Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex

In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly-d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3β (GSK-3β) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.     

Brain injury activates microglia that induce neural stem cell proliferation ex vivo and promote differentiation of neurosphere-derived cells into neurons and oligodendrocytes

Brain damage, such as ischemic stroke, enhances proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ). To date, no reliable in vitro systems, which can be used to unravel the potential mechanisms underlying this lesion-induced effect, have been established. Here, we developed an ex vivo method to investigate how the proliferation of NSPCs changes over time after experimental stroke or excitotoxic striatal lesion in the adult rat brain by studying the effects of microglial cells derived from an injured brain on NSPCs. We isolated NSPCs from the SVZ of brains with lesions and analyzed their growth and differentiation when cultured as neurospheres. We found that NSPCs isolated from the brains 1-2 weeks following injury consistently generated more and larger neurospheres than those harvested from naive brains. We attributed these effects to the presence of microglial cells in NSPC cultures that originated from injured brains. We suggest that the effects are due to released factors because we observed increased proliferation of NSPCs isolated from non-injured brains when they were exposed to conditioned medium from cultures containing microglial cells derived from injured brains. Furthermore, we found that NSPCs derived from injured brains were more likely to differentiate into neurons and oligodendrocytes than astrocytes. Our ex vivo system reliably mimics what is observed in vivo following brain injury. It constitutes a powerful tool that could be used to identify factors that promote NSPC proliferation and differentiation in response to injury-induced activation of microglial cells, by using tools such as proteomics and gene array technology.     

Combination of media, biomaterials and extracellular matrix proteins to enhance the differentiation of neural stem/precursor cells into neurons

The purpose of this study was to induce the differentiation of neural stem/precursor cells (NSPC) more towards neurons than glial cells by the combination of media, biomaterials and extracellular matrix (ECM) proteins. Considering the role of serum, 10% fetal bovine serum or its fractions were added to DMEM/F12 medium to examine the effect of the differentiation-promoting potential on cultured NSPC isolated from embryonic rat cerebral cortex. The NSPC were cultured for 7 days, after which differentiation was assayed using immunocytochemistry for lineage specific markers. It was demonstrated that molecules promoting neuron differentiation were present in serum with molecular weight <100 kDa, which could dominate the differentiation of NSPC principally into neurons in the presence of basic fibroblast growth factor. In contrast, NSPC were induced to differentiate predominantly into glial cell phenotypes in the presence of whole serum components. Based on medium containing serum fraction, semi-quantification showed that the MAP2-positive percentage of the immunoreactive ratio within migrated cells could be promoted over 85% by combining poly(ethylene-co-vinyl alcohol) biomaterial and fibronectin matrix protein. These results are very encouraging, since an environment favorable for neuronal differentiation should be useful in the development of strategies for controlling the behavior of NSPC in neuroscience research.     

Effect of inflammatory cytokines on major histocompatibility complex expression and differentiation of human neural stem/progenitor cells

To develop transplantation of neural stem/progenitor cells (NSPCs) as a successful treatment of neurodegenerative disorders, the possible induction of an inflammatory response following implantation needs to be taken into consideration. Inflammatory cytokines can upregulate major histocompatibility complex (MHC) expression on transplanted cells, thereby rendering them more susceptible to graft rejection. Furthermore, cytokines also have a profound effect on cell differentiation, migration, and proliferation, which can greatly affect the outcome of transplantation. Here we studied the effect of three inflammatory cytokines, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6), from three different species (human, monkey, rat) on expression of MHC molecules and differentiation of two human NSPC lines derived from striatum and hippocampus. Human and monkey IFN-gamma strongly upregulate MHC expression in both NSPC lines in a dose-dependent manner, whereas rat IFN-gamma has an effect on MHC expression only in hippocampal cells. Furthermore, TNF-alpha, but not IL-6, upregulates MHC expression in both NSPC lines. Differentiation of NSPCs in the presence of cytokines showed that IFN-gamma increased the neuronal yield threefold in striatal NSPC cultures and increased the number of oligodendrocytes twofold in hippocampal NSPC cultures. Addition of TNF-alpha enhanced gliogenesis in both cell lines, whereas IL-6 stimulated neurogenesis. Human NSPC lines' response to cytokines is therefore species specific and also dependent on the NSPCs' region of origin. The successful translation of different cell lines from animal models to clinical trials could be substantially influenced by the species-specific regulation of MHC and differentiation as reported here. Disclosure of potential conflicts of interest is found at the end of this article.     

