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发表于 2009-3-5 00:02 |只看该作者 |倒序浏览 |打印

作者：Peter E. Halla,b, Justin D. Lathiaa,b,c, Nigel G. A. Millera, Maeve A. Caldwellb, Charles Ffrench-Constanta,b

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      【摘要】
   The identification of markers for the isolation of human neural stem cells (hNSCs) is essential for studies of their biology and therapeutic applications. This study investigated expression of the integrin receptor family by hNSCs as potential markers. Selection of 6hi or &szlig;1hi cells by fluorescence-activated cell sorting led to an enrichment of human neural precursors, as shown by both neurosphere forming assays and increased expression of prominin-1, sox2, sox3, nestin, bmi1, and musashi1 in the &szlig;1hi population. Cells expressing high levels of &szlig;1 integrin also expressed prominin-1 (CD133), a marker previously used to isolate hNSCs, and selection using integrin &szlig;1hi cells or prominin-1hi cells was found to be equally effective at enriching for hNSCs from neurospheres. Therefore, integrin subunits 6 and &szlig;1 are highly expressed by human neural precursors and represent convenient markers for their prospective isolation. , D; S8 Z; A7 \0 ^/ z0 @# M5 c
      【关键词】 Fluorescence-activated cell sorting Neurosphere Prominin Laminin Extracellular matrix
   INTRODUCTION# r% l! N( d5 x

Human neural stem cells (hNSCs) have the ability to form all the major cell types of the central nervous system, making them candidates for cell-based therapies in neurodegenerative disorders, such as Parkinson¡¯s disease or multiple sclerosis. However, two factors have hampered the development of clinical applications, such as cell transplants. First, the very limited availability of fetal tissue containing large numbers of hNSCs. Second, the inability to purify these cells from more differentiated types. The first can be overcome by the expansion of primary cells, as human neural stem cells can be maintained in vitro within free-floating aggregates, termed neurospheres. The second factor, however, remains a key problem, especially as neurospheres represent a complex mixture of neural stem cells (NSCs) and more differentiated cell types. Cell markers appropriate for selection strategies are therefore required. The intracellular location of factors enriched in NSCs, including Musashi1, Nestin, and Sox1, has limited their usefulness as prospective markers , few markers have been developed for hNSCs.

