Mild Hypoxia Enhances Proliferation and Multipotency of Human Neural Stem Cells

Background

Neural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it''s estimated to be 2.5 to 3%.

Methodology/Principal Findings

We investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of β-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation.

Conclusions/Significance

These findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson''s disease, multiple sclerosis, and Alzheimer''s disease.     

Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.     

Tenuigenin Promotes Proliferation and Differentiation of Hippocampal Neural Stem Cells

The present study was to investigate the influence of tenuigenin, an active ingredient of Polygala tenuifolia Willd, on the proliferation and differentiation of hippocampal neural stem cells in vitro. Tenuigenin was added to a neurosphere culture and neurosphere growth was measured using MTT assay. The influence of tenuigenin on the proliferation of neural progenitors was examined by Clone forming assay and BrdU detection. In addition, the differentiation of neural stem cells was compared using immunocytochemistry for β III-tubulin and GFAP. The results showed that addition of tenuigenin to the neural stem cell medium increased the number of newly formed neurospheres. More neurons were also obtained when tenuigenin was added in the differentiation medium. These findings suggest that tenuigenin is involved in regulating the proliferation and differentiation of hippocampal neural stem cells. This result may be one of the underlying reasons for tenuigenin’s nootropic and anti-aging effects.     

乳鼠海马神经干细胞的体外分离、扩增和分化研究

探讨海马神经干细胞（neuralstemcells,NSCs）在体外分离扩增和诱导分化的可行性。无菌条件下分离新生（24h）SD大鼠海马神经干细胞,采用无血清培养和胎牛血清诱导分化。免疫荧光染色技术分别检测诱导前细胞巢蛋白（Nestin）的表达,以及分化细胞的神经元特异性烯醇化酶（neuron specific enolase,NSE）、胶质纤维酸性蛋白（glialfibrillaryacidicprotein,GFAP）的表达,以鉴定细胞类型。流式细胞仪检测神经干细胞分化前后增殖能力的变化。结果显示：从乳鼠海马分离培养的细胞生长状态良好,具有克隆增殖能力,并呈Nestin表达阳性,分化后可出现NSE及GFAP表达阳性的细胞。流式细胞仪检测显示：诱导前,细胞增殖活跃,S＋G2／M期细胞为（36．27±1．99）％,而分化各阶段（3,7,10d）S＋G2／M期细胞比例与诱导前（Ctrl）相比则明显下降（尸〈0．05）,分别为（26．39±1．10）％、（26．33±1．33）％和（24．54±1．12）％。这些结果表明乳鼠海马存在神经干细胞,并具有自我更新和多向分化的潜能,可用于基础和临床的相关研究。     

APP5肽模拟物P165对体外培养大鼠海马神经干细胞增殖和分化的影响

目的研究一种小分子多肽─APP5肽的模拟物P165对体外培养的大鼠胚胎海马神经干细胞（neuralstem cells,NSCs）增殖和分化的影响,以期能找到一种可代替神经营养因子的小分子物质,能够促进NSCs的增殖或分化,为将来的临床应用提供理论依据。方法（1）原代培养SD大鼠胚胎脑海马NSCs;（2）利用5-溴脱氧尿嘧啶核苷（BrdU）和神经元、星型胶质细胞、少突胶质细胞的特异性标记物微管相关蛋白2（MAP2）、胶质纤维酸性蛋白（GFAP）、2,3-环核苷酸-3磷酸二酯酶（CNPase）对培养的NSCs进行鉴定;（3）将培养的NSCs分为对照组、血清组、APP5肽反序列组和P165组,观察各组细胞形态的变化;（4）将培养的NSCs分为对照组、APP5肽反序列组和P165组,利用细胞计数,测定干细胞克隆形成率、干细胞克隆形成大小的方法分析P165对海马NSCs增殖的影响。结果（1）海马神经干细胞呈神经球聚集生长,BrdU染色阳性;加入血清后神经球周围有细胞呈放射状向四周生长,并带有突起。染色呈MAP2、GFAP或CNPase阳性;（2）海马NSCs加入P165及其反序列后细胞形态上与对照组相比没有明显改变;（3）与对照组相比,加P165后海马NSCs数量明显增加,克隆形成率和克隆形成的直径均有明显的增加,并有统计学差异。结论P165能够促进海马NSCs的增殖,但并不促进其分化。     

