Transplantation of Cultured Human Neural Progenitor Cells into Rat Brain: Migration and Differentiation

We studied the fate in vitro cultured human stem/progenitor cells after transplantation into rat brain. The cells from human fetuses at 8-12 weeks' gestation were cultured in vitro for 14 days and transplanted into the brain of 10-day-old and adult rats. Microscopic examination showed that human stem/progenitor cells migrated into various regions of rat brain. Immunohistochemical assay demonstrated that some cells differentiated into astrocytes and neurons, while others retained the embryonic phenotype.     

Immunohistochemical Study of Fetal Stem/Progenitor Cells from Human Brain Transplanted into Traumatized Spinal Cord of Adult Rats

Neural stem/progenitor cells from human fetal brain were grown in a tissue culture and transplanted into traumatized spinal cord of adult rats. The behavior and differentiation of transplanted cells were studied morphologically by means of histological and immunohistochemical methods and confocal microscopy. Human neural stem/progenitor cells were viable for not less than 3 months. They migrated and differentiated into neurons and glia in the traumatized spinal cord of adult rats.     

Lectins as a Tool for Detecting Neural Stem/Progenitor Cells in the Adult Mouse Brain

Glycoconjugates are biopolymers that are broadly distributed in the central nervous system, including the cell surface of neural stem cells or neural precursor cells (NSCs/NPCs). Glycoconjugates can be recognized by carbohydrate‐binding proteins, lectins. Two lectins, Phaseolus vulgaris lectin agglutinin E‐form (PHA‐E4) and wheat germ agglutinin (WGA) have been reported to be useful in isolating NSCs/NPCs by fluorescence‐activated cell sorting (FACS) or immunopanning methods. In this study, we analyzed the lectin‐binding properties of NSCs/NPCs in two neurogenic regions of the adult mouse brain to determine whether PHA‐E4 and WGA exhibit specific binding patterns on sections and whether there are other lectins presenting the binding pattern similar to those of PHA‐E4 and WGA in lectin histochemistry. Among nine types of lectins, peanut agglutinin was localized to the white matter and four lectins bound to cells within the subventricular zone (SVZ) of the lateral ventricle. Lectin histochemistry combined with immunohistochemistry demonstrated that one lectin, Ricinus communis agglutinin, specifically detected type A neuronal precursors and that the remaining three lectins, Agaricus bisporus agglutinin (ABA), PHA‐E4, and WGA, recognized type B NSCs and type C transient amplifying cells in the SVZ. These three lectins also recognized type 1 quiescent neural progenitors and type 2a amplifying neural progenitors in the subgranular layer of the dentate gyrus. Lectin histochemistry of the neurosphere culture also yielded similar results. These observations suggest that, in addition to PHA‐E4 and WGA, ABA lectin may also be applicable in FACS or immunopanning for the isolation of NSCs/NPCs. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Transplantation of Cultured Neural Cells from Human Fetuses into the Brain of Rats Exposed to Acute Hypoxia

Neural stem cells of human brain were cultured for a long time and successfully transplanted into the brain of rats exposed to acute hypoxia. Stem and committed cells, neuroblasts, and astrocytes were revealed in transplants by immunohistochemical assay. The transplants and brain tissue were not separated with a glial barrier. Human neuroblasts widely migrated into regions of neuronal degeneration in the host brain.     

In Vitro Development of Neural Progenitor Cells from Human Embryos

Behavior of stem/progenitor cells from the brain of human embryos during in vitro culturing was studied. Cultured cells from human embryonic brain developed and formed neurospheres heterogeneous by their cell composition. In a serum-containing medium some cells underwent differentiation by the neuronal pathway, while others remained in the stem state.     

