Optimized Flow Cytometric Analysis of Central Nervous System Tissue Reveals Nove - 《stemcells》干细胞学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 27 下一页

返回列表

查看: 469664\|回复: 260	go Optimized Flow Cytometric Analysis of Central Nervous System Tissue Reveals Nove [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 00:53 |只看该作者 |倒序浏览 |打印

作者：David M. Panchisiona, Hui-Ling Chena, Francesca Pistollatoa, Daniela Papinib, Hsiao-Tzu Nic, Teresa S. Hawleyd作者单位：aCenter for Neuroscience Research, Children ! \6 p% t6 ~- H( u6 Q
   8 @7 H0 d- g- [" N' r





         - m5 v% n' k& k9 R+ q
         , ~$ Y+ {( Y$ i( M6 ~3 }
                  & z- x- ?, [' M5 L
      ; g6 r3 p( {$ j0 ]2 j; z
      , K' C! ?5 x* I% E, [3 s' b
        J/ z0 j' t" y8 ^# r
      【摘要】3 f* E( I( V( @
   Although flow cytometry is useful for studying neural lineage relationships, the method of dissociation can potentially bias cell analysis. We compared dissociation methods on viability and antigen recognition of mouse central nervous system (CNS) tissue and human CNS tumor tissue. Although nonenzymatic dissociation yielded poor viability, papain, purified trypsin replacement (TrypLE), and two purified collagenase/neutral protease cocktails (Liberase-1 or Accutase) each efficiently dissociated fetal tissue and postnatal tissue. Mouse cells dissociated with Liberase-1 were titrated with antibodies identifying distinct CNS precursor subtypes, including CD133, CD15, CD24, A2B5, and PSA-NCAM. Of the enzymes tested, papain most aggressively reduced antigenicity for mouse and human CD24. On human CNS tumor cells, CD133 expression remained highest after Liberase-1 and was lowest after papain or Accutase treatment; Liberase-1 digestion allowed magnetic sorting for CD133 without the need for an antigen re-expression recovery period. We conclude that Liberase-1 and TrypLE provide the best balance of dissociation efficiency, viability, and antigen retention. One implication of this comparison was confirmed by dissociating E13.5 mouse cortical cells and performing prospective isolation and clonal analysis on the basis of CD133/CD24 or CD15/CD24 expression. Highest fetal expression of CD133 or CD15 occurred in a CD24hi population that was enriched in neuronal progenitors. Multipotent cells expressed CD133 and CD15 at lower levels than did these neuronal progenitors. We conclude that CD133 and CD15 can be used similarly as selectable markers, but CD24 coexpression helps to distinguish fetal mouse multipotent stem cells from neuronal progenitors and postmitotic neurons. This particular discrimination is not possible after papain treatment.
, `# g: U* Z. u. @* w4 W0 ]8 n
Disclosure of potential conflicts of interest is found at the end of this article.   h- Z2 [1 ?" Y5 q# l2 \7 a
      【关键词】 Brain Brain tumors Flow cytometry Cell viability Stem cells Progenitor cells
   INTRODUCTION
* `7 q2 X$ V9 {; S& i. R- s3 T
The analysis of neuroepithelial precursor cells is indispensable for studying the mechanisms controlling proliferation, mitotic arrest, and lineage commitment in the nervous system .! D  K; @4 [/ p

Cell-surface antigen-based selection has allowed prospective isolation of subclasses of CNS and neural crest cells ; both cell types appear to be limited in self-renewal potential.# j2 c5 T; Z' b, ~4 J; C

A critical step in flow cytometric analysis of the nervous system is the dissociation of tissue into single cells ) solutions have been introduced to eliminate undefined factors and reduce lot-to-lot variability. However, the comparative effect of these different dissociation methods on the analysis of important selectable neural antigens is poorly understood.
/ u; y7 x# B+ h* N) F
Here, we compared a number of these dissociation methods on neural tissue for cell viability, dissociation efficiency, and retention of CD133, CD15, and CD24 antigenicity. We found that papain, Liberase-1, TrypLE, and Accutase efficiently dissociate mouse CNS tissue and human CNS tumors into single cells. Liberase-1 and TrypLE provide the best balance of dissociation efficiency, viability, and antigen retention. Using Liberase-1 dissociation, we characterize novel patterns of selectable marker coexpression in mouse fetal cortex; lateral, medial, and caudal ganglionic eminence; and postnatal subventricular zone. We also show that CD133 and CD15 expression closely parallel each other in mouse brain cells and can be used similarly for prospective isolation. However, the highest mean levels of CD133 and CD15 are expressed in neuronal progenitors rather than multipotent stem cells of the fetal mouse forebrain. Selection using CD24 coexpression enhances the discrimination of these populations, but this type of selection is limited by the manner of dissociation.6 e# O# w( ]6 L3 O3 o/ U# I

MATERIALS AND METHODS
4 V* ~+ S* A8 K; m. M; X
Tissue Dissociation* |, n# