胎鼠脊髓源性神经干细胞分离培养与鉴定

目的:研究胎鼠的脊髓源性神经干细胞的分离培养方法并观察其增殖和分化能力。方法:利用显微操作技术分离获得胎鼠脊髓组织、无血清培养技术和酶消化法结合机械法传代培养神经干细胞、免疫细胞化学方法鉴定神经干细胞和分化情况。结果:建立了胎鼠脊髓源性神经干细胞的分离、培养和鉴定的方法,观察到了脊髓源性神经干细胞具有较强的增殖能力,在添加有5ng／mlEGF和5ng／mlbFGF的无血清培养液中可贴壁分化为神经元、少突细胞和星形胶质细胞。结论:在体外培养条件下分离培养的胎鼠脊髓源性神经干细胞具有干细胞的特性即较强的增殖能力和多向分化潜能。     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

Regulation of neural stem cell by bone morphogenetic protein (BMP) signaling during brain development

Neurogenesis is the process in which neurons are generated from neural stem/progenitor cells (NSCs/NPCs). It involves the proliferation and neuronal fate specification/differentiation of NSCs, as well as migration, maturation and functional integration of the neuronal progeny into neuronal network. NSCs exhibit the two essential properties of stem cells: self-renewal and multipotency. Contrary to previous dogma that neurogenesis happens only during development, it is generally accepted now that neurogenesis can take place throughout life in mammalian brains. This raises a new therapeutic potential of applying stem cell therapy for stroke, neurodegenerative diseases and other diseases. However, the maintenance and differentiation of NSCs/NPCs are tightly controlled by the extremely intricate molecular networks. Uncovering the underlying mechanisms that drive the differentiation, migration and maturation of specific neuronal lineages for use in regenerative medicine is, therefore, crucial for the application of stem cell for clinical therapy as well as for providing insight into the mechanisms of human neurogenesis. Here, we focus on the role of bone morphogenetic protein (BMP) signaling in NSCs during mammalian brain development.     

Interleukin-15 regulates proliferation and self-renewal of adult neural stem cells

The impact of inflammation is crucial for the regulation of the biology of neural stem cells (NSCs). Interleukin-15 (IL-15) appears as a likely candidate for regulating neurogenesis, based on its well-known mitogenic properties. We show here that NSCs of the subventricular zone (SVZ) express IL-15, which regulates NSC proliferation, as evidenced by the study of IL-15-/- mice and the effects of acute IL-15 administration, coupled to 5-bromo-2'-deoxyuridine/5-ethynyl-2'-deoxyuridine dual-pulse labeling. Moreover, IL-15 regulates NSC differentiation, its deficiency leading to an impaired generation of neuroblasts in the SVZ-rostral migratory stream axis, recoverable through the action of exogenous IL-15. IL-15 expressed in cultured NSCs is linked to self-renewal, proliferation, and differentiation. IL-15-/- NSCs presented deficient proliferation and self-renewal, as evidenced in proliferation and colony-forming assays and the analysis of cell cycle-regulatory proteins. Moreover, IL-15-deficient NSCs were more prone to differentiate than wild-type NSCs, not affecting the cell population balance. Lack of IL-15 led to a defective activation of the JAK/STAT and ERK pathways, key for the regulation of proliferation and differentiation of NSCs. The results show that IL-15 is a key regulator of neurogenesis in the adult and is essential to understanding diseases with an inflammatory component.     

Maintenance and Neuronal Cell Differentiation of Neural Stem Cells C17.2 Correlated to Medium Availability Sets Design Criteria in Microfluidic Systems

Background

Neural stem cells (NSCs) play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes.

Methodology/Principal Findings

A series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (MF, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement) successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. MF smaller than 8.3×10⁴ µm³/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When MF was equal or greater than 8.3×10⁴ µm³/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. MF had minimal relation with the average neurite length.

Significance

MFs can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also offers insight about consumption rate of serum molecules involved in maintaining the stemness of NSCs.     

Effects of Oxygen Concentration on the Proliferation and Differentiation of Mouse Neural Stem Cells In Vitro

Background and purpose Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. Methods NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. Results The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. Conclusions These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.     