Formation of Neuroepithelial Structures in Culture of Neural Stem Cells from Human Brain

Dissociated fetal brain cells in a floating culture form clusters and then neurospheres, some of which contain structures shaped as cell “rosettes”. The cells in these “rosettes” are arranged radially around the central cavity, in which their apical processes form desmosome-like contacts. Mitotic division of cells in the “rosettes” is associated with migration of the nuclei, similarly to division of neuroepithelial cells in the neural tube during normal embryogenesis. These cells express nestin, a marker of neural stem cells. The cells in “rosettes” found after transplantation have similar characteristics. __________ Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 3, pp. 131–135, September, 2005     

Drosophila Neural Stem Cells in Brain Development and Tumor Formation

Neuroblasts, the neural stem cells in Drosophila, generate the complex neural structure of the central nervous system. Significant progress has been made in understanding the mechanisms regulating the self-renewal, proliferation, and differentiation in Drosophila neuroblast lineages. Deregulation of these mechanisms can lead to severe developmental defects and the formation of malignant brain tumors. Here, the authors review the molecular genetics of Drosophila neuroblasts and discuss some recent advances in stem cell and cancer biology using this model system.     

Morphofunctional Study of the Therapeutic Efficacy of Human Mesenchymal and Neural Stem Cells in Rats with Diffuse Brain Injury

We studied the effect of transplantation of human stem cells from various tissues on reparative processes in the brain of rats with closed craniocerebral injury. Combined treatment with standard drugs and systemic administration of xenogeneic stem cells had a neuroprotective effect. The morphology of neurons rapidly returned to normal after administration of fetal neural stem cells. Fetal mesenchymal stem cells produced a prolonged effect on proliferative activity of progenitor cells in the subventricular zone of neurogenesis. Adult mesenchymal stem cells had a strong effect on recovery of the vascular bed in ischemic regions. Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 23-37, 2009     

人动员外周血成体干/祖细胞定向诱导分化为血管内皮前体细胞的免疫表型分析

观察动员后的人外周血成体干/祖细胞体外定向诱导分化为血管内皮前体细胞（EPC）的免疫表型特征及变化。采用健康成人经粒细胞集落刺激因子（rhG-CSF）动员后的外周血成体干/祖细胞,经贴壁培养法定向分化为EPC,之后通过流式细胞术检测EPC表型。结果显示,培养后的贴壁细胞具有内皮细胞特征,能够与I型荆豆凝集素（UEA-I）结合。流式细胞术检测UEA-I结合率为92.1%,内皮细胞标志物CD31、KDR、CD62E均有不同程度增加,而粒-巨噬细胞集落生成单位（CFU-GM）及CD34表达明显减低,表明诱导分化后细胞的免疫表型发生了相应的变化。     

联合应用EGF和NGF对成年大鼠海马神经干细胞分化的影响

目的探讨联合应用表皮生长因子(EGF)和神经生长因子(NGF)对成年大鼠海马神经干细胞(NSCs)分化的影响。方法用含碱性成纤维生长因子(bFGF)、表皮生长因子、B27的无血清细胞培养技术体外培养成年大鼠海马神经干细胞,单克隆培养细胞行Nestin免疫细胞化学染色,诱导分化1w后细胞行GFAP和NSE免疫细胞化学染色;根据培养基中所加营养因子的不同将第4代细胞分为4组培养:EGF组、EGF+NGF组、NGF组、对照组,此4组细胞培养1w后行NSE免疫细胞化学染色,计数阳性细胞比例后进行统计学分析。结果单克隆培养后克隆球表达Nestin,诱导分化1w后细胞表达NSE、GFAP。与空白对照组相比,EGF组、NGF组和EGF+NGF组细胞分化为神经元的比例较高(P<0.05),其中EGF+NGF组细胞的比例最高。结论单独或联合应用EGF、NGF可以促进成年大鼠海马神经干细胞向神经元分化。     

Low Doses of Bisphenol A Disrupt Neuronal Differentiation of Human Neuronal Stem/Progenitor Cells

