Intraocular injection of folate antagonist methotrexate induces neuronal differentiation of embryonic stem cells transplanted in the adult mouse retina

Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuronal cells. However, the integrated ES cells also have a tumorigenic effect just because they have the ability for multipotential differentiation to various types of tissues. Thus, control of neoplastic potentials of ES cells is very important for the treatment of degenerative or injured diseases. Mouse ES cells carrying the sequence for the green fluorescent protein (GFP) gene were transplanted into adult mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-d-aspartate (NMDA) administration. One week after the ES cell injection, folate antagonist methotrexate (MTX) was injected intravitreally. Eyes were retrieved 4 weeks after ES cell transplantation for histologic analyses. Conventional histological analysis was performed by hematoxylin and eosin staining with the use of paraffin-embedded sections. Neuronal differentiation and teratogenic potential of ES cells were demonstrated by immunohistochemistry. The proliferative activity of transplanted cells was detected by mitotic index, proliferating cell nuclear antigen index and AgNOR count. The incorporation of transplanted ES cells in MTX-treated and non-treated retinas at 4 weeks after transplantation was observed in 8/16 eyes (50%) and 8/16 eyes (50%), respectively. Transplanted ES cells in MTX-treated retina showed increased neuronal differentiation and decreased expression of teratogenic markers, compared with ES cells in non-treated retina. The proliferative activity of transplanted ES cells in MTX-treated retina was lower than that in non-treated retina. These results suggest that intravitreal MTX treatment following transplantation can induce neuronal differentiation in the transplanted ES cells and decrease their proliferative activity.     

Induced neuronal differentiation of human embryonic stem cells

Human embryonic stem (ES) cells are pluripotent cells capable of forming differentiated embryoid bodies (EBs) in culture. We examined the ability of growth factors under controlled conditions to increase the number of human ES cell-derived neurons. Retinoic acid (RA) and nerve growth factor (betaNGF) were found to be potent enhancers of neuronal differentiation, eliciting extensive outgrowth of processes and the expression of neuron-specific molecules. Our findings show that human ES cells have great potential to become an unlimited cell source for neurons in culture. These cells may then be used in transplantation therapies for neural pathologies.     

Phosphatase and tensin homolog deletion enhances neurite outgrowth during neural stem cell differentiation

Neural stem cell (NSC) transplantation has emerged as a promising approach for the treatment of neurological disorders such as cerebral ischemia. As the majority of newly generated cells from exogenous NSCs fail to integrate into the ischemic brain and establish functional synaptic networks, NSC transplantation for ischemic stroke experiences limited neurological function recovery. Augment of endogenous neurite growth in the process of NSC differentiation is an avenue to promote synaptic networks. Phosphatase and tensin homolog (PTEN), a tumor suppressor, has been established to regulate axon growth in the adult central nervous system. The aim of this study was to explore the role of PTEN on neurite growth during NSC differentiation. Our results revealed that the protein expression of PTEN was significantly increased during NSC differentiation, whereas the expression of phosphorylated S6 ribosomal (p-S6R) was markedly decreased. Small interfering RNA knockdown of PTEN in NSCs can accelerate neurite outgrowth during NSC differentiation. These results indicated a remarkable effect of PTEN inhibition on neuronal process after NSC differentiation, and identified a novel route to promote endogenous neurite growth in differentiated NSCs, which may facilitate the application of NSC transplantation in ischemic stroke.     