One method used to identify potential markers is to examine those cell surface receptors likely to be required for stem cell maintenance in vivo. The NSCs are localized to the ventricular and subventricular zone of the developing brain, where specialized areas called niches are responsible for maintaining the multipotency and self-renewal capacity of NSCs . In this study, we therefore asked whether integrins are highly expressed by hNSCs and provide a marker for their isolation.. s$ \# z" @1 q3 G4 }
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MATERIALS AND METHODS" q  X( y5 i" K
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Cell Culture1 }$ j! a2 d4 q2 `+ f6 t
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Human fetal tissue (8¨C10 weeks postconception) was collected in accordance with the arrangements for informed consent recommended by the Polkinghorne Committee . In brief, fresh tissue was initially dissociated in trypsin and seeded at a density of 200,000 cells per milliliter in a T75 flask containing Dulbecco¡¯s modified Eagle¡¯s medium/Ham¡¯s F-12 medium (3:1; Gibco, Paisley, U.K., http://www.invitrogen.com), supplemented with N2 (1:100; Gibco), epidermal growth factor (EGF), fibroblast growth factor (FGF)-2 (both at 20 ng/ml; R&D Systems Inc., Abingdon, U.K., http://www.rndsystems.com), and heparin (5 µg/ml; Sigma-Aldrich, Poole, U.K., http://www.sigmaaldrich.com). Spheres had grown to a radius of >0.35 mm after 14 days of expansion, and passaging of cells was routinely undertaken at this time point by sectioning spheres into 200-µm sections using a McIlwain tissue chopper, after which they were reseeded into fresh growth medium. Cultures were fed every 4¨C5 days by replacing half the medium. To induce differentiation, growth factors were withdrawn, and neurospheres were plated on poly(D-lysine) and laminin-1 (10 µg/ml; Sigma-Aldrich)-coated eight-well chamber slides (Nunc, Roskilde, Denmark, http://www.nuncbrand.com) for 7 days prior to immunostaining.$ g) L: o. e& b) U8 F% j" i
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Semiquantitative Reverse Transcription-Polymerase Chain Reaction
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Total RNA was extracted from neurospheres using RNeasy minikit (Qiagen, Crawley, U.K., http://www1.qiagen.com) before being treated with RNase-free DNase I (Roche Diagnostics, Lewes, U.K., http://www.roche-applied-science.com) to remove genomic DNA contamination. cDNA was generated from 1 µg of total RNA using a First Strand cDNA Synthesis kit (GE Healthcare Life Sciences, Little Chalfont, U.K., http://www.amersham.com). Each polymerase chain reaction (PCR) consisted of 1/30th reverse transcription reaction, 1x PCR buffer (Qiagen), 0.25 mM each dNTP (GE Healthcare Life Sciences), 2 µM each primer, and 1.25 units of Taq polymerase (Qiagen). PCR mixtures then were denatured at 95¡ãC for 2 minutes before being cycled at 94¡ãC for 1 minute, the annealing temperature for 1 minute, and 72¡ãC for 1 minute. A final extension of 72¡ãC for 10 minutes occurred after cycling. Details of the primer sequences, annealing temperatures, and cycle numbers are provided in supplementary online Table 1. The PCR products were separated by electrophoresis on 2.5% agarose gels.
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Immunohistochemistry
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Neurospheres were fixed in 4% paraformaldehyde for 1 hour at room temperature before being washed with phosphate-buffered saline (PBS) and placed in 25% sucrose overnight at 4¡ãC. Spheres were then cut into 14-µm sections using a Leica CM3000 cryostat (Leica, Milton Keynes, U.K., http://www.leica.co.uk). Sections were treated with 10% normal goat serum (DakoCytomation, Ely, U.K., http://www.dakocytomation.co.uk) and 0.2% Triton X-100 in PBS for 1 hour at room temperature, before being incubated with anti-nestin (1:500; a kind gift from Dr. H. Okano, Tokyo, Japan) or anti-ß1 integrin (1:200; clone P5D2; Chemicon, Chandlers Ford, U.K., http://www.chemicon.com) overnight at 4¡ãC. Subsequently, the sections were incubated with the appropriate Alexa Fluor-conjugated secondary antibody (Molecular Probes Inc., Leiden, The Netherlands, http://www.probes.invitrogen.com), diluted 1:200 in Hoechst 33258, for 2 hours at room temperature. Sections were viewed under a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Welwyn Garden City, U.K., http://www.zeiss.co.uk), and images were captured using Openlab software (Improvision, Coventry, U.K., http://www.improvision.co.uk). Differentiated neurospheres were fixed as described above and then labeled with anti-ßIII tubulin (1:500; Tuj1; Sigma-Aldrich) and anti-glial fibrillary acidic protein (1:500; DakoCytomation) followed by appropriate secondary antibodies as described above.) R5 u5 O4 T" u+ T  w2 B4 A

Flow Cytometry and Neurosphere Formation Assay' y& t/ e- M+ H- l
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Neurospheres were dissociated with Accutase (Chemicon) before being resuspended in PBS and stained with antibodies against ß1 integrin (clone TDM29-fluorescein isothiocyanate; Chemicon) or prominin-1 (clone 293C3-phycoerythrin; Miltenyi Biotec, Bisley, U.K., http://www.miltenyibiotec.com) at 4¡ãC for 1 hour. For 6 integrin staining, cells were incubated with the primary antibody (clone NKI-GoH3; Chemicon) for 1 hour at 4¡ãC before addition of the R-phycoerythrin-conjugated secondary antibody (1:300; Molecular Probes) for 45 minutes at 4¡ãC. All primary antibodies were used at 2 µg/106 cells. Dead cells were excluded by propidium iodide staining (2.5 µg/ml; Sigma-Aldrich), and doublets were identified by the pulse-width parameter. The specificity of each antibody was confirmed by the lack of significant labeling if the fluorochrome-conjugated anti-integrin or anti-CD133 antibody was omitted, as shown in Figures 2E and 4A. The cells were sorted using a MoFlo flow cytometer (DakoCytomation) into 96-well plates (Nunc) at a density of either 250 or 500 live cells per well (750 or 1,500 cells per cm2) or, in the experiments using a limiting dilution assay to determine the frequency of neurosphere-forming cells, progressively decreasing numbers of cells per well. Each well contained 200 µl of neurosphere medium, supplemented with 2% B27 (Gibco) in place of the N2. Cells were fed every 4¨C5 days by replacing 50% of the medium, and the number of neurospheres formed was counted after 21 days in culture.
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Statistical Analysis