Bisphenol A (BPA) is an endocrine disrupting chemical. Human epidemiological studies have suggested that adverse neurobehavioral outcomes are induced by fetal exposure to BPA. The remarkable differences in the corticogenesis between human and agyrencephalic mammals are an increase in the intermediate progenitor cells (IPCs) and a following increase in the subplate thickness. It is uncertain whether low doses of BPA (low-BPA) affect human early corticogenesis when basal progenitor cells (BPs) produce IPCs resulting in amplified neurogenesis. In this study, human-derived neuronal stem/progenitor cells were exposed to low-BPA or the vehicle only, and the resultant cell type-specific molecular changes and morphology were analyzed. We focused on stem cells immunoreactive for SOX2, BPs for NHLH1, and immature neurons for DCX. SOX2-positive cells significantly decreased at day in vitro (DIV) 4 and 7, whereas NHLH1-positive cells tended to be higher, while DCX-positive cells significantly increased at DIV7 when exposed to 100 nM of BPA compared with the vehicle. Morphologically DCX-positive cells showed a decrease in unipolar cells and an increase in multipolar cells when exposed to 100 nM of BPA compared with the vehicle. These results provide insights into the in vivo effect of low-BPA on neuronal differentiation in the human fetal corticogenesis.     

缺氧对新生大鼠海马神经干细胞增殖和分化的影响

目的探讨单纯性缺氧对新生大鼠海马神经干细胞(NSCs)增殖和分化的影响。方法将36只新生1d大鼠随机均分为N组(正常对照组)、Q2组(缺氧2h组)、Q5组(缺氧5h组)。将海马组织在无血清培养液(均含有碱性成纤维生长因子bFGF、表皮生长因子EGF和白细胞抗原B27)中,分别进行原代培养、单克隆培养和传代培养,单克隆培养细胞进行巢蛋白(Nestin)和Hoechst33342免疫荧光双标染色;传代培养的细胞传三代诱导分化,诱导分化3d的细胞分别行神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和Hoechst3342免疫荧光双标染色,计数NSE阳性细胞比例,进行统计学分析。结果单克隆培养的细胞均呈Nestin阳性表达,诱导分化的细胞部分呈NSE或GFAP阳性表达。Q2组、N组、Q5组细胞原代培养克隆球直径达2μm的时间分别为7d、10d、13d。传3代后,对各组细胞进行诱导分化,Q2组细胞诱导分化为神经元的比例明显高于N组,Q5组的细胞诱导分化为神经元的比例明显低于N组。结论短时间缺氧可促进在体海马神经干细胞增殖和诱导其向神经元分化,长时间缺氧则抑制在体神经干细胞增殖和向神经元分化。     

胰岛素对大鼠神经干细胞增殖和分化的影响

目的探讨胰岛素对大鼠神经干细胞(NSCs)增殖和分化的影响。方法培养大鼠大脑皮层NSCs,用胰岛素作用于培养的NSCs,3H-TdR掺入检测NSCs的增殖,免疫细胞化学和流式细胞仪检测NSCs的分化。结果胰岛素能明显促进NSCs对3H-TdR的摄入,明显促进细胞的增殖,并且胰岛素促细胞增殖作用具有一定的量效关系;免疫细胞化学和流式细胞仪结果显示,胰岛素对NSCs向神经元的分化有明显促进作用,并且NSCs分化后多巴胺能神经元数目明显增加。结论胰岛素可能具有促进NSCs增殖和向神经元包括多巴胺能神经元分化的作用。     

