神经干细胞研究进展

神经干细胞研究是当今生命科学研究的热点之一。神经干细胞是神经系统发育过程中保留下来的具有自我更新和多分化潜能的原始细胞。随着对神经干细胞认识的不断深入,其临床应用前景与价值得到了越来越多研究者的肯定。从神经干细胞的生物学特征、来源、培养鉴定、分化及应用等几个方面对目前的研究做一概述。     

Proteomics of neural stem cells

The isolation of neural stem cells from fetal and adult mammalian CNS and the demonstration of functional neurogenesis in adult CNS have offered perspectives for treatment of many devastating hereditary and acquired neurological diseases. Due to this enormous potential, neural stem cells are a subject of extensive molecular profiling studies with a search for new markers and regulatory pathways governing their self-renewal as opposed to differentiation. Several in-depth proteomic studies have been conducted on primary or immortalized cultures of neural stem cells and neural progenitor cells, and yet more remains to be done. Additionally, neurons and glial cells have been obtained from embryonic stem cells and mesenchymal stem cells, and proteins associated with the differentiation process have been characterized to a certain degree with a view to further investigations. This review summarizes recent findings relevant to the proteomics of neural stem cells and discusses major proteins significantly regulated during neural stem cell differentiation with a view to their future use in cell-based regenerative and reparative therapy.     

The neural stem cell niche

The neural stem cell niche defines a zone in which stem cells are retained after embryonic development for the production of new cells of the nervous system. This continual supply of new neurons and glia then provides the postnatal and adult brain with an added capacity for cellular plasticity, albeit one that is restricted to a few specific zones within the brain. Critical to the maintenance of the stem cell niche are microenvironmental cues and cell-cell interactions that act to balance stem cell quiescence with proliferation and to direct neurogenesis versus gliogenesis lineage decisions. Ultimately, based on the location of the niche, stem cells of the adult brain support regeneration in the dentate gyrus of the hippocampus and the olfactory bulb through neuron replacement. Here, we provide a summary of the current understanding of the organization and control mechanisms of the neural stem cell niche.     

Counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm(2) quadrant (0.1 microl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in microl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.     

Passaging human neural stem cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology. This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research. The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003). We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days. Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).     

The culture of neural stem cells

A stem cell has three important features. Firstly, the ability of self‐renewal: making identical copies of itself. Secondly, multipotency, generating all the major cell lineages of the host tissue (in the case of embryonic stem cells—pluripotency). Thirdly, the ability to generate/regenerate tissues. Thus, the study of stem cells will help unravel the complexity of tissue development and organisation, and will also have important clinical applications. Neural stem cells (NSCs) are present during embryonic development and in certain regions of the adult central nervous system (CNS). Mobilizing adult NSCs to promote repair of injured or diseased CNS is a promising approach. Since NSCs may give rise to brain tumor, they represent in vitro models for anti‐cancer drug screening. To facilitate the use of NSCs in clinical scenarios, we need to explore the biology of these cells in greater details. One clear goal is to be able to definitively identify and purify NSCs. The neurosphere‐forming assay is robust and reflects the behavior of NSCs. Clonal analysis where single cells give rise to neurospheres need to be used to follow the self‐renewal and multipotency characteristics of NSCs. Neurosphere formation in combination with other markers of NSC behavior such as active Notch signaling represents the state of the art to follow these cells. Many issues connected with NSC biology need to be explored to provide a platform for clinical applications. Important future directions that are highlighted in this review are; identification of markers for NSCs, the use of NSCs in high‐throughput screens and the modelling of the central nervous development. There is no doubt that the study of NSCs is crucial if we are to tackle the diseases of the CNS such as Parkinson's and Alzheimer's. J. Cell. Biochem. 106: 1–6, 2009. © 2008 Wiley‐Liss, Inc.     