Fusion of neural stem cells in culture

An important issue in stem cell biology relates to mechanisms of cellular plasticity. Specifically, could any observed multipotency of, e.g., adult stem cells arise from true transdifferentiation or as a result of cell-cell fusion? We studied this issue using a culture paradigm of astrocyte monolayers and multipotent neurospheres generated from neonatal cerebellar cortex and the subventricular zone (SVZ). Based on fluorescence in situ hybridization (FISH), cells from these cultures were found to contain an abnormal number of sex chromosomes, suggesting that cellular fusion is a common in vitro occurrence. A Cre/lox recombination method was also exploited to further confirm the evidence of fusion. Next, we assessed the potential of fusogenic microglial involvement by combining CD11b immunolabeling with FISH sex chromosome analysis. Differentiating neurospheres were also studied from the PU.1 knockout mouse that lacks cells of myeloid origin, presumed to be a source of central nervous system microglia. Very few cells immunopositive for the microglial marker CD11b were found to be aneuploid, and there was no difference in fusion frequency between PU.1+/+ and PU.1-/- neurospheres. These results, together, suggest that stem and/or progenitor cells that generate neurons and glia in culture possess the ability to generate fused polyploidal cells, but microglial participation is not a requirement for fusion to occur. In addition to caution that should be exerted during the interpretation of in vitro neural cell plasticity, the data also suggest that novel therapeutic treatments could be designed that exploit cellular fusion in rescue paradigms for degenerating neuronal populations.     

Characterization of neuronal/glial differentiation of murine adipose-derived adult stromal cells

Neural tissue has limited capacity for intrinsic repair after injury, and the identification of alternate sources of neuronal stem cells has broad clinical potential. Preliminary studies have demonstrated that adipose-derived adult stromal (ADAS) cells are capable of differentiating into mesenchymal and non-mesenchymal cells in vitro, including cells with select characteristics of neuronal/glial tissue. In this study, we extended these observations to test the hypothesis that murine (mu) ADAS cells can be induced to exhibit characteristics of neuronal and glial tissue by exposure to a cocktail of induction agents. We characterized the differentiation of muADAS cells in vitro using immunohistochemistry and immunoblotting, and examined whether these cells respond to the glutamate agonist N-methyl-D-aspartate (NMDA). We found that induced muADAS cells express proteins indicative of neuronal/glial cells, including nestin, GFAP, S-100, NeuN, MAP2, tau, and beta-III tubulin. Induced muADAS cells express gamma-aminobutyric acid (GABA), the NR-1 and NR-2 subunits of the glutamate receptor, GAP-43, synapsin I, and voltage-gated calcium channels. Finally, induced muADAS cells demonstrate decreased viability in response to NMDA. These findings suggest that muADAS cells can be induced to exhibit several phenotypic, morphologic, and excitotoxic characteristics consistent with developing neuronal and glial tissue.     

Stromal cell-derived inducing activity does not promote dopaminergic differentiation, but enhances differentiation and proliferation of neural stem cell-derived astrocytes

Previous evidence has shown that stromal cell-derived inducing activity (SDIA), produced by the mouse PA6 stromal cell line, promotes dopaminergic differentiation of mouse, monkey and human embryonic stem cells in vitro. To examine whether PA6 stromal cells can enhance the yield of dopaminergic differentiation from neural progenitors, we generated neurospheres from embryonic day 11.5 (E11.5) (midbrain and forebrain) and E14.5 (ventral mesencephalon and cortex) rat embryos and allowed them to differentiate in co-culture with PA6 cells or poly-l-lysine/laminin-coated dishes. We observed that SDIA did not promote dopaminergic differentiation of E11.5 and E14.5 neurospheres but more prominently, enhanced astrocyte differentiation, cell survival and astrocyte proliferation. Our results suggest that PA6 cells do not have a general capacity to promote differentiation into dopaminergic neurons from all types of stem cells, but that they may specifically induce dopaminergic differentiation of highly uncommitted stem cells such as embryonic stem cells.     