Identification of astrocyte-expressed factors that modulate neural stem/progenitor cell differentiation

Multipotent neural stem/progenitor cells (NSPCs) can be isolated from many regions of the adult central nervous system (CNS), yet neurogenesis is restricted to the hippocampus and subventricular zone in vivo. Identification of the molecular cues that modulate NSPC fate choice is a prerequisite for their therapeutic applications. Previously, we demonstrated that primary astrocytes isolated from regions with higher neuroplasticity, such as newborn and adult hippocampus and newborn spinal cord, promoted neuronal differentiation of adult NSPCs, whereas astrocytes isolated from the nonneurogenic region of the adult spinal cord inhibited neural differentiation. To identify the factors expressed by these astrocytes that could modulate NSPC differentiation, we performed gene expression profiling analysis using Affymetrix rat genome arrays. Our results demonstrated that these astrocytes had distinct gene expression profiles. We further tested the functional effects of candidate factors that were differentially expressed in neurogenesis-promoting and -inhibiting astrocytes using in vitro NSPC differentiation assays. Our results indicated that two interleukins, IL-1beta and IL-6, and a combination of factors that included these two interleukins could promote NSPC neuronal differentiation, whereas insulin-like growth factor binding protein 6 (IGFBP6) and decorin inhibited neuronal differentiation of adult NSPCs. Our results have provided further evidence to support the ongoing hypothesis that, in adult mammalian brains, astrocytes play critical roles in modulating NSPC differentiation. The finding that cytokines and chemokines expressed by astrocytes could promote NSPC neuronal differentiation may help us to understand how injuries induce neurogenesis in adult brains.     

Differences in the effect on neural stem cells of fetal bovine serum in substrate-coated and soluble form

The influence of fetal bovine serum (FBS) adsorbed to poly(ethylene-co-vinyl alcohol) (EVAL) and polyvinyl alcohol (PVA) substrates (coated FBS) and FBS present in the culture medium (soluble FBS) on the behavior of embryonic rat cerebral cortical neural stem cells was studied at neurosphere level. When both coated FBS and soluble FBS were not present in the culture system, the fate and behavior of neurospheres were mediated mainly by the substrates used. When neurospheres were cultured either on FBS-coated EVAL or FBS-coated PVA substrates in the serum-free medium, the most striking morphological characteristic of neurospheres was that these neurosphere-forming cells attached and were induced to differentiate into process-bearing cell phenotypes predominantly; however, the differentiated cell phenotypes were dissimilar on these two substrates. On the contrary, when neurospheres were cultured in the medium containing 10% FBS, the neurosphere-forming cells were induced into protoplasmic cells typically but no difference in differentiated cell phenotypes on EVAL and PVA substrates was observed. Interestingly, instead of promoting process outgrowth under serum-free medium condition, coated FBS enhanced migration of differentiated protoplasmic cells when soluble FBS were present. These results inform that the substrates, coated serum, and soluble serum within the culture environment together can significantly alter cell behavior and morphological differentiation and will therefore be an important clue for the development of biomaterials to regulate the potential of the CNS neural stem cells.     

The effects of peptide modified gellan gum and olfactory ensheathing glia cells on neural stem/progenitor cell fate