For the analysis of expression (PCR or FACS), three fetal brain samples were analyzed, and a representative experiment is shown. For the cell sorting studies, eight experimental replicates were performed for each of at least two fetal brain samples tissues, and data are presented as mean ¡À SE. All statistics were calculated using Student¡¯s t test, with p

RESULTS AND DISCUSSION
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Human neural stem cells, like rodent NSCs, can be maintained in vitro as free-floating aggregates, termed neurospheres. These spheres grow from single cells following dissociation at each passage in culture, and when withdrawn from mitogenic growth factors, each sphere will generate neurons, astrocytes, and oligodendrocytes. As such, these single cells have stem cell properties, and currently, the identification of NSCs is predominantly based on assays defining NSCs as neurosphere-initiating cells . To investigate a role of integrins as markers for hNSCs, it was first necessary to define which subunits are expressed. Using a reverse transcription-polymerase chain reaction (RT-PCR)-based approach, Figure 1 shows the integrin subunits found to be consistently expressed by human neurospheres, regardless of the growth factors used (EGF, FGF-2, or a combination) or the length of time in culture. The mRNA for  subunits 1¨C3, 5¨C10, and V were expressed in all samples (n = 3), along with ß subunits 1, 5, and 8, whereas integrins ß4 and ß6 were never seen.* H2 f9 y% C: ?( j5 _
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Figure 1. Expression of integrin subunits by human neurospheres. Reverse transcription-polymerase chain reaction (PCR) analysis of mRNA prepared from human neurospheres. The numbers over each lane indicate the specific  and ß subunits expressed (i.e., 1 is 1, 2 is 2, etc.). Note that 6, V, ß1, ß5, and ß8 were detected after only 30 PCR cycles, whereas other subunits required 40 cycles. Also, note that splice variants of 3 and 6 are expressed. Size markers are shown in base pairs on the left.7 \! {* ^4 e5 y5 s% N, s2 I

In light of the number of integrin subunits that associate with ß1 expressed in human neurospheres by RT-PCR, we next asked whether the ß1 integrin subunit was highly expressed on hNSCs. Three sets of experiments were conducted to examine this. Nestin is an intermediate filament protein that is highly expressed by neural stem cells . In agreement, here we found that ß1 integrin and nestin are more highly expressed at the edge of human neurospheres, showing that human neurospheres also have a distinct three-dimensional architecture and demonstrating that hNSCs do express high levels of ß1 integrin (Fig. 2A).

Figure 2. Integrin ß1 is highly expressed by human neural stem cells. (A): Cryostat section of a human neurosphere stained for Hoechst 33258 (to label nuclei), ß1 integrin, and nestin. A merged image is shown in the lower right panel. Expression of both proteins is colocalized at the edge of the neurosphere. Scale bar = 10 µm. (B): ß1(hi), 6(hi), and 6(hi) ß1(hi) were sorted at 250 live cells into a 96-well plate, and the number of neurospheres forming was quantified 21 days later. "Unsorted" refers to sorting live cells with no gating on ß1 or 6 expression. Neurosphere formation was significantly increased relative to the unsorted population for all conditions, although no significant additive effect of combining selection for ß1(hi) and 6(hi) was found. *, p