诱导成人骨髓基质细胞成为神经干细胞及其分化的实验研究

目的　体外诱导成人骨髓基质细胞 (BMSCs)转化为神经干细胞 (NSCs)进而分化为神经元和胶质细胞。方法　以含有碱性成纤维细胞生长因子 (b FGF)或表皮生长因子 (EGF)加 b FGF或 b FGF、EGF加全反式维甲酸 (ATRA)的培养液培养 BMSCs,进行显微镜观察 ,纤维连接蛋白 (fibronectin)、I型胶原 (collagen )和神经上皮干细胞蛋白 (nestin)免疫组织化学染色和神经元特异烯醇化酶 (NSE) ,胶质纤维酸性蛋白 (GFAP)免疫荧光染色。结果　经诱导物诱导 72 h后 ,fibronectin和 collagen I免疫阳性细胞减少。nestin免疫阳性细胞增多。 7d后 ,其又减少。同时 NSE和 GFAP免疫阳性细胞增多。细胞分化后 ,NSE阳性细胞最多占细胞总数的 2 4 .76 %±2 .72 % ,同时 GFAP阳性细胞占细胞总数的 36 .5 8%± 3.2 6 %。结论　 EGF、b FGF、ATRA及适宜的培养液可使BMSCs定向 ,转化为 NSCs,进而分化为神经元和胶质细胞。     

Fluoxetine promotes gliogenesis during neural differentiation in mouse embryonic stem cells

Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed for treatment of mood disorders and depression, even during pregnancy and lactation. SSRIs are thought to be much safer than tricyclic antidepressants, with a low risk of embryonic toxicity. Several recent studies, however, have reported that fetal exposure to SSRIs increases the risk of adverse effects during fetal and neonatal development. This is consistent with our previous finding that fluoxetine, a prototypical SSRI, profoundly affected the viability of cultured embryonic stem (ES) cells as well as their ability to differentiate into cardiomyocytes. Furthermore, we found that fluoxetine induced fluctuations in ectodermal marker gene expression during ES cell differentiation, which suggests that fluoxetine may affect neural development. In the present study, we investigated the effects of fluoxetine on the process of differentiation from ES cells into neural cells using the stromal cell‐derived inducing activity (SDIA) method. Fluoxetine treatment was found to enhance the expression of glial marker genes following neural differentiation, as observed by immunocytochemical analysis or quantitative RT‐PCR. The promoter activity of glial marker genes was also significantly enhanced when cells were treated with fluoxetine, as observed by luciferase reporter assay. The expression of neuronal markers during ES cell differentiation into neural cells, on the other hand, was inhibited by fluoxetine treatment. In addition, FACS analysis revealed an increased population of glial cells in the differentiating ES cells treated with fluoxetine. These results suggest that fluoxetine could facilitate the differentiation of mouse ES cells into glial cell lineage, which may affect fetal neural development. © 2010 Wiley‐Liss, Inc.     

突触蛋白-Ⅰ在胚胎干细胞体外神经分化过程中的表达变化

目的探讨突触蛋白-I（synapsin-I）在胚胎干细胞（ESCs）体外神经分化过程中的表达变化及作用。方法采用“五步法”和维甲酸（RA）法两种途径体外诱导ESCs向神经细胞分化，并以另一种可向神经细胞分化的肿瘤细胞-PC12细胞的诱导过程作参照，从不同的途径、不同的细胞进行比较．通过免疫组织化学、RT-PCR、Western blot方法观察这一过程中synapsin-I的表达变化．找出synapsin-I在ESCs向神经细胞分化过程中表达变化的共同规律。结果结合形态学和其它神经特异性指标的变化，synapsin-I在ESCs和PC12细胞向神经细胞分化的过程中具有早期即有表达，后逐渐升高，至分化成熟阶段达最高，后期又逐渐下降的变化规律。结论在ESCs的分化过程中，synapsin-I的表达存在特定的时空规律，并与ESCs的形态学改变相关，提示synapsin-I可能对ESCs在神经分化过程中的形态分化起着重要的作用。     