The regenerative capacity of injured adult central nervous system (CNS) tissue is very limited. Specifically, traumatic spinal cord injury (SCI) leads to permanent loss of motor and sensory functions below the site of injury, as well as other detrimental complications. A potential regenerative strategy is stem cell transplantation; however, cell survival is typically less than 1%. To improve cell survival, stem cells can be delivered in a biomaterial matrix that provides an environment conducive to survival after transplantation. One major challenge in this approach is to define the biomaterial and cell strategies in vitro. To this end, we investigated both peptide-modification of gellan gum and olfactory ensheathing glia (OEG) on neural stem/progenitor cell (NSPC) fate. To enhance cell adhesion, the gellan gum (GG) was modified using Diels-Alder click chemistry with a fibronectin-derived synthetic peptide (GRGDS). Amino acid analysis demonstrated that approximately 300 nmol of GRGDS was immobilized to each mg of GG. The GG-GRGDS had a profound effect on NSPC morphology and proliferation, distinct from that of NSPCs in GG alone, demonstrating the importance of GRGDS for cell-GG interaction. To further enhance NSPC survival and outgrowth, they were cultured with OEG. Here NSPCs interacted extensively with OEG, demonstrating significantly greater survival and proliferation relative to monocultures of NSPCs. These results suggest that this co-culture strategy of NSPCs with OEG may have therapeutic benefit for SCI repair.     

Distinctive effects of arachidonic acid and docosahexaenoic acid on neural stem /progenitor cells

Arachidonic acid (ARA) and docosahexaenoic acid (DHA), which are the dominant polyunsaturated fatty acids in the brain, have crucial roles in brain development and function. Recent studies have shown that ARA and DHA promote postnatal neurogenesis. However, the direct effects of ARA on neural stem/progenitor cells (NSPCs) and the effects of ARA and DHA on NSPCs at the neurogenic and subsequent gliogenic stages are still unknown. Here, we analyzed the effects of ARA and DHA on neurogenesis, specifically maintenance and differentiation, using neurosphere assays. We confirmed that primary neurospheres are neurogenic NSPCs and that tertiary neurospheres are gliogenic NSPCs. Regarding the effects of ARA and DHA on neurogenic NSPCs, ARA and DHA increased the number of neurospheres, whereas neither ARA nor DHA had a detectable effect on NSPCs in the differentiation condition. In gliogenic NSPCs, DHA increased the number of neurospheres, whereas ARA had no such effect. In contrast, ARA increased the number of astrocytes, whereas DHA increased the number of neurons in the differentiation condition. These results suggest that ARA promotes the maintenance of neurogenic NSPCs and might induce the glial differentiation of gliogenic NSPCs and that DHA promotes the maintenance of both neurogenic and gliogenic NSPCs and might lead to the neuronal differentiation of gliogenic NSPCs.     

成年小鼠神经干细胞体外培养、鉴定及分化

目的:建立一种简便易行的从成年小鼠脑组织分离、培养和鉴定神经干细胞(neural stem cells,NSCs)的方法,为相关研究提供新的研究手段。方法:采用机械分离和酶消化结合方法分离成年昆明种小鼠的脑组织,用无血清培养基悬浮培养;倒置相差显微镜观察细胞形态;MTT法(四甲基偶氮唑盐微量酶反应比色法)观察NSCs的自我增殖能力;免疫荧光细胞化学技术检测NSCs标志物巢蛋白(Nestin)的表达;多聚赖氨酸铺板和撤除培养基中FGF和EGF的条件下,给予5%血清和1μm维甲酸诱导NSCs分化,免疫荧光细胞化学技术检测GFAP(标记胶质细胞),β-tubulin Ⅲ(标记神经元)蛋白的表达来测定NSCs的分化能力。结果:从成年小鼠脑组织分离的细胞,在无血清培养液中可形成神经球,并可在体外扩增和连续传代,免疫荧光细胞化学表明神经球Nestin阳性表达,在给予血清和维甲酸条件下神经球可表达GFAP和β-tubulin Ⅲ。结论:本研究成功建立了体外培养成年小鼠脑组织分离和培养NSCs的方法,培养的NSCs具有自我更新、增殖及多向分化潜能。此方法是一个稳定、简便易行的方法,可广泛用于神经干细胞相关的基础和应用研究。     

一种诱导神经干细胞向神经元定向分化的方法

目的 介绍一种诱导神经干细胞向神经元定向分化的方法 . 方法 采用选择性无血清培养液培养大鼠神经干细胞.纯化培养2或3代后,将其接种于条件培养液中,诱导其向神经元定向分化.用倒置显微镜观察形态学变化,用NSE免疫细胞化学法检测其向神经元分化情况. 结果 条件培养液可有效诱导神经干细胞向神经元定向分化.将神经球消化成单细胞后接种,不但分化过程比神经球接种更同步,而且可提高其向神经元分化的比例. 结论 本方法 可稳定高效地诱导神经干细胞向神经元定向分化.     