锂对造血干细胞和神经干细胞作用影响的研究进展

锂在现代精神病学中使用超过65年,其构成了双相情感障碍（BD）长期治疗的基础。锂的许多生物学特性已经被证实,包括抗病毒、血液系统和神经系统保护作用。本文系统综述了锂对造血干细胞（HSCs）、神经干细胞（NSCs）以及诱导多能干细胞（iPSCs）作用影响的研究进展及其目前已证实的分子机制。自20世纪70年代以来,锂对保持HSCs和生长因子高水平的作用已被报道。锂可以改善HSCs的归巢能力、形成菌落的能力和自我更新的能力。关于锂对神经发生影响的研究表明,锂可促进海马齿状回的干细胞增殖,并导致施旺氏细胞有丝分裂活性增强。锂被证实与神经保护和神经营养作用相关,具体作用反映在锂可改善突触的可塑性,促进细胞存活,抑制细胞凋亡等。在临床研究中发现,锂离子的治疗可增加大脑灰质的成分,尤其作用在额叶、海马和杏仁核等位置。锂对干细胞的作用涉及多条介质和信号通路,其中最重要的介质和信号通路被认为是糖原合成酶激酶-3（GSK-3）和Wnt/β-catenin通路,另外包括调节cAMP、蛋白激酶B、磷脂酰肌醇3-激酶（pi3k）和肌醇单磷酸酶（IMP）水平的信号通路等也与锂作用有紧密的联系。锂在现阶段被利用于治疗BD和降低痴呆症患病风险的临床实验中,并对神经退行性疾病发挥有益作用。除此之外,为了研究的发病机制和锂离子在其中的作用机制,从BD患者中获得的iPSCs也被广泛应用。     

Nutrient control of neural stem cells

The physiological status of an organism is able to influence stem cell behaviour to ensure that stem cells meet the needs of the organism during growth, and in response to injury and environmental changes. In particular, the brain is sensitive to metabolic fluctuations. Here we discuss how nutritional status is able to regulate systemic and local insulin/IGF signalling so as to control aspects of neural stem behaviour. Recent results have begun to reveal how systemic signals are relayed to neural stem cells through local interactions with a glial niche. Although much still remains to be discovered, emerging parallels between the regulation of Drosophila and mammalian stem cells suggest a conserved mechanism for how the brain responds to changes in nutritional state.     

Effects of aspirin and clopidogrel on neural stem cells

Cerebral infarction causes severe morbidity and mortality. Most patients with cerebral infarction should take antiplatelet drugs daily, so the effects of those drugs on the regeneration of the brain need to be investigated. Aspirin and clopidogrel are the most widely used antiplatelet drugs for the prevention of ischemic stroke. We investigated the effects of aspirin and clopidogrel on neural stem cells (NSCs). NSCs were dissociated from fetal rat cortex and cultured with basic fibroblast growth factor and N2 medium. To measure the effects of aspirin and clopidogrel on NSCs, NSCs were treated with several concentrations of aspirin, clopidogrel bisulfate, and clopidogrel resinate for 24 h. After the treatment, we measured cell viability by cell counting kit-8, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, flow cytometry, and lactate dehydrogenase assay. To evaluate their effects on NSC proliferation, we performed BrdU cell proliferation assay and colony-forming unit assay. We compared the intracellular protein level in the NSCs treated with aspirin and two types of clopidogrel, by proteomics analysis. Various viability tests showed that clopidogrel resinate and clopidogrel bisulfate did not affect the viability and proliferation of NSCs whereas aspirin decreased them even at low concentrations which are clinically relevant. Moreover, through the proteomics, it was confirmed that the toxicity of aspirin to NSCs might be associated with the alteration of several intracellular proteins. Taken together, these results suggest that clopidogrel resinate and clopidogrel bisulfate are safe but aspirin could be toxic to NSCs. Therefore, when these antiplatelet agents are prescribed over the long-term, the finding that aspirin could be toxic to NSCs should be considered.     

Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells

Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells–derived neural crest stem cells (iPSCs–NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs–NCSCs were examined respectively in this study. LIPUS at 500 mW cm^?2 enhanced the viability and proliferation of iPSCs–NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100β and GFAP in iPSCs–NCSCs whereas after 7 days expression of only NF-M, S100β and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs–NCSCs in vitro for peripheral nerve tissue engineering.     