L-型钙通道对大鼠胚胎海马神经干细胞增殖和分化的调控

为鉴定大鼠胚胎海马神经干细胞(NSCs)是否表达功能性的L-型钙通道,L-型钙通道是否参与了对大鼠胚胎NSCs增殖和分化调控.分离孕15天Wistar大鼠胚胎海马组织,制成单细胞悬液,利用无血清培养技术,在添加bFGF、EGF、N-2和B27 supplement的DMEM/F12培养液中进行培养.采用细胞免疫荧光法对原代至第5代细胞进行鉴定,均有巢蛋白(nestin)的表达,第3代nestin阳性细胞比例达97%.把培养的细胞诱导分化5天后,这些细胞表现为神经元和星形胶质细胞的形态,且分别呈Ⅲ型β-微管蛋白(Tuj1)阳性和胶质纤维酸性蛋白(GFAP)阳性;细胞免疫印迹结果显示,NSCs表达L-型钙通道的Cav1.2α1C亚单位,而无Cav1.3α1D亚单位的表达;利用全细胞膜片钳技术在NSCs上记录到了L-型钙电流,证明了NSCs所表达的L-型钙通道具有功能.进一步对细胞进行药理学干预,发现L-型钙通道的激活不仅可以促进胚胎NSCs的增殖,而且使增殖的NSCs向神经元分化的比例显著增加.以上结果表明,Wistar大鼠胚胎海马NSCs表达功能性的L-型钙通道;L-型钙通道参与了胚胎NSCs增殖和分化的调控.     

Long-Term Potentiation Promotes Proliferation/Survival and Neuronal Differentiation of Neural Stem/Progenitor Cells

Neural stem cell (NSC) replacement therapy is considered a promising cell replacement therapy for various neurodegenerative diseases. However, the low rate of NSC survival and neurogenesis currently limits its clinical potential. Here, we examined if hippocampal long-term potentiation (LTP), one of the most well characterized forms of synaptic plasticity, promotes neurogenesis by facilitating proliferation/survival and neuronal differentiation of NSCs. We found that the induction of hippocampal LTP significantly facilitates proliferation/survival and neuronal differentiation of both endogenous neural progenitor cells (NPCs) and exogenously transplanted NSCs in the hippocampus in rats. These effects were eliminated by preventing LTP induction by pharmacological blockade of the N-methyl-D-aspartate glutamate receptor (NMDAR) via systemic application of the receptor antagonist, 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP). Moreover, using a NPC-neuron co-culture system, we were able to demonstrate that the LTP-promoted NPC neurogenesis is at least in part mediated by a LTP-increased neuronal release of brain-derived neurotrophic factor (BDNF) and its consequent activation of tropomysosin receptor kinase B (TrkB) receptors on NSCs. Our results indicate that LTP promotes the neurogenesis of both endogenous and exogenously transplanted NSCs in the brain. The study suggests that pre-conditioning of the host brain receiving area with a LTP-inducing deep brain stimulation protocol prior to NSC transplantation may increase the likelihood of success of using NSC transplantation as an effective cell therapy for various neurodegenerative diseases.     

