脑损伤模型，免疫组织化学方法动态检测巢蛋白（Nestin）和5溴脱氧尿苷（BrdU）的表达。BrdU标记方法确定增列殖的前体细胞；Nestin的表达用于确定神经前体细胞。结果 同正常对照组相比较，伤侧皮层、海马及室下区的Nestin阳性细胞数于伤后1d明显增多，7d达高峰,30d消失；BrdU阳性细胞数于作后3d达高峰，而7d以后逐渐减小，室下区BrdU阳性细胞及Nestin阳性细胞经胼胝体向对侧迁移。结论 液压冲击性脑损伤可激发成年大鼠神经前体细胞增殖及迁移。     

Human immunodeficiency virus type 1 Tat modulates proliferation and differentiation of human neural precursor cells: implication in NeuroAIDS

Human immunodeficiency virus type 1 (HIV-1) and viral proteins affect neuronal survival and neuron-glial cell interactions, which culminate in neurological disorders. HIV-1 infects regions of neurogenesis in human adult and pediatric brain. However, little is known about the effect of HIV-1 or viral proteins on the properties of human neural precursor cells (hNPCs), particularly neurogenesis, hence a detailed investigation on these lines is warranted. Human neural precursor cells were cultured in presence and absence of HIV-1B transactivating protein Tat to investigate if HIV-1 viral protein alters the properties of human neural precursor cells. Cellular and molecular approaches were adopted to study the effect of HIV-1B transactivating protein Tat on proliferation and differentiation potential of human fetal brain-derived NPCs. Cell proliferation assays such as BrdU and Ki67 staining and pathway-specific cDNA and protein arrays were used in the study. Data reveal that HIV-1B Tat protein severely affects proliferation of hNPCs, as evident by lower incorporation of BrdU and Ki67 staining as well as neurosphere assay. HIV-1 Tat substantially attenuated neurogenesis, as evident by the smaller numbers of Tuj-1- and doublecortin-positive cells differentiated from hNPCs, without affecting their viability. These data suggest that HIV-1 Tat alters the properties of human neural precursor cells via attenuation of the cell cycle regulatory unit cyclin D1 and the mitogen-activated protein kinase (MAPK) pathway, particularly extracellular signal-related kinase 1/2 (ERK1/2). The study provides new insights into cellular and molecular mechanisms that may modulate human neural precursor cell properties in HIV/AIDS (acquired immunodeficiency syndrome) individuals. Validation with autopsy brain samples is necessary to further substantiate these important observations.     

Association between integrin-dependent migration capacity of neural stem cells in vitro and anatomical repair following transplantation

In previous transplantation studies using neural stem cell lines immortalized by the temperature-sensitive SV40 large T-antigen, we have shown that animals with experimental hippocampal lesions resulting from four vessel occlusion recover spatial memory functions more effectively when grafted with the MHP36 cell line than with the MHP15 cell line [Gray et al. (1999). Philos. Trans. R. Soc. London Biol. Sci. 354:1407-1421]. In the present study, we have investigated the cellular and molecular basis of these differences in repair capacity both in vivo and in vitro. Using the same model of hippocampal damage we have shown that following transplantation MHP36 cells migrate and align within the damaged CA1 of the ipsilateral hippocampus. MHP15 cells, in contrast, migrate in a more indiscriminate pattern that does not reflect the anatomy of the region. To analyze the migratory properties of these two cell lines in more detail, we performed migration assays at a nonpermissive temperature on the extracellular matrix substrates laminin, fibronectin, and vitronectin. These showed that MHP36 cells have a greater migration potential than the MHP15 cells. While the pattern of cell surface extracellular matrix receptors of the integrin family was identical in both cell lines, the different degrees of migration on vitronectin were both blocked by inhibitors of alphaV integrins. Differences in integrin signaling therefore contribute to the greater migration potential of the repairing MHP36 cell line.     