头针诱导神经干细胞的增殖分化

目的研究脑缺血再灌注大鼠神经干细胞（neural stem cells,NSCs）增殖、分化情况及头针对其影响。方法将Wistar大鼠70只,随机分为假手术组10只、模型组和头针组各30只;模型组和头针组采用线栓法制作大脑中动脉闭阻（middle cerebralartery occlusion,MCAO）模型,各组大鼠又按照缺血时间（7、14、28d）分为3个亚组,每个时相点10只。头针组大鼠于缺血再灌注成功后即行头针治疗,每日1次,直至处死前;各组大鼠处死前1d腹腔注5-溴脱氧尿核苷（bromodeoxyuridine,BrdU）液;对各亚组大鼠行神经功能缺损评分（neurological severity score,NSS）,免疫荧光法计数各组大鼠海马（dentate gyrus,DG）BrdU阳性细胞和BrdU/NeuN阳性双标细胞。结果第28d头针组的NSS显著低于模型组（P〈0.05）。大鼠缺血再灌注后各组阳性细胞有明显表达,而头针组在不同时相的阳性细胞数较同时相模型组有明显增加。结论头针能促进神经干细胞的增殖和分化,改善神经功能缺损评分,从而促进神经功能的修复。     

突触蛋白-Ⅰ反义寡核苷酸对胚胎干细胞体外神经分化过程的影响

目的 探讨突触蛋白-Ⅰ在体外诱导胚胎干细胞(ESC)向神经细胞分化过程中的作用,寻求这一过程的可调控点或调控切入点.方法 采用"五步法"体外诱导ESC向神经细胞分化,于不同诱导阶段转染突触蛋白-Ⅰ反义寡核苷酸,观察转染后ESC的形态学、分化效率及其他神经特异性蛋白表达的变化.同时以突触蛋白-Ⅰ反义寡核苷酸对PC12细胞诱导过程的影响作参照.结果 胚胎干细胞分化的第3阶段反义链组的突起伸长速度较正常组和正义链组减慢,分化的神经前体细胞[nestin(+)]比例较正常组和正义链组明显减少(68.5%±4.2% vs 76.2%±5.1%和75.8%±4.9%,P＜0.05).第4阶段反义链组所扩增的神经前体细胞[nestin(+)]比例较正常组和正义链组明显减少(75.1%±4.7% vs 90.2%±4.3%和88.7%±4.5%,P＜0.01).第5阶段反义链组细胞之间的联系较正常组和正义链组减少,神经元样细胞[MAP2(+)]的比例较正常组和正义链组减少(30.7%±3.2% vs 41.2%±2.7%和40.5%±2.4%,P＜0.05).PC12细胞反义链组于诱导第1、4、7、10天细胞突起长度均较正常组和正义链组短,细胞分化率(0.33%±0.46%、9.78%±3.47%、45.3%±7.98%和34.2%±5.89%)显著低于正常组(1.81%±0.40%、45.13%±4.17%、90.26%±4.68%和84.66%±4.81%)和正义链组(P＜0.01).结论 抑制突触蛋白-Ⅰ的表达可导致胚胎干细胞向神经细胞分化的进程滞后和神经分化效率降低,提示突触蛋白-Ⅰ在胚胎干细胞的体外神经分化过程中各阶段均起着重要的作用,可能参与了其中的调控机制.     

七氟烷促进成年大鼠海马神经干细胞增殖

目的 观察七氟烷对体外培养的成年大鼠海马神经干细胞(NSCs)是否具有增殖作用及可能的分子机制. 方法 常规培养成年NSCs并应用免疫荧光染色巢蛋白进行鉴定.将培养2h的NSCs分别放入含不同浓度(1.3%、1.9%、2.7%、3.3%)七氟烷的密闭容器中,作用2 h后按常规方法 继续培养24h.应用DAPI染色检测NSCs数目的 变化.用Western blot法检测细胞环磷腺苷反应元件结合蛋白(CREB)、磷酸化CREB(pCREB)水平的变化. 结果与空白对照组比较,1.9%、2.7%、3.3%七氟烷组细胞数升高,而且浓度越大,作用越明显,差异均有统计学意义(P<0.05);Western blot检测发现空白对照组和1.3%、1.9%七氟烷组细胞均表达CREB和pCREB,但1.9%七氟烷组细胞pCREB/CREB水平较高,与空白对照组和1.3%七氟烷组比较差异有统计学意义(P<0.05). 结论 七氟烷可促进NSCs的增殖能力,其机制可能与提高了细胞中CREB的磷酸化水平有关.     