神经干细胞移植的临床研究进展

神经系统损伤会导致脑内神经干细胞（neural stem cells,NSCs）的扩增以实现自我修复功能,而通过外源细胞移植的方式来加速这一进程,可能是一种更有效的治疗手段。当前,神经干细胞临床研究所面临的主要问题是如何评价细胞在移植后的行为和功能。该文综述了近几年使用神经干细胞移植治疗几种主要神经系统疾病的临床研究成果,并着重关注了干细胞移植后的示踪研究。     

A unified hypothesis on the lineage of neural stem cells

For many years, it was assumed that neurons and glia in the central nervous system were produced from two distinct precursor pools that diverged early during embryonic development. This theory was partially based on the idea that neurogenesis and gliogenesis occurred during different periods of development, and that neurogenesis ceased perinatally. However, there is now abundant evidence that neural stem cells persist in the adult brain and support ongoing neurogenesis in restricted regions of the central nervous system. Surprisingly, these stem cells have the characteristics of fully differentiated glia. Neuroepithelial stem cells in the embryonic neural tube do not show glial characteristics, raising questions about the putative lineage from embryonic to adult stem cells. In the developing brain, radial glia have long been known to produce cortical astrocytes, but recent data indicate that radial glia might also divide asymmetrically to produce cortical neurons. Here we review these new developments and propose that the stem cells in the central nervous system are contained within the neuroepithelial --> radial glia --> astrocyte lineage.     

Effects of sulforaphane on neural stem cell proliferation and differentiation

Sulforaphane (SFN) is a natural organosulfur compound with anti‐oxidant and anti‐inflammation properties. The objective of this study is to investigate the effect of SFN on the proliferation and differentiation of neural stem cells (NSC). NSCs were exposed to SFN at the concentrations ranging from 0.25 to 10 µM. Cell viability was evaluated with MTT assay and lactate dehydogenase (LDH) release assay. The proliferation of NSCs was evaluated with neurosphere formation assay and Ki‐67 staining. The level of Tuj‐1 was evaluated with immunostaining and Western blot to assess NSC neuronal differentiation. The expression of key proteins in the Wnt signaling pathway, including β‐catenin and cyclin D1, in response to SFN treatment or the Wnt inhibitor, DKK‐1, was determined by Western blotting. No significant cytotoxicity was seen for SFN on NSCs with SFN at concentrations of less than 10 µM. On the contrary, SFN of low concentrations stimulated cell proliferation and prominently increased neurosphere formation and NSC differentiation to neurons. SFN treatment upregulated Wnt signaling in the NSCs, whereas DKK‐1 attenuated the effects of SFN. SFN is a drug to promote NSC proliferation and neuronal differentiation when used at low concentrations. These protective effects are mediated by Wnt signaling pathway.     

神经干细胞研究介绍

神经干细胞研究是近年脑科学研究的热点，本文综述了神经干细胞的分离培养方法、脑内迁移路线、发育分化的影响因素以及可能的应用前景。     

Radial glia and neural stem cells

During the last decade, the role of radial glia has been radically revisited. Rather than being considered a mere structural component serving to guide newborn neurons towards their final destinations, radial glia is now known to be the main source of neurons in several regions of the central nervous system, notably in the cerebral cortex. Radial glial cells differentiate from neuroepithelial progenitors at the beginning of neurogenesis and share with their ancestors the bipolar shape and the expression of some molecular markers. Radial glia, however, can be distinguished from neuroepithelial progenitors by the expression of astroglial markers. Clonal analyses showed that radial glia is a heterogeneous population, comprising both pluripotent and different lineage-restricted neural progenitors. At late-embryonic and postnatal stages, radial glial cells give rise to the neural stem cells responsible for adult neurogenesis. Embryonic pluripotent radial glia and adult neural stem cells may be clonally linked, thus representing a lineage displaying stem cell features in both the developing and mature central nervous system. This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) NUSUG grant (In vivo screening for genes implicated in glioma formation and development of new animal models of glial tumors) and by Fondazione CARIGE grant (Basi molecolari e cellulari dei gliomi: individuazione di marcatori diagnostici e di nuovi bersagli terapeutici).     

神经营养因子与神经干细胞

生长因子在神经干细胞的增殖,分化和存活过程中有重要作用。神经营养因子是其中的一类,它包括神经生长因子（NGF）家族,胶质源性神经营养因子（GDNF）家族和其它神经营养因子。NGF家族包括NGF,BDNF,NT－3,NT－4／5和NT－6。这一家族可促进epidermic growth facter(EGF)反应 海马及前脑室管膜下区神经干细胞的存活和分化。GDNF家族包括GDNF,NTN,PSP和ART。GDNF家族促神经发育的作用主要在外周,它促进肠神经嵴前体细胞的存活和增殖,且对外周感觉神经的发育至关重要。其它生长因子如bFGF和EGF,它们能促进神经干细胞增殖和存活;CNTF和LIF等在神经干细胞的分化中也有重要作用。