脑梗死大鼠神经前体细胞增殖水平的研究

目的研究脑梗死病灶周围及海马处神经前体细胞增殖水平的动态变化。方法采用易卒中型肾性高血压大鼠(RHRSP)，电凝大脑中动脉(MCA)主干制成脑梗死(MCAO)模型。行大鼠神经功能评定，免疫组化观察并计数梗死灶边缘、对侧镜区及双侧海马5-溴脱氧尿核苷(Bromodeoxyuridine，BrdU)标记的细胞。结果MCAO后大鼠神经功能评分减低，5d时恢复正常。MCAO后梗死灶边缘、对侧镜区及双侧海马均有BrdU阳性细胞分布，且病灶侧多于病灶对侧，集中分布于病灶周围。结论脑缺血可诱导神经前体细胞增殖并移向病灶，可能成为脑梗死恢复的重要物质基础。     

Human fetal neural precursor cells can up-regulate MHC class I and class II expression and elicit CD4 and CD8 T cell proliferation

The use of allogeneic fetal neural precursor cells (NPCs) as a cell replacement therapy in neurodegenerative disorders holds great promise. However, previous studies concerning the possibility of alloimmune rejection of the transplanted cells have been inconclusive. Here, we used flow cytometry to quantify the expression of major histocompatibility complex (MHC) molecules by human NPCs, obtained from the cortex or ventral mesencephalon of fetuses with gestational ages between 7 and 11 weeks. MHC class I was undetectable on the surface of freshly isolated primary fetal tissue from either location, but increased over time in proliferating NPC cultures; after 7days in vitro, MHC class I was detectable on most cells. Following differentiation, MHC class I expression persisted on non-neuronal cells. MHC class II levels remained low at all time points but were inducible by pro-inflammatory cytokines, whereas the co-stimulatory molecules, CD80 and CD86, remained undetectable. Nonetheless, CD4+ and CD8+ T cells proliferated when peripheral blood mononuclear cells (PBMCs) were cultured with allogeneic NPCs. Weaker responses were obtained when NPCs were co-cultured with purified allogeneic responder T cells, suggesting that indirect allorecognition contributed significantly to PBMC responses. In conclusion, differentiating human NPCs are immunogenic in vitro, suggesting that they may trigger immune rejection unless transplant recipients are immunosuppressed.     

Human neural stem cell transplantation reduces spontaneous recurrent seizures following pilocarpine-induced status epilepticus in adult rats

Transplantation of neural stem cells (NSCs) can replace lost neurons and improve the functional deficits. Cell transplantation strategies have been tried in the epileptic disorder, but the effect of exogenous NSCs is unknown. In this study, we attempted to test the anti-epileptogenic effect of NSCs in adult rats with status epilepticus. Experimental status epilepticus was induced by lithium-pilocarpine injection, and beta galactosidase-encoded human NSCs were transplanted intravenously on the next day of status epilepticus. Spontaneous recurrent seizures were monitored with Racine's seizure severity scale. Immunohistochemistry with anti-beta gal, Tuj-1, NeuN, GFAP, CNPase, GluR2, parvalbumin, and GABA were performed and extracellular field excitatory postsynaptic potentials (fEPSP) were recorded. Human NSCs suppressed spontaneous recurrent seizure formation and transplanted NSCs were differentiated into GABA-immunoreactive interneurons in the damaged hippocampus. Amplitude of fEPSP in the hippocampal CA1 was reduced, which was reversed by picrotoxin. These findings suggest that NSCs could be differentiated into inhibitory interneurons and decrease neuronal excitability, which could prevent spontaneous recurrent seizure formation in adult rats with pilocarpine-induced status epilepticus.     

远志皂苷元促进人神经前体细胞系的体外增殖

Senegenin,an effective component of Polygala tenuifolia root extract,promotes proliferation and differentiation of neural progenitor cells in the hippocampus.However,the effects of senegenin on mesencephalon-derived neural progenitor cells remain poorly understood.Cells from a ventral mesencephalon neural progenitor cell line(ReNcell VM) were utilized as models for pharmaceutical screening.The effects of various senegenin concentrations on cell proliferation were analyzed,demonstrating that high senegenin concentrations(5,10,50,and 100 μmo/L),particularly 50 μmol/L,significantly promoted proliferation of ReNcell VM cells.In the mitogen-activated protein kinase signal transduction pathway,senegenin significantly increased phosphorylation levels of extracellular signal-regulated kinases.Moreover,cell proliferation was suppressed by extracellular signal-regulated kinase inhibitors.Results suggested that senegenin contributed to in vitro proliferation of human neural progenitor cells by upregulating phosphorylation of extracellular signal-regulated kinase.     