体外培养胚鼠中脑神经干/祖细胞超微结构分析

目的:研究并分析神经干细胞的超微结构和生物学特征。方法:无血清培养大鼠胚胎中脑组织中神经干细胞,用血清诱导分化。扫描及透射电镜观察神经球及单个神经干/祖细胞的超微结构。结果:培养的神经干/祖细胞呈神经球样生长,经血清诱导可分化为神经元样和神经胶质细胞样细胞。扫描电镜下神经球表面的细胞直径约为5～10μm,常3～5个以上彼此粘附聚集成簇,松散不规则分布。透射电镜下神经球由神经干细胞和无定形结构的物质构成,根据电子密度不同分为明细胞和暗细胞。神经球内部大部分相邻细胞排列紧密,部分细胞可见有贯通两相邻细胞膜的通道;少部分相邻细胞排列疏松,并见部分相邻细胞间有壁呈腺泡样结构的囊泡的释放活动。神经球内干/祖细胞核质比高,多种细胞器不发达,高尔基复合体、核糖体、溶酶体等在不同神经干/祖细胞中的数量和分布不一致。多见自噬小体,髓样体。大部分细胞为单核,罕见双核、肾形核,常染色质多分布均匀,异染色质少,多见单个核仁。结论:胚鼠中脑神经干/祖细胞球内紧密排列的相邻细胞间有贯通两胞膜的孔道,排列疏松的相邻细胞间有腺泡样结构的囊泡的释放活动。研究结果为进一步研究多因子对神经干/祖细胞的发育增殖、定向分化的调控及其相关途径奠定了一定基础。     

新生小鼠海马神经干细胞体外培养条件下的增殖与分化

背景：神经干细胞移植可用于中枢神经损伤的临床治疗。 目的：观察新生小鼠海马神经干细胞在体外培养条件下的增殖和分化。 方法：从新生昆明小鼠海马取材,采用机械分离法对原代细胞进行无血清培养,采用机械法复合酶消化法对原代细胞进行传代,以体积分数为10%胎牛血清诱导分化。免疫荧光行巢蛋白、β-微管蛋白Ⅲ和胶质纤维酸性蛋白染色,对培养的细胞进行鉴定。CCK-8法检测神经干细胞增殖能力。 结果与结论：从新生小鼠海马分离得到的细胞具有连续传代形成克隆球的能力,克隆球呈巢蛋白免疫反应阳性;加入胎牛血清可诱导分化为β-微管蛋白Ⅲ和胶质纤维酸性蛋白阳性细胞。提示实验成功建立了体外培养新生小鼠海马组织分离和培养神经干细胞的方法,培养的神经干细胞在体外培养条件下保持着自我增殖和分化的潜能。     