大鼠骨髓间充质干细胞向神经细胞和星形胶质细胞分化的研究

目的探讨体外诱导骨髓间充质干细胞(BMSCs)分化成神经细胞或星形胶质细胞。方法使用四种不同的诱导方式来处理BMSCs:-巯基乙醇(BME)、维甲酸(RA)、无血清培养基以及和新生鼠的脑星形胶质细胞共培养。结果四种方法处理后,BMSCs都能表达神经细胞标示物MAP2或星形胶质细胞标志物GFAP,并伴随着明显的细胞形态的改变。用RA诱导以及在无血清的培养基中培养,BMSCs主要分化成神经元样细胞,表达神经元的标志物MAP2,并且BME能促进这个过程。但在和星形胶质细胞共培养后,BMSCs主要分化成GFAP+细胞。结论BMSCs依赖于不同的诱导条件,具有分化成神经细胞或星形胶质细胞的可塑性。     

红景天苷对新生大鼠海马区神经干细胞分化的研究

目的研究红景天苷药物血清诱导新生大鼠海马神经干细胞向神经元方向分化,并促进所分化神经元细胞发育的作用。方法从新生24h内的Wistar大鼠脑中分离扩增获得大量神经干细胞后,加入低、中、高剂量的红景天苷药物血清及对照血清,观察其对神经干细胞分化为神经元的影响,并通过免疫细胞化学染色检测神经干细胞分化为神经元的状况。结果各药物实验组诱导神经干细胞分化为NSE阳性细胞的个数面积与周长明显高于对照组(P<0.05),分化为GFAP阳性细胞的个数明显低于对照组,且呈量效依赖关系。结论红景天苷药物学清在体外可促进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,对所分化的神经元细胞有促生长发育作用。     

异丙酚促进体外培养的成年大鼠海马神经干细胞增殖

目的异丙酚对于成年神经干细胞增殖能力的影响目前尚不明确,本研究对异丙酚对体外培养的大鼠成年神经干细胞的作用及其可能的分子机制进行了研究。方法体外培养的大鼠成年神经干细胞给予BrdU后,分别用10、50、100μg/ml的异丙酚进行处理,并同时设立溶媒对照和空白对照。24h后,采用免疫染色显示BrdU阳性细胞,DAPI复染细胞核。细胞存活率采用细胞计数法和MTT法测定。人工计数BrdU阳性细胞和固缩细胞核的比例。用Fura2-AM检测胞浆内Ca2＋浓度。分别用免疫荧光和免疫印迹法检测细胞内CREB和磷酸化CREB的表达位置和水平。结果低浓度（10μg/ml）异丙酚不能促进大鼠成年神经干细胞的增殖,而中浓度（50μg/ml）和高浓度异丙酚（100μg/ml）则呈剂量依赖性的促进其增殖。异丙酚提高了BrdU阳性细胞的比例。细胞死亡率在处理前后维持在低水平上（〈2%）。异丙酚显著提高了大鼠成年神经干细胞胞浆内的游离Ca2＋浓度。异丙酚处理前后,大鼠成年神经干细胞均能表达CREB和磷酸化CREB,但是异丙酚干预后,大鼠成年神经干细胞内磷酸化CREB表达水平显著增高。结论异丙酚可促进体外培养的大鼠成年神经干细胞的增殖水平,这是细胞存活率升高的主要原因,可能与胞浆内Ca2＋浓度以及CREB磷酸化水平的升高有关。     

pCREB is Involved in Neural Induction of Mouse Embryonic Stem Cells by RA

Mouse embryonic stem (ES) cells can be induced by various chemicals to differentiate into a variety of cell types in vitro. In our study, retinoic acid (RA), one of the most important inducers, used at a concentration of 5 μM, was found to induce the differentiation of ES cells into neural progenitor cells (NPCs). During embryoid body (EB) differentiation, the level of active cyclic AMP response element‐binding protein (CREB) was relatively high when 5 μM RA treatment was performed. Inhibition of CREB activity committed EBs to becoming other germ layers, whereas increased expression of CREB enhanced NPC differentiation. Moreover, RA increased the expression of active CREB by enhancing the activity of JNK. Our research suggests that CREB plays a role in RA‐induced NPC differentiation by increasing the expression of active JNK. Anat Rec, 291:519–526, 2008. © 2008 Wiley‐Liss, Inc.     

