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Neurosphere Assays: Growth Factors and Hormone Differences in Tumor and Nontumor

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发表于 2009-3-4 23:57 |显示全部帖子
作者:Kaisorn Chaichana, Grettel Zamora-Berridi, Joaquin Camara-Quintana, Alfredo Quiones-Hinojosa作者单位:Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 0 y5 W& o+ N$ u" ?
                  
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          【摘要】3 m0 [, l/ h1 ]( v. J. Y
      The "no new neuron" dogma that the brain is quiescent throughout adult life has been challenged by the discovery of cells with stem cell-like qualities of self-renewal and multipotency in the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus in adults. This self-renewing capacity also makes these neural stem cells a possible source of brain tumors, which was supported by the discovery of self-sustaining brain tumor stem-like cells in cancers such as glioblastoma multiforme. Neurosphere assays are the standard for studying these stem-like cells in both normal and cancer tissues. Despite the importance of these assays, there is no standardized protocol to allow for a comparison of results because several studies use different growth factors and hormones at different concentrations. The primary objective of this study is to review the literature for both nontumor and tumor studies to assess their respective neurosphere assay components. We found significant variation in assay components, namely hormones and growth factors, as well as their respective concentrations. This illustrates the need for a standardized protocol to allow proper comparison among studies and a better assessment of the effects of different factors. ! C, G) Z: T7 @* b& S
          【关键词】 Neurosphere assay Neural stem cells Brain tumor stem cells
( w$ T* q4 r: u% f$ X                  INTRODUCTION
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The dogmatic view of the brain as immutable and incapable of neurogenesis was first challenged by Altman in the 1960s, .( ]& Y/ [2 G/ j7 M
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This has led to the discovery of a cell population within brain tumors that displays many key features of NSCs, . Therefore, BTSCs may represent a new target for cancer therapy.
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Advances in stem cell research have created a need for novel experimental techniques to study these cells. Currently, neurosphere assays remain the standard for identifying stem cell populations, allowing for isolation and characterization of NSCs (Fig. 1) and, most recently, BTSCs . The components of these culture systems, however, are not uniform and vary significantly between studies. This review provides a comprehensive summary of the different neurosphere culture media that have been used to study NSCs and BTSCs, and describes their various components.; W+ H7 |# t5 L
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Figure 1. Neurospheres derived from human subventricular zone.(A): Low-magnification image from a light microscope demonstrating neurospheres created from differentiated human subventricular zone astrocytes.(B): Higher magnification image of some of the neurospheres in(A). The neurosphere assay used to create these neurospheres, consisted of Dulbecco's modified Eagle's medium/F-12 with fibroblast growth factor (20 ng/ml) and epidermal growth factor (10 ng/ml), as well as insulin, progesterone, transferrin, selenium, putrescine, and glutamine.
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1 H5 R" Y1 S* j) R/ hMATERIALS AND METHODS
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- N( P# X/ \8 b6 V/ f$ q* LThe PubMed database current to May 2006 was filtered using the search terms "neurosphere," "assay," "neural stem cells," or "brain tumor stem cells," and limited to English-language papers. Eleven non-brain tumor and nine brain tumor neurosphere assay studies were selected for inclusion into the review for comparison. Studies were excluded if they cited another study's protocol or did not clearly state the components of their neurosphere media. The culture media and cell types were obtained from the methods section of each paper and tabulated (Tables 1 and 2 for comparison.
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4 v6 ]6 |* X  S0 i; j  KTable 1. Studies with non-brain tumor neurosphere assays
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: k6 `2 s! B) `; eTable 2. Studies with tumor stem cells and their respective culture media
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) ]5 }& ?  {2 W1 jRESULTS
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; a7 s1 X9 ], j0 P3 N$ ?/ dNontumor Neurosphere Studies& V% x% |( m5 D& |, w
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Eleven studies were obtained with non-brain tumor neurosphere assays from 1992 to 2006 (Table 1) .& C/ h3 L& z- p- p