IL-6促进骨髓源性神经干细胞增殖的实验研究

目的研究白细胞介素6(IL-6)及其受体对体外培养的大鼠骨髓源性神经干细胞增殖的影响及信号转导机制。方法采用密度梯度离心法分离骨髓基质细胞,应用特制的神经干细胞培养基诱导培养6d后收集贴壁细胞传代培养,用AlamarBlue摄入法检测不同浓度的IL-6及IL-6受体对大鼠骨髓源性神经干细胞增殖的影响,Westernblot方法检测不同剂量IL-6对干细胞中STAT3表达的影响。结果IL-6能够促进干细胞的增殖,随着药物剂量的增加,增殖作用明显增加,呈明显剂量依赖性,其中20、100、200ng/mL组均与对照组有显著差异(P<0.01),但2ng/mL与对照组、100ng/mL与200ng/mL组之间无显著差异(P>0.05),20ng/mL与100ng/mL组之间有差异(P<0.05)。IL-6受体可抑制干细胞的增殖,单独应用IL-6受体,100ng/mL、200ng/mL组与对照组均有显著差异(P<0.05)。而IL-6与IL-6受体联合应用可促进干细胞的增殖,但增殖作用比单独应用IL-6组差,两组相比有显著性差异(P<0.05)。随IL-6浓度的增加,干细胞中的STAT3表达增加,20、100、200ng/mL组与对照组相差显著(P<0.01)。结论100ng/mL组的IL-6对骨髓源性神经干细胞有较好的增殖促进作用;IL-6受体可抑制骨髓源性神经干细胞增殖;联合应用IL-6和IL-6受体增殖效果不如单独应用IL-6,提示骨髓源性神经干细胞表面自身具有IL-6受体;IL-6通过增加STAT3表达促进骨髓源性神经干细胞增殖。     

脑源性神经生长因子基因转染对脊髓神经干细胞分化的影响

目的 研究BDNF基因转染小鼠脊髓源性NSCs向神经元分化情况.方法 选取体外培养E14小鼠胚胎脊髓来源NSCs,构建整合有BDNF基因的逆转录病毒载体,感染体外培养的NSCs,诱导其向神经元分化.采用免疫细胞化学方法鉴定,确定NSCs的分化比例.结果 转染后诱导分化24 h后可见部分细胞贴壁分化,48 h后转染细胞大部分贴壁.BDNF转染NSCs分化为神经元比例较未转染NSCs明显增高,差异有统计学意义(P＜0.05).结论 逆转录病毒载体介导BDNF基因转染NSCs可促进细胞分化,且分化多为神经元方向.

Abstract：

Objective To study the differentiation potential of mouse spinal cord derived neural stem cells (NSCs) into neurons after being transfected with BDNF gene in vitro. Methods Spinal cord derived NSCs from the E14 fetus mouse were isolated and cultured in vitro; the retrovirus containing pLXSN-BDNF gene was established and transfected into thc above NSCs, and thea, spinal cord derived NSCs were induced to be differentiated into neuron-like cells. Immunohistochemistry was employed to detect and calculate the ratio of differentiation of NSCs into neurons. Results The NSCs cultured in vitro partly adhered to the wall and differentiated within 24 h of transfection with BDNF gene, and most of the NSCs adhered to the wall differentiated within 48 h of transfection. The level of neurons from spinal cord derived NSCs modified by BDNF gene was markedly increased as compared with that that from normal spinal cord derived NSCs (P＜0.05). Conclusion NSCs transfected by retroviral pLXSN-BDNF can promote the cell differentiation. BDNF gene can increase greatly the percentage of neurons in the course of inducing the differentiation of mouse NSCs.     