星形胶质细胞条件培养液促进新生大鼠海马神经干细胞分化

目的探讨星形胶质细胞条件培养液(ACM)对神经干细胞(NSCs)体外分化的影响。方法行新生大鼠大脑皮质星形胶质细胞的纯化培养后收集ACM,将新生大鼠海马NSCs单克隆培养后行nestin免疫细胞荧光染色,诱导分化5d后行神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、半乳糖脑苷脂(GALC)免疫细胞荧光染色;将单克隆培养的NSCs分为对照组和实验组,对照组为单纯NSCs培养液,实验组根据NSCs培养液与ACM比的不同分3组:A组(2:1),B组(1:1),C组(1:2)。各组培养1周,采用NSE免疫细胞荧光检测方法标记神经元并计数和统计学分析。结果纯化的星形胶质细胞胶质纤维酸性蛋白抗体标记阳性率为96.5%;单克隆培养的海马NSCs呈nestin阳性,诱导后呈NSE、GFAP和GALC阳性表达。ACM培养的海马NSCs各组分化为神经元的比例明显高于对照组(<0.01),其中A组的比例最高。结论ACM可以促进新生大鼠海马NSCs向神经元分化。     

Combinated Transplantation of Neural Stem Cells and Collagen Type I Promote Functional Recovery After Cerebral Ischemia in Rats

Using tissue engineering, a complex of neural stem cells (NSCs) and collagen type I was transplanted for the therapy of cerebral ischemic injury. NSCs from E14 d rats were dissociated and cultured by neurosphere formation in serum‐free medium in the presence of basic fibroblast growth factor (bFGF), then seeded onto collagen to measure cell adhesive ability. BrdU was added to the culture medium to label the NSCs. Wistar rats (n=100) were subjected to 2‐hour middle cerebral artery occlusion. After 24 hours of reperfusion, rats were assigned randomly to five groups: NSCs‐collagen repair group, NSCs repair group, unseeded collagen repair group, MCAO medium group, and sham group. Neurological, immunohistological and electronic microscope assessments were performed to examine the effects of these treatments. Scanning electronic microscopy showed that NSCs assemble in the pores of collagen. At 3, 7, 15, and 30 d after transplantation of the NSC‐collagen complex, some of the engrafted NSCs survive, differentiate and form synapses in the brains of rats subjected to cerebral ischemia. Six d after transplantation of the NSC‐collagen complex into the brains of ischemic rats, the collagen began to degrade; 30 d after transplantation, the collagen had degraded completely. The implantation of NSCs and type I collagen facilitated the structural and functional recovery of neural tissue following ischemic injury. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

不同鼠龄生后大鼠海马神经干细胞的传代和分化

目的研究不同鼠龄大鼠海马神经干细胞(NSCs)的传代增殖和分化情况。方法将生后SD大鼠分为3d、10d、20d 3组,应用胰酶消化法将海马组织制成单细胞悬液,用含碱性成纤维生长因子(bFGF)、表皮生长因子(EGF)、B27的无血清细胞培养技术进行体外培养,单克隆培养后通过Nestin、NSE、GFAP免疫细胞化学染色鉴定神经干细胞、神经元、星形胶质细胞;细胞传3代后,在各组培养基中加入终浓度为10%的血清诱导分化,1周后进行NSE免疫细胞化学染色,计数阳性细胞比例,进行统计学分析。结果3组SD大鼠海马组织细胞,单克隆培养后表达Nestin,诱导分化后分别表达NSE和GFAP。生后3d大鼠神经干细胞在传1-6代时每代均快于生后10d和20d大鼠,传6代后,3组细胞传代时间间隔几近一致;生后3d组大鼠海马神经干细胞分化为神经元的比例较其它2组高(P<0.05)。结论生后3d大鼠神经干细胞增殖速度和分化为神经元的数量都高于10d和20d大鼠。