- d0 ~, q5 d2 n" ?The components that were added to the media varied from study to study. The use of growth factors was not uniform. Five of the studies used both epidermal growth factor (EGF) and fibroblast growth factor (FGF) at varying concentrations between 10 and 20 ng/ml ).
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Tumor Neurosphere Studies1 O. G* J" q( F$ T8 v. p! C9 e0 t

% }# A+ y: x9 {" ?7 k9 a8 L- KNine studies were obtained with brain tumor neurosphere assays from 1999 to 2006 (Table 2). .
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1 {  e! j8 f, E" q# M; {/ g" B; vAs with nontumor neurosphere assays, the components added to brain tumor neurosphere media were not uniform across the studies. The studies used different types and concentrations of growth factors. Seven studies used both EGF and FGF at concentrations between 10 and 50 ng/ml ).
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DISCUSSION
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6 }7 i* ~0 a# sThe no new neuron doctrine that the brain is quiescent throughout adult life and devoid of neural stem cells has dominated neuroscience thinking for centuries. This began to change with Altman's discovery of new neurons in rats in 1966, . This has led to an era in neuroscience research in which neural stem cells are being studied actively for both their regenerative capacity and their devastating neoplastic potential.  ^( s1 K: X' A8 S1 j. ^9 L4 J% P
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Neurosphere assays were initially used by Reynolds and Weiss in 1992 to isolate neurospheres from the mouse striatum . In contrast, non-stem cells lack these features of self-renewal and multipotency and are theoretically eliminated with serial passages.
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Neurosphere assays, despite their utility, also have some limitations. First, neurosphere assays are an in vitro phenomenon and do not occur in vivo .
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Despite the importance of neurosphere assays, there is no standardized protocol to allow for a comparison of results. Reynolds' and Weiss' original studies used a serum-free medium containing 20 ng/ml of EGF .
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+ `2 w. I: B9 e2 W( wEach of these components may have different and significant effects on the creation of neurospheres and, most importantly, on the interpretation of data. NSCs and BTSCs are usually grown on serum-free medium because serum irreversibly differentiates stem cells . Therefore, the use of mitogens and/or hormones can have adverse effects on experimental results.: Z& k0 @1 W. y- D; U9 v; L4 O1 R- R

1 \. l" L4 w* W4 P0 C! y2 y8 [A number of micronutrients are also included in some neurosphere assays. Selenium, or sodium selenite, has anticancer properties of an unknown mechanism .& _6 _) |  G- ^- S' A, o

0 ^1 L; m) U. W( y+ m( bCONCLUSION
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The discovery of these NSCs and BTSCs opens the door to potential treatments for degenerative diseases such as Parkinson and devastating cancers such as GBM, which has a mean lifespan of 14.6 months after diagnosis . The best means of assessing these types of stem cells is through neurosphere assays. To effectively perform these studies and compare results, however, there must be a standardized protocol and/or at least a discussion related to this issue when authors compare their data to other studies to allow proper comparison among studies and potentially a better assessment of the effects of different factors. The wide variation in media components used by each group confounds data interpretation and limits effective sharing of information in the research community. Progress in this area will help us move forward in our study of novel therapies for neurological diseases.
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DISCLOSURES6 r9 ^) G0 w6 ?6 q; q( x
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The authors indicate no potential conflicts of interest.
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来几句吧  

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中国干细胞行业门户第一站
楼上的稍等啦  
佰通生物

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干细胞之家微信公众号
脂肪干细胞

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拿把椅子看表演

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我回不回呢 考虑再三 还是不回了吧 ^_^  

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发表于 2015-7-1 07:43 |显示全部帖子
小生对楼主之仰慕如滔滔江水连绵不绝,海枯石烂,天崩地裂,永不变心.  

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发表于 2015-8-16 15:54 |显示全部帖子
嘿嘿......哈哈......呵呵.....哟~呼  

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72 
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72  
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发表于 2015-9-2 08:52 |显示全部帖子
楼主福如东海,万寿无疆!  

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发表于 2015-9-11 11:10 |显示全部帖子
不错啊! 一个字牛啊!  
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