不同电刺激对卒中后大鼠神经前体细胞增殖的影响

目的 探讨不同部位电刺激治疗对脑卒中后大鼠神经前体细胞增殖的作用.方法 选择2～3月龄,体质量80～120 g雄性SD大鼠95只,随机分为梗死灶周围电刺激组(A组)、肢体电刺激组(B组)、对照组(C组)和假手术组(D组).建立大脑中动脉闭塞模型,在术后24 h开始对两电刺激组大鼠头皮和肢体电刺激治疗.在梗死后7d、14d、21 d、28 d以横木行走试验(BWT)进行神经功能评分,同时进行室管膜和室管膜下5-溴脱氧尿嘧啶核苷(Brdu)的免疫组织化学检测.结果 A组及B组缺血后室管膜Brdu的表达与C组比较在7、14d差异均有统计学意义(P＜0.05),MCAO后7d二者相比Brdu 在室管膜和室管膜下区的表达差异有统计学意义(P＜0.05);MCAO后21dA组Brdu的表达和C组差异有统计学意义(P＜0.05),但此时B组和C组差异无统计学意义(P＞0.05);在MCAO后第1天及第28天三者之间的Brdu均有表达但差异无统计学意义(P＞0.05).结论 电刺激促进MCAO后神经干细胞的增殖,但梗死灶周围头皮电刺激治疗较肢体电刺激治疗引起的神经干细胞增殖明显.     

Trophic and immunoregulatory properties of neural precursor cells: Benefit for intracerebral transplantation

Intracerebral xenotransplantation of porcine fetal neuroblasts (pNB) is considered as an alternative to human neuroblasts for the treatment of neurodegenerative diseases. However, pNB are systematically rejected, even in an immunoprivileged site such as the brain. Within this context, neural stem/precursor cells (NSPC), which were suggested as exhibiting low immunogenicity, appeared as a useful source of xenogeneic cells. To determine the advantage of using porcine NSPC (pNSPC) in xenotransplantation, pNB and pNSPC were grafted into the striatum of rats without immunosuppression. At day 63, all the pNB were rejected while 40% of the rats transplanted with pNSPC exhibited large and healthy grafts with numerous pNF70-positive cells. The absence of inflammation at day 63 and the occasional presence of T cells in pNSPC grafts evoked a weak host immune response which might be partly due to the immunosuppressive properties of the transplanted cells. T cell proliferation assays confirmed such a hypothesis by revealing an inhibitory effect of pNSPC on T cells through a soluble factor. In addition to their immunosuppressive effect, in contrast to pNB, very few pNSPC differentiated into tyrosine hydroxylase-positive neurons but the cells triggered an intense innervation of the striatum by rat dopaminergic fibers coming from the substantia nigra. Further experiments will be required to optimize the use of pNSPC in regenerative medicine but here we show that their immunomodulatory and trophic activities might be of great interest for restorative strategies. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."     

Human neural stem cell differentiation following transplantation into spinal cord injured mice: association with recovery of locomotor function

Abstract

Stem cells are under intense investigation as potential therapeutics for central nervous system (CNS) injury and disease. However, several reports have suggested that stem cells grown as neurospheres and transplanted into an injured environment preferentially differentiate into astrocytes, contributing to glial scar. Further, the relationship between functional recovery and cell transplantation has not been empirically investigated in early studies. Using severe combined immunodeficient (scid) mice to minimize xenograft rejection, we report that prospectively isolated human fetal CNS-derived stem cells grown as neurospheres (hCNS-SCns) survive, migrate and express differentiation markers for neurons and oligodendrocytes after long-term engraftment in spinal cord injured (SCI) NOD-scid mice. Only rarely do these cells differentiate into glial fibrillary acidic protein (GFAP)-positive astrocytes, with no apparent contribution to glial scar. hCNS-SCns engraftment was associated with recovery of locomotor function. After long-term engraftment and stable behavioral plateaus in recovery were achieved (4 months post-transplantation), locomotor improvements were abolished by selective ablation of human cells with diphtheria toxin (DT). These data suggest that hCNS-SCns survival is required for locomotor recovery, possibly via differentiation and integration of human cells in the mouse host or continuous supply of trophic or other support necessary for gains in host cell function.