脑络欣通和左归丸药物血清体外培养神经干细胞的增殖与分化

BACKGROUND:Because of limited source and a relatively weak ability of differentiation and proliferation, how to take positive and effective measures to promote neural stem cell proliferation, differentiation has become the focus of research. OBJECTIVE:To investigate the effects of drug-contained sera of Naoluo Xintong versus Zuogui pill on the proliferation and differentiation of in vitro cultured rat neural stem cells. METHODS:Embryonic neural stem cells of Sprague-Dawley rats were isolated and cultured in vitro, and then were co-cultured with the serum medium containing 10% Naoluo Xintong and 10% Zuogui pill, respectively. Comparative observations were performed between two groups by inverted microscope and immunofluorescence staining. RESULTS AND CONCLUSION:Under the inverted microscope, the cells began to grow in cluster and gather into a ball, but the diameter was relatively small after 24-hour culture; the neurospheres expended further, with relatively regular shape, but no neurosphere differentiation appeared after 48 hours of culture. The average prominent length of the neurospheres in the Zuogui pill group was significantly greater than that in the Naoluo Xintong group after 5 days of culture (P < 0.05). The rate of rat neural stem cells differentiating into MAP-2-positive cells in the Zuogui pill group was significantly lower than that in the Naoluo Xintong group (P < 0.05), but the rate of differentiated cells positive for glial fibrillary acidic protein in the Zuogui pill group was significantly higher than that in the Naoluo Xintong group (P < 0.05). After 48 hours of culture, neurospheres were cultured in the different drug-contained media, and 12 hours later, the neurospheres adhered to the wall, and a small amount of cell migration occurred. Then, cell migration began to increase with time. Under the immunofluorescence staining: prominent neurons with long protrusions were increased in both two groups, but there were no significant differences in the proportion of neurons and astrocytes between two groups (P > 0.05). These findings suggest that drug-contained sera of Naoluo Xintong and Zuogui pill can both not only promote the proliferation and differentiation of in vitro cultured rat neural stem cells, but also provide a suitable microenvironment for neural cell proliferation. Additionally, there are significant differences between the two drugs. Consequently, it is feasible to induce neural cell proliferation and differentiation by Naoluo Xintong and Zuogui pill.     

The effect of chitosan and PVDF substrates on the behavior of embryonic rat cerebral cortical stem cells

In this study, the behavior of neural stem cells from embryonic rat cerebral cortex were compared on the chitosan and poly(vinylidene fluoride) (PVDF) substrates at single-cell and neurosphere level. It was found that chitosan and PVDF substrates inhibited the proliferation and differentiation of single neural stem cells. It seemed that single-cell cultures on both substrates show cells remained dormant. However, neurospheres could exhibit different or similar behavior on these two substrates, which is dependent on the presence or absence of serum. More cells migrated outside from the neurospheres and longer processes extended from differentiated cells on chitosan than on PVDF when neurospheres were cultured in the serum-free medium. On the contrary, when serum was added to the culture system, chitosan and PVDF could induce the neurosphere-forming cells into an extensive cellular substratum of protoplasmic cells upon which process-bearing cells spread. In addition, based on the immunocytochemical analysis, the percentages of differentiated cell phenotypes of neurospheres cultured on chitosan and PVDF substrates became similar in the presence of serum. Therefore, it is reasonable to suggest that biomaterials may stimulate or inhibit the proliferation and differentiation of neural stem cells according to the complex environmental conditions. The information presented here should be useful for the development of biomaterials to regulate the preservation, proliferation, and differentiation of neural stem cells.     

Development of human brain neural/progenitor cells after transplantation into the brain of adult rats

The purpose of the present study was the investigation of human neural stem/progenitor cells (SPC) cultured in vitro, with special reference to their capacity for grafting, migration and differentiation after transplantation into adult rat brain. SPC were isolated from the brain of 9-week-old human embryos and were cultured in a selective medium for 3 weeks. For transplantation, cell suspension or whole neurospheres were used; they were studied 4 weeks following the transplantation in hippocampus, striatum and lateral ventricle of adult rat brain. For the analysis of transplanted SPC, various histological and immunohistochemical staining methods were applied (bisbenzidine, BrdU, antibodies against human nuclei, vimentin, beta-tubulin, neurofilaments, GFAP), that allowed an independent evaluation of their state and differentiation. Transplanted human brain SPC were shown to survive well for one month in all the areas of adult rat brain without immunosuppression. Cells from suspension transplants actively migrated and differentiated into neurons and glial cells. Meanwhile, cell migration from the transplanted whole neurospheres was limited or absent due to the formation of glial barrier.     