AcSDKP Regulates Cell Proliferation through the PI3KCA/Akt Signaling Pathway

The natural tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) is generated from the N-terminus of thymosin-β4 through enzymatic cleavage by prolyl oligopeptidase (POP). AcSDKP regulation of proliferation of different cells is implicated in hematopoiesis and angiogenesis. This tetrapeptide present in almost all cells was recently detected at elevated concentrations in neoplastic diseases. However, previously reported in vitro and in vivo studies indicate that AcSDKP does not contribute to the pathogenesis of cancers. Here we show that exogenous AcSDKP exerts no effect on the proliferation of actively dividing malignant cells. Using S17092, a specific POP inhibitor (POPi), to suppress the biosynthesis of AcSDKP in U87-MG glioblastoma cells characterized by high intracellular levels of this peptide, we found that all tested doses of POPi resulted in an equally effective depletion of AcSDKP, which was not correlated with the dose-dependent decreases in the proliferation rate of treated cells. Interestingly, addition of exogenous AcSDKP markedly reversed the reduction in the proliferation of U87-MG cells treated with the highest dose of POPi, and this effect was associated with activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. However, extracellular-regulated protein kinase (ERK) activation was unaltered by S17092 and AcSDKP co-treatment. Knockdown of individual PI3K catalytic subunits revealed that p110α and p110β contributed differently to AcSDKP regulation of U87-MG cell proliferation. Disruption of p110α expression by small interfering RNA (siRNA) abrogated AcSDKP-stimulated Akt phosphorylation, whereas knockdown of p110β expression exhibited no such effect. Our findings indicate for the first time that the PI3KCA/Akt pathway mediates AcSDKP regulation of cell proliferation and suggest a role for this ubiquitous intracellular peptide in cell survival.     

Differential Centrifugation in Culture and Differentiation of Rat Neural Stem Cells

(1) The study of neural stem cells (NSC) has attracted much attention in recent years because of their therapeutic potential. However, the problem in culture and differentiation of NSC was how to obtain single cell suspension that preserves the function of NSC, and remove the debris caused by mechanical dissociation. In the present study, we try to find a simple and effective way to address the problem, i.e. differential centrifugation. (2) After a gentle mechanical dissociation using Pasteur pipette, the suspension was first centrifuged at 100 g for 5 min, and then recentrifuged at 400 g for 6 min. Finally, the two deposits were resuspended and seeded into culture flask respectively. The suspension from the second deposit was allowed for further culture and differentiation. Immunofluorescence technique was used to identify neural stem cell, neuron, astrocyte, and oligodendrocyte. (3) After the second differential centrifugation, single cell suspension was obtained with 2–3 cell clusters, and the cells not only grew to form neurospheres, but also differentiated into neurons, astrocytes, and oligodendrocytes. (4) Differential centrifugation is a simple and effective way to obtain single cell suspension, which will help make large-scale production of neurodifferentiated cells more effective.     

Melatonin Regulates the Viability and Differentiation of Rat Midbrain Neural Stem Cells

(1) Neurogenesis driven by neural stem cells (NSCs) is regulated by physiological and pathological factors. Melatonin (MT) has profound neurotrophic and neuroprotective effects. Hence, we studied the role of MT in regulating the viability and differentiation of NSCs derived from rat ventral midbrain. (2) NSCs were isolated from the rat ventral midbrain. The viability of NSCs was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium assay. The differentiation of NSCs was examined by analyzing the expression of the neural markers, MT receptors, brain derived neurotropic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) with semi-quantitative RT-PCR, immunofluorescence cytochemistry, and Western blot. (3) Our results showed that MT could promote the viability of NSCs. In addition, MT could significantly elevate the mRNA and protein levels of tyroxine hydroxylase (TH), a marker of dopaminergic neurons, and decrease the expression of the astrocytes maker glial fibrillary acidic protein (GFAP). MT also increased the production of BDNF and GDNF in the cultured NSCs. Meanwhile, we first found that two subtypes of MT receptors, MT1 and MT2, were expressed in the ventral midbrain NSCs. (4) These results demonstrated that MT could induce NSCs to differentiate into dopaminergic neurons and decrease astrocyte production. These findings also suggest that MT could offer a beneficial tool in guiding directional differentiation of NSCs.     