Autophagy activator promotes neuronal differentiation of adult adipose-derived stromal cells

Preliminary research from our group found altered autophagy intensity during adipose-derived stromal cell differentiation into neuronal-like cells, and that this change was associated with morphological changes in differentiated cells. This study aimed to verify the role of rapamycin, an autophagy activator, in the process of adipose-derived stromal cell differentiation into neuronal-like cells. Immunohistochemical staining showed that expression of neuron-specific enolase and neurofilament-200 were gradually upregulated in adipose-derived stromal cells after 5 mM β-mercaptoethanol induction, and the differentiation rate gradually increased with induction time. Using transmission electron microscopy, induced cells were shown to exhibit cytoplasmic autophagosomes, with bilayer membranes, and autolysosomes. After rapamycin (200 μg/L) induction for 1 hour, adipose-derived stromal cells began to extend long processes, similar to the morphology of neuronal-like cells, while untreated cells did not exhibit similar morphologies until 3 hours after induction. Moreover, the differentiation rate was significantly increased after rapamycin treatment. Compared with untreated cells, expression of LC3, an autophagy protein, was also significantly upregulated. Positive LC3 expression tended to concentrate at cell nuclei with increasing induction times. Our experimental findings indicate that autophagy can significantly increase the speed of adipose-derived stromal cell differentiation into neuronal-like cells.     

神经干细胞分化过程中c-myc内含子结合蛋白1与c-myc基因表达研究

目的研究神经干细胞分化为胶质细胞过程中c-myc内含子结合蛋白1(MIBP1)基因与c-myc基因的表达变化。方法从孕16d胚胎鼠脑组织中分离神经干细胞,以专用培养基IMDM(含有B27添加剂,重组人上皮生长因子、重组人碱性成纤维细胞生长因子-2、L-谷氨酸和黄体酮)进行培养。应用胶原及含10%胎牛血清、抗生素的分化培养基,诱导细胞向胶质细胞及神经元方向分化,第1天细胞贴壁启动分化,第7天明显分化,第14天进入终末分化。经过振荡处理,进一步得到纯化星形胶质细胞,采用胶质纤维酸性蛋白免疫组化方法鉴定细胞分化。分别提取分化前及分化后第1、7和14天神经干细胞的RNA,应用逆转录-多聚酶链反应方法检测神经干细胞中MIBP1与c-myc基因的表达,以β-actin作为内参照物,应用图像分析仪对电泳条带进行定量分析,观察两基因在神经干细胞分化中的作用。结果在神经干细胞分化过程中,MIBP1基因表达逐渐增强,其中未分化神经干细胞为(63.53±11.97)%,培养第1、7和14天表达率分别为(70.04±16.10)%、(79.09±15.40)%和(99.65±0.54)%。而c-myc基因表达呈逐渐降低,未分化神经干细胞为(99.76±7.62)%;培养第1、7和14天表达率分别为(75.20±12.33)%、(45.65±10.11)%和(33.31±4.34)%。两基因分别与相应未分化组比较,差异具有极显著性意义(均P<0.001)。星形胶质细胞纯化前后MIBP1基因表达量无明显变化。结论在神经干细胞分化为胶质细胞的过程中MIBP1基因表达逐渐增强,同时c-myc基因表达受到抑制。提示MIBP1基因与胶质细胞的分化过程相关,可能通过抑制c-myc基因的表达而实现调控作用。     

鼠脑梗死后自体神经干细胞的原位增殖、分化及其可塑性

目的研究成年大鼠脑梗死后自体神经干细胞的原位增殖、分化及其可塑性。方法雄性Wistar大鼠共90只,分对照组(n=10)、脑梗死后1d组(n=16)、脑梗死后3d组(n=16)、脑梗死后7d组(n=16)、脑梗死后14d组(n=16)、脑梗死后28d组(n=16)。用免疫组织化学方法动态检测BrdU、GFAP、NeuN、PSA-NCAM的表达。BrdU确定神经干细胞的增殖,GFAP、NeuN确定神经干细胞的分化,PSA-NCAM确定神经干细胞的可塑性。结果与对照组相比,大鼠海马BrdU 细胞数在脑梗死后1d组开始增加,7d组达到高峰,28d组接近正常水平;BrdU /GFAP 细胞数在脑梗死前后无明显变化;BrdU /NeuN 细胞数在脑梗死后14d组开始增加,28d组最多;BrdU /PSA-NCAM 细胞数在脑梗死后7d组开始增加,14d组达到高峰,28d组开始下降,但仍高于对照组,大约占同期BrdU阳性细胞数60%。结论大鼠脑梗死激活自体神经干细胞原位增殖,大多数增殖的神经干细胞分化成神经元并且具有可塑性。     