Second, we asked whether cells expressing high levels of ß1 integrin showed the property of NSCs of being able to generate neurospheres. We used the neurosphere-forming assay to examine an integrin ß1hi population (the top 10% of cells as determined by ß1 integrin expression levels) compared with the total population. Labeled cells dissociated from neurospheres grown from human fetal tissue and passaged by chopping as described under Materials and Methods (primary neurospheres) were sorted by FACS into 96-well plates at a very low density of 1.25 cells per microliter (Fig. 2B) . Twenty-one days later, the number of secondary neurospheres was counted. The results showed that selecting for the integrin ß1hi cells within the cells dissociated from the primary neurospheres led to a 2.1-fold increase in the number of secondary neurospheres compared with the number formed when starting with an unselected population of primary neurosphere cells (p   a, O1 ?% f+ A( o

Third, ß1hi and ß1lo cells were evaluated for their expression of a panel of NSC markers (Fig. 2D). The PCR screen verified that ß1hi cells were enriched for well-established markers of rodent NSCs, with the mRNA for prominin-1, nestin, sox2, sox3, musashi1, and bmi1 being upregulated compared with the ß1lo cells. No change was seen with sox1 and musashi2, consistent with their expression in both precursors and differentiated neuronal subtypes . Furthermore, the neuronal marker, ßIII tubulin, was more highly expressed in the ß1lo population (Fig. 2D). Together, these findings indicate that hNSCs express high levels of ß1 integrin, whereas differentiated human neural cell types downregulate their expression of ß1 integrin." c' `4 a. w; k" w5 R7 ]
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Next, we asked which ß1 heterodimer was highly expressed on hNSCs. An attractive candidate is 6ß1, a receptor for laminins expressed in the basal lamina found in many stem cell niches .

An important technical issue when sorting for cells expressing high levels of markers such as integrins is that the flow cytometer measures total fluorescence. This means, as shown in supplementary online Figure 2, that large cells are more likely to appear in the markerhi category, as they will have an increased total fluorescence compared with a small cell for an equal marker density on the cell surface. Therefore, it is possible that selecting for ß1hi cells merely chooses large cells, and as increased cell size has been shown to select for rodent NSCs , the enrichment for hNSCs we observe may simply reflect this selection for size. To exclude this possibility, cells of one particular size were sorted for ß1hi and ß1lo (Fig. 3). Again, selection of ß1hi cells led to a significant enrichment in the neurosphere-forming cell (p
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Figure 3. Elevated expression of ß1 integrin in human neural stem cells is independent of cell size. To demonstrate that the enrichment seen with ß1hi cells was not simply a consequence of selecting for large cells, cells of medium (corresponding to the median 10%) or large (top 10%) size were selected and further sorted for high or low ß1 expression. Note that increased neurosphere formation was observed when selecting for integrin ß1hi cells, regardless of the cell size. *, p
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Currently, the most effective marker for hNSCs is prominin-1, a pentatransmembrane domain protein of unknown function (CD133) . The efficiency of sorting for hNSCs using integrin ß1 was compared with the use of prominin-1 by sorting cells into 10% bins based on expression levels of either marker and performing a neurosphere-forming assay (Fig. 4B). The results indicate that ß1 integrin is as effective as prominin-1 for purifying hNSCs, leading to 2.8- and 2.4-fold increases in neurosphere numbers, respectively (p = .49; n = 3).) O2 J2 k- u' G. [) f1 R( X. L: C

Figure 4. Integrin ß1 is co-expressed with prominin-1 (CD133). (A): Flow cytometry analysis showing control, unstained cells (left) or the co-expression of prominin-1 and integrin ß1 (right). (B): Cells were sorted according to their expression levels of either integrin ß1 or prominin-1 and analyzed in a neurosphere forming assay. Bin 91¨C100 represents the cells expressing the highest levels of a marker; 1¨C10 represents the lowest. A population of live cells without selection for integrin or prominin-1 expression is also presented for comparison. Cells expressing the highest levels of ß1 integrin or prominin-1 were found to form the most neurospheres. Note that integrin ß1 is as effective as prominin-1 at enriching for hNSCs. All points, except for bins 51¨C60 and 61¨C70, were significantly different from the whole population for both markers (p
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From the findings reported here, we conclude that integrin 6ß1 is highly expressed by human neural stem cells and can be used as a marker in FACS to enrich for them. These are notable findings for two reasons. First, the extent of this enrichment from neurospheres is equal to that achieved by selecting for prominin-1, currently the most effective marker for selecting hNSCs. Second, they add to the evidence that integrins play an important role in stem cell biology. Previous reports have identified 6 and ß1 integrin subunits as being highly expressed by rodent stem cells in testes, liver, skin, and brain , these findings indicate that integrins are likely to have a fundamental role in the signaling pathways regulating stemness in human and rodent stem cells.
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DISCLOSURES