Angiopoietin-Like 4 Confers Resistance to Hypoxia/Serum Deprivation-Induced Apoptosis through PI3K/Akt and ERK1/2 Signaling Pathways in Mesenchymal Stem Cells

Angiopoietin-like 4 (ANGPTL4) is a potential anti-apoptotic agent for various cells. We examined the protective effect of ANGPTL4 on hypoxia/serum deprivation (SD)-induced apoptosis of MSCs, as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured in vitro. Apoptosis was induced by hypoxia/SD for up to 24 hr, and assessed by flow cytometry and TUNEL assay. Expression levels of Akt, ERK1/2, focal adhesion kinase (FAK), Src, Bcl-2, Bax, cytochrome C and cleaved caspase-3 were detected by Western blotting. Integrin β1 mRNA was detected by qRT-PCR. Mitochondrial membrane potential was assayed using a membrane-permeable dye. Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat ANGPTL4 in a concentration dependent manner. Moreover, ANGPTL4 decreased the hypoxia/SD-induced caspase-3 cleavage and the cytochrome C release, but increased the Bcl-2/Bax ratio and the mitochondrial membrane potential. Decreased expression of integrin β1, the ANGPTL4 receptor was observed during hypoxia/SD conditions, however, such decrease was reversed by ANGPTL4. In addition, ANGPTL4 induced integrin β1-associated FAK and Src phosphorylation, which was blocked by anti-integrin β1 antibody. ANGPTL4 also reversed the hypoxia/SD-induced decrease of Akt and ERK 1/2 phosphorylation, and the effect of ANGPTL4 was abolished by inhibitors of either integrins, ERK1/2, or phosphatidylinositol 3-kinase (PI3K). Blocking integrinβ1, Akt or ERK largely attenuated anti-apoptotic effect of ANGPTL4. ANGPTL4 protects MSCs from hypoxia/SD-induced apoptosis by interacting with integrins to stimulate FAK complex, leading to downstream ERK1/2 and PI3K/Akt signaling pathways and mimicking the pathway in which MSCs contact with the extracellular matrix.     

Notch信号通路对神经干细胞增殖分化的作用

神经干细胞作为一种具有自我更新能力和多向分化潜能的细胞,它的增殖和分化受到多种源于自身或外在、邻近或远程细胞信号通路的调控,各种细胞因子及胞间通讯在神经干细胞的增殖和分化中发挥着重要的作用。近年来的多种研究表明,Notch信号通路正是这样一种可以通过相邻细胞的配体与受体相互作用,从而传递信号,进一步发挥其生物学功能的重要信号通路。该通路参与了神经干细胞维持自我形态及向多种具有不同功能的神经细胞分化的过程．对于研究神经干细胞的增殖和分化具有巨大的意义。该文将就当前Notch信号通路对神经干细胞增殖分化影响的相关研究进行简要综述。     

Fragile X Mental Retardation Protein Regulates Proliferation and Differentiation of Adult Neural Stem/Progenitor Cells

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP is an RNA–binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs). We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3β. Dysregulation of GSK3β led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis.     

IGF-1 Reduces BACE-1 Expression in PC12 Cells via Activation of PI3-K/Akt and MAPK/ERK1/2 Signaling Pathways

Insulin-like growth factor 1 (IGF-1) stimulates α-secretase processing of amyloid precursor protein (APP) and decreases Aβ production. Little is known about the relationship between IGF-1 and β-site amyloid precursor protein cleaving enzyme 1 (BACE-1), the protease essential for the production of β-amyloid peptides (Aβ). Here, we investigated the effect of IGF-1 on BACE-1 in PC12 cells. Quantitative polymerase chain reaction analysis and western blot showed that treatment of cells with IGF-1 significantly decreased the levels of BACE-1 mRNA and protein. Furthermore, IGF-1 increased the phosphorylation of Akt and ERK1/2. The presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 and the mitogen-activated protein kinase kinases (MEK) inhibitor PD98059 blocked the effect of IGF-1 on BACE-1. Our data indicated that IGF-1-induced reduction of BACE-1 might involve the PI3-K/Akt and MAPK/ERK1/2 signaling pathways.