Astrocyte‐derived interleukin‐6 promotes specific neuronal differentiation of neural progenitor cells from adult hippocampus

The purpose of this study was to investigate the ability of astrocyte‐derived factors to influence neural progenitor cell differentiation. We previously demonstrated that rat adult hippocampal progenitor cells (AHPCs) immunoreactive for the neuronal marker class III β‐tubulin (TUJ1) were significantly increased in the presence of astrocyte‐derived soluble factors under noncontact coculture conditions. Using whole‐cell patch‐clamp analysis, we observed that the cocultured AHPCs displayed two prominent voltage‐gated conductances, tetraethyl ammonium (TEA)‐sensitive outward currents and fast transient inward currents. The outward and inward current densities of the cocultured AHPCs were approximately 2.5‐fold and 1.7‐fold greater, respectively, than those of cells cultured alone. These results suggest that astrocyte‐derived soluble factors induce neuronal commitment of AHPCs. To investigate further the activity of a candidate neurogenic factor on AHPC differentiation, we cultured AHPCs in the presence or absence of purified rat recombinant interleukin‐6 (IL‐6). We also confirmed that the astrocytes used in this study produced IL‐6 by ELISA and RT‐qPCR. When AHPCs were cultured with IL‐6 for 6–7 days, the TUJ1‐immunoreactive AHPCs and the average length of TUJ1‐immunoreactive neurites were significantly increased compared with the cells cultured without IL‐6. Moreover, IL‐6 increased the inward current density to an extent comparable to that of coculture with astrocytes, with no significant differences in theoutward current density, apparent resting potential, or cell capacitance. These results suggest that astrocyte‐derived IL‐6 may facilitate AHPC neuronal differentiation. Our findings have important implications for understanding injury‐induced neurogenesis and developing cell‐based therapeutic strategies using neural progenitors. © 2010 Wiley‐Liss, Inc.     

Analysis of the expression and function of BRINP family genes during neuronal differentiation in mouse embryonic stem cell‐derived neural stem cells

We previously identified a novel family of genes, BRINP1, 2, and 3, that are predominantly and widely expressed in both the central nervous system (CNS) and peripheral nervous system (PNS). In the present study, we analyzed the expression pattern of three BRINP genes during differentiation of mouse embryonic stem (ES) cell‐derived neural stem cells (NSCs) and their effects on the cell‐cycle regulation of NSCs. While there was no significant expression of any BRINP‐mRNA expressed in mouse ES cells, BRINP 1 and 2‐mRNAs was expressed at high levels in the ES cell‐derived neural stem cells. Upon differentiation into neuronal cells in the presence of retinoic acid and BDNF, all three types of BRINP‐mRNA were induced with a similar time course peaking at day three of treatment. Upon differentiation into astroglial cells in the presence of serum, BRINP1‐mRNA was slightly up‐regulated, while BRINP2‐ and BRINP3‐mRNAs were almost abolished in the astrocytes. While 69.2, 26.1, and 7.7% of cells in a population of NSCs in the exponentially growing phase were in the G1, S and G2 phases, respectively, over‐expression of any one of the three BRINP genes completely abolished cells in the G2 phase and significantly reduced the cells in S phase to 11.8–13.8%. Based on these results, the physiological roles of induced BRINP genes in the cell‐cycle suppression of terminally differentiated post‐mitotic neurons are discussed. © 2009 Wiley‐Liss, Inc.