C.F.C. has consulted for Geron within the last 2 years.7 o2 B8 c- {6 J+ A* z

ACKNOWLEDGMENTS

This study was supported by the Multiple Sclerosis Society (P.E.H.), the NIH-Cambridge Graduate Partnership Program (J.D.L.), the Royal Society (M.A.C.), and the Wellcome Trust (N.G.A.M. and C. ff.-C.).1 i) G9 k0 I1 Z7 _6 g; U
      【参考文献】0 k0 r* C0 k, u, ]
: D6 [# }. h. p' x8 J8 p" }
* w- e* Y* D8 p% w2 y- M
Sakakibara S, Imai T, Hamaguchi K et al. Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev Biol 1996;176:230¨C242.  U3 L: {; z5 b! X

Kaneko Y, Sakakibara S, Imai T et al. Musashi1: An evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev Neurosci 2000;22:139¨C153.
' R  s- _; N( m% @( O) t+ V/ t% ]3 |
Hockfield S, McKay RD. Identification of major cell classes in the developing mammalian nervous system. J Neurosci 1985;5:3310¨C3328.
! b+ C  z' s) P2 n
Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990;60:585¨C595.
8 K; Y0 N, H0 L# Y* t% b; n4 h, R
Pevny LH, Sockanathan S, Placzek M et al. A role for SOX1 in neural determination. Development 1998;125:1967¨C1978.0 @5 L' U) O) q2 F) G, G

Keyoung HM, Roy NS, Benraiss A et al. High-yield selection and extraction of two promoter-defined phenotypes of neural stem cells from the fetal human brain. Nat Biotechnol 2001;19:843¨C850., U7 {+ E0 K& @

Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241:58¨C62.2 {5 f5 T% x+ F. ?
' p2 M8 Y. X. P1 C4 j
Baum CM, Weissman IL, Tsukamoto AS et al. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A 1992;89:2804¨C2808.3 E# q' h  D, m( J
2 q0 ]" B1 N; N! W* C
Murayama A, Matsuzaki Y, Kawaguchi A et al. Flow cytometric analysis of neural stem cells in the developing and adult mouse brain. J Neurosci Res 2002;69:837¨C847.4 }0 F& A" H( B

Rietze RL, Valcanis H, Brooker GF et al. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 2001;412:736¨C739.
! N0 j* {# f0 B
Capela A, Temple S. LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal. Neuron 2002;35:865¨C875.

Temple S. The development of neural stem cells. Nature 2001;414:112¨C117.0 X4 J$ B$ C4 G9 h! j

Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 2001;414:98¨C104.

Hynes R. Integrins: Bidirectional, allosteric signaling machines. Cell 2002;110:673¨C687.
# ]8 V+ e* A& V, n% t) Y5 J' s; z' g
Jones PH, Watt FM. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 1993;73:713¨C724." |$ T# [% v  C1 ?

Campos LS, Leone DP, Relvas JB et al. Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance. Development 2004;131:3433¨C3444., |/ w/ C7 H8 G5 U6 k

Nagato M, Heike T, Kato T et al. Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers. J Neurosci Res 2005;80:456¨C466.: s  F/ `  L# Y6 E# @  q

Ivanova NB, Dimos JT, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601¨C604.
7 n" |9 i9 t/ U: i
Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": Transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597¨C600.

Fujikawa T, Hirose T, Fujii H et al. Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting. J Hepatol 2003;39:162¨C170.9 Z; J0 r# C4 q# k7 y
' C* O4 c7 T" l" x
Suzuki A, Zheng Y, Kondo R et al. Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology 2000;32:1230¨C1239., \' K1 r% ~* ~% }$ f. W
& o( ]  q1 [) z" C
Kubota H, Avarbock MR, Brinster RL. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc Natl Acad Sci U S A 2003;100:6487¨C6492.