In vitro differentiated neural stem cells express functional glial glutamate transporters

The possibility to isolate stem cells from the adult central nervous system and to maintain and propagate these cells in vitro has raised a general interest with regards to their use in cell replacement therapy for degenerative brain diseases. Considering the critical role played by astrocytes in the control of glutamate homeostasis, we have characterised the expression of functional glutamate transporters in neural stem cells exposed to selected culture conditions favouring their differentiation into astrocytes. Commonly, neural stem cells proliferate in suspension as neurospheres in serum-free medium. The addition of serum or a supplement of growth factors (G5) to the culture medium was found to trigger cell adhesion on coated surfaces and to favour their differentiation. Indeed, after 7 days in these conditions, the vast majority of the cells adopted markedly distinct morphologies corresponding to protoplasmic (with serum) or fibrous (with G5 supplement) astrocytes and approximately 35–40% acquired the expression of the glial fibrillary acidic protein (GFAP). Immunocytochemical analysis also revealed that the treatments with serum or with the G5 supplement triggered the expression of the glial glutamate transporters GLT-1 (35 and 21%, respectively) and GLAST (29 and 69%, respectively). This effect was correlated with a robust increase in the Na⁺-dependent [³H]-D-aspartate uptake, which was partially inhibited by dihydrokainate, a selective blocker of GLT-1. Together, these results indicate that in vitro differentiation of cultured neural stem cells can give rise to distinct populations of astrocytes expressing functional glutamate transporters.     

体外诱导成人成肌细胞转化为神经前体细胞

目的探讨成人成肌细胞在体外能否转化为神经前体细胞。方法从成人正常颞肌分离单细胞,体外培养及克隆纯化,培养至第三代时加入含有碱性成纤维细胞生长因子、表皮生长因子和白血病抑制因子的无血清培养液诱导分化,观察诱导后细胞的形态变化,并进行免疫细胞化学鉴定和RT-PCR分析。结果成肌细胞经诱导后聚集成球,悬浮生长。免疫细胞化学染色见细胞球表达巢蛋白,5-溴脱氧尿嘧啶核苷掺入试验阳性;细胞分化后表达神经丝蛋白68,胶质纤维酸性蛋白和半乳糖脑苷脂。RT-PCR分析结果与免疫细胞化学染色结果一致。结论成人成肌细胞在体外一定条件下诱导能够转化为神经前体细胞。     

人胚胎纹状体来源神经干细胞的体外培养

背景：目前神经干细胞多由动物获得,不适合人类临床移植治疗。 目的：探索体外环境下人胚胎纹状体来源神经干细胞的培养方法,同时观察其生物学特性。 方法：取经水囊引产的孕8-16周人胚胎纹状体,体外用无血清DMEM培养基进行培养,待细胞形成神经球后进行传代,并应用含体积分数10%胎牛血清的DMEM/ F12培养液进行诱导分化。 结果与结论：体外培养的人胚胎纹状体来源神经干细胞生长迅速,表达神经干细胞标志物nestin。克隆形成实验显示细胞克隆形成率为6.0%-7.0%;BrdU掺入实验显示细胞增殖率为37.9%。免疫荧光染色显示经诱导分化的细胞表达神经元标志物Ⅲ型β微管蛋白、星形胶质细胞标志物胶质纤维酸性蛋白及神经干细胞标志物nestin,但不表达少突胶质细胞标志物髓鞘碱性蛋白。可见人胚胎纹状体来源神经干细胞在体外无血清条件下可保持其生物学特点,具有自我更新能力,经胎牛血清诱导后可向神经元及星形胶质细胞分化。     

In vivo assessment of guided neural stem cell differentiation in growth factor immobilized chitosan-based hydrogel scaffolds

In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 μm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.     

冬虫夏草含药血清诱导神经干细胞定向分化的影响

目的探讨冬虫夏草中含药血清诱导神经干细胞（NSCs）定向分化的作用。方法取新生24 h内的Wistar大鼠大脑海马区分离培养神经干细胞三代后,加入低、中、高剂量冬虫夏草含药血清与对照血清进行培养,观察其对神经干细胞影响,通过MTT检测神经细胞生长曲线和免疫组化法检测神经干细胞向神经元分化的情况。结果新生大鼠海马分离培养的神经克隆球,经免疫细胞化学染色检测为Nestin阳性细胞。神经干细胞贴壁分化的细胞呈NSE、GFAP阳性。冬虫夏草低、中剂量组诱导神经干细胞分化为神经元的阳性细胞明显高于对照组,且呈量效依赖关系,高剂量组与对照组没有明显差别。结论冬虫夏草含药血清可体外诱导神经干细胞向神经元方向分化,在一定范围内存在量效依赖关系。