Plant-derived human recombinant growth factors and serum albumin maintain stemness of human-induced pluripotent stem cells

Stem cells are an important therapeutic source for recovery and regeneration, as their ability of self-renewal and differentiation offers an unlimited supply of highly specialized cells for therapeutic transplantation. Growth factors and serum are essential for maintaining the characteristics of stem cells in culture and for inducing differentiation. Because growth factors are produced mainly in bacterial (Escherichia coli) or animal cells, the use of such growth factors raises safety concerns that need to be resolved for the commercialization of stem cell therapeutics. To overcome this problem, studies on proteins produced in plants have been conducted. Here, we describe the functions of plant-derived fibroblast growth factor 2 (FGF2) and human serum albumin in the maintenance and differentiation of human-induced pluripotent stem cells (hiPSCs). Plant-derived FGF2 and human epidermal growth factor EGF were able to differentiate hiPSCs into neural stem cells (NSCs). These NSCs could differentiate into neuronal and glial cells. Our results imply that culturing stem cells in animal-free culture medium, which is composed of plant-derived proteins, would facilitate stem cell application research, for example, for cell therapy, by reducing contamination risk.     

Inhibition of Mitochondrial Complex III Blocks Neuronal Differentiation and Maintains Embryonic Stem Cell Pluripotency

The mitochondrion is emerging as a key organelle in stem cell biology, acting as a regulator of stem cell pluripotency and differentiation. In this study we sought to understand the effect of mitochondrial complex III inhibition during neuronal differentiation of mouse embryonic stem cells. When exposed to antimycin A, a specific complex III inhibitor, embryonic stem cells failed to differentiate into dopaminergic neurons, maintaining high Oct4 levels even when subjected to a specific differentiation protocol. Mitochondrial inhibition affected distinct populations of cells present in culture, inducing cell loss in differentiated cells, but not inducing apoptosis in mouse embryonic stem cells. A reduction in overall proliferation rate was observed, corresponding to a slight arrest in S phase. Moreover, antimycin A treatment induced a consistent increase in HIF-1α protein levels. The present work demonstrates that mitochondrial metabolism is critical for neuronal differentiation and emphasizes that modulation of mitochondrial functions through pharmacological approaches can be useful in the context of controlling stem cell maintenance/differentiation.     

Muscarinic acetylcholine receptors involved in the regulation of neural stem cell proliferation and differentiation in vitro

Neural stem cells (NSCs) are currently considered powerful candidates for cell therapy in neurodegenerative disorders such as Parkinson's disease. However, it is not known when and how NSCs begin to differentiate functionally. Recent reports suggest that classical neurotransmitters such as acetylcholine (Ach) are involved in the proliferation and differentiation of neural progenitor cells, suggesting that neurotransmitters play an important regulatory role in development of the central nervous system (CNS). We have shown by calcium imaging and immunochemistry that proliferation and differentiation are enhanced by M2 muscarinic Ach receptors (mAchR) expressed on the NSC surface and on their neural progeny. Moreover, atropine, an mAchR antagonist, blocks the enhancement and inhibits the subsequent differentiation of NSCs. Further understanding of this neural-nutrition role of Ach might elucidate fetal brain development, the brain's response to injury, and learning and memory.     

Melatonin antagonizes interleukin‐18‐mediated inhibition on neural stem cell proliferation and differentiation

Neural stem cells (NSCs) are self‐renewing, pluripotent and undifferentiated cells which have the potential to differentiate into neurons, oligodendrocytes and astrocytes. NSC therapy for tissue regeneration, thus, gains popularity. However, the low survivals rate of the transplanted cell impedes its utilities. In this study, we tested whether melatonin, a potent antioxidant, could promote the NSC proliferation and neuronal differentiation, especially, in the presence of the pro‐inflammatory cytokine interleukin‐18 (IL‐18). Our results showed that melatonin per se indeed exhibited beneficial effects on NSCs and IL‐18 inhibited NSC proliferation, neurosphere formation and their differentiation into neurons. All inhibitory effects of IL‐18 on NSCs were significantly reduced by melatonin treatment. Moreover, melatonin application increased the production of both brain‐derived and glial cell‐derived neurotrophic factors (BDNF, GDNF) in IL‐18‐stimulated NSCs. It was observed that inhibition of BDNF or GDNF hindered the protective effects of melatonin on NSCs. A potentially protective mechanism of melatonin on the inhibition of NSC's differentiation caused IL‐18 may attribute to the up‐regulation of these two major neurotrophic factors, BNDF and GNDF. The findings indicate that melatonin may play an important role promoting the survival of NSCs in neuroinflammatory diseases.     