Shinohara T, Avarbock MR, Brinster RL. Beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999;96:5504¨C5509.
, ~5 ]! w" ?2 h1 A9 w
Polkinghorne J. Review of the guidance on the research use of fetuses and fetal material. London, U.K.: Her Majesty¡¯s Stationary Office, 1989;.
# k/ ~% i* K& `1 ^( H, x$ L$ \
Department of Health. Guidance on the supply of fetal tissue for research, diagnosis and therapy. London, U.K.: Her Majesty¡¯s Stationary Office, 1995;.6 H+ g/ d5 R" H  C5 `! J

Svendsen CN, ter Borg MG, Armstrong RJ et al. A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods 1998;85:141¨C152.: r) t& H5 e  t% V( G3 B1 X# J

Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255:1707¨C1710.

Tropepe V, Sibilia M, Ciruna BG et al. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 1999;208:166¨C188.

Ekonomou A, Kazanis I, Malas S et al. Neuronal migration and ventral subtype identity in the telencephalon depend on SOX1. PLOS Biol 2005;3:e186.

Sakakibara S, Nakamura Y, Satoh H et al. RNA-binding protein Musashi2: Developmentally regulated expression in neural precursor cells and subpopulations of neurons in mammalian CNS. J Neurosci 2001;21:8091¨C8107.9 r& p5 s; X( I" h
; c4 r9 w' ~  H8 q1 u5 \3 r5 r. q
Yurchenco PD, O¡¯Rear JJ. Basal lamina assembly. Curr Opin Cell Biol 1994;6:674¨C681.

Jacques TS, Relvas JB, Nishimura S et al. Neural precursor cell chain migration and division are regulated through different beta1 integrins. Development 1998;125:3167¨C3177.8 j5 I# F) |0 p+ M; T) `; A* Y

Leone DP, Relvas JB, Campos LS et al. Regulation of neural progenitor proliferation and survival by beta1 integrins. J Cell Sci 2005;118:2589¨C2599.

Uchida N, Buck DW, He D et al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 2000;97:14720¨C14725.
9 k+ [; N' Y( i$ {7 i5 [& j) n
Corbeil D, Roper K, Fargeas CA et al. Prominin: A story of cholesterol, plasma membrane protrusions and human pathology. Traffic 2001;2:82¨C91.6 o/ U( {  t: s3 [% A( m, d
! x4 s6 D2 V4 H2 o; u
Richardson GD, Robson CN, Lang SH et al. CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci 2004;117:3539¨C3545.
/ q: g  P7 E7 F
Yu Y, Flint A, Dvorin EL et al. AC133¨C2, a novel isoform of human AC133 stem cell antigen. J Biol Chem 2002;277:20711¨C20716./ V) W: H# i* b* {) S+ e* l4 i
/ ]4 E* V, B; L6 x! p# F0 p
Blanpain C, Lowry WE, Geoghegan A et al. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 2004;118:635¨C648.
9 p5 `) [% p5 ^$ v/ s
Wagers AJ, Weissman IL. Differential expression of alpha2 integrin separates long-term and short-term reconstituting Lin-/loThy1.1(lo)c-kit  Sca-1  hematopoietic stem cells. STEM CELLS 2006;24:1087¨C1094.2 k- x, p, Y- V

Corbel C, Vaigot P, Salaun J. (alpha)IIb Integrin, a novel marker for hemopoietic progenitor cells. Int J Dev Biol 2005;49:279¨C284.
7 C4 {8 J: a( A, @3 d! [7 }
Pajoohesh-Ganji A, Pal-Ghosh S, Simmens SJ et al. Integrins in slow-cycling corneal epithelial cells at the limbus in the mouse. STEM CELLS 2006;24:1075¨C1086.6 x* B9 l9 x; @2 i
! o" c% [9 O" }6 F7 A
Fujimoto K, Beauchamp RD, Whitehead RH. Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression. Gastroenterology 2002;123:1941¨C1948.

Li A, Simmons PJ, Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A 1998;95:3902¨C3907.

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