Kuwanon V Inhibits Proliferation,Promotes Cell Survival and Increases Neurogenesis of Neural Stem Cells

Neural stem cells (NSCs) have the ability to proliferate and differentiate into neurons and glia. Regulation of NSC fate by small molecules is important for the generation of a certain type of cell. The identification of small molecules that can induce new neurons from NSCs could facilitate regenerative medicine and drug development for neurodegenerative diseases. In this study, we screened natural compounds to identify molecules that are effective on NSC cell fate determination. We found that Kuwanon V (KWV), which was isolated from the mulberry tree (Morus bombycis) root, increased neurogenesis in rat NSCs. In addition, during NSC differentiation, KWV increased cell survival and inhibited cell proliferation as shown by 5-bromo-2-deoxyuridine pulse experiments, Ki67 immunostaining and neurosphere forming assays. Interestingly, KWV enhanced neuronal differentiation and decreased NSC proliferation even in the presence of mitogens such as epidermal growth factor and fibroblast growth factor 2. KWV treatment of NSCs reduced the phosphorylation of extracellular signal-regulated kinase 1/2, increased mRNA expression levels of the cyclin-dependent kinase inhibitor p21, down-regulated Notch/Hairy expression levels and up-regulated microRNA miR-9, miR-29a and miR-181a. Taken together, our data suggest that KWV modulates NSC fate to induce neurogenesis, and it may be considered as a new drug candidate that can regenerate or protect neurons in neurodegenerative diseases.     

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.     

Potential of embryonic and adult stem cells in vitro

Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissue-specific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.     

GDNF对神经干细胞增殖、分化的影响

神经干细胞(neural stem cells,NSCs)具有如下特点:(1)可以向神经组织分化或源自神经系统的一部分。(2)具备维持和更新的自主能力。(3)可通过细胞分裂增殖。以上特点决定了它的应用价值,被公认为治疗阿尔茨海默氏病,帕金森氏症,脊髓损伤,中风等神经退行性疾病的最佳方案。用干细胞治疗癌症,免疫相关性疾病,和其他疾病被认为是很有创新的新疗法,可能有一天会扩展到修复和补充大脑损伤。胶质细胞源性神经营养因子(glial Cell line一derived neurotrophic factor,GDNF)为TGF一β超家族的一员,具有很强神经保护作用,大量实验研究证实GDNF可促进帕金森病大鼠模型的中脑神经干细胞定向分化为多巴胺能神经元,同时大量实验发现其可促进神经干细胞增殖及分化,为神经干细胞的应用奠定了基础。     

Evaluation of the differentiation status of neural stem cells based on cell morphology and the expression of Notch and Sox2

Neural stem cells (NSCs) isolated from a variety of sources are being developed as cellular therapies aimed at treating neurodegenerative diseases. During NSC culture and expansion it is important the cells do not differentiate prematurely because this may have an unfavorable effect on product quality and yield. In our study, we evaluated the use of Notch and Sox2 as markers for undifferentiated human and mouse NSCs. The expression of Notch2 and Sox2 during extensive-passage, low-oxygen culture and differentiation conditions were analyzed to confirm that the presence of these signature proteins directly correlates with the ability of NSCs to form new neurospheres and differentiate into multiple cell types. Using expression of Notch1, Notch2 and Sox2 as a reference, we then used flow cytometry to identify a specific morphological profile for undifferentiated murine and human NSCs. Our studies show that Notch and Sox2 expression, along with flow cytometry analysis, can be used to monitor the differentiation status of NSCs grown in culture for use in cellular therapies.     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。     

Brg1 is required for murine neural stem cell maintenance and gliogenesis

Epigenetic alterations in cell-type-specific gene expression control the transition of neural stem cells (NSCs) from predominantly neurogenic to predominantly gliogenic phases of differentiation, but how this switch occurs is unclear. Here, we show that brahma-related gene 1 (Brg1), an ATP-dependent chromatin remodeling factor, is required for the repression of neuronal commitment and the maintenance of NSCs in a state that permits them to respond to gliogenic signals. Loss of Brg1 in NSCs in conditional brg1 mutant mice results in precocious neuronal differentiation, such that cells in the ventricular zone differentiate into post-mitotic neurons before the onset of gliogenesis. As a result, there is a dramatic failure of astrocyte and oligodendrocyte differentiation in these animals. The ablation of brg1 in gliogenic progenitors in vitro also prevents growth-factor-induced astrocyte differentiation. Furthermore, proteins implicated in the maintenance of stem cells, including Sox1, Pax6 and Musashi-1, are dramatically reduced in the ventricular zones of brg1 mutant mice. We conclude that Brg1 is required to repress neuronal differentiation in NSCs as a means of permitting glial cell differentiation in response to gliogenic signals, suggesting that Brg1 regulates the switch from neurogenesis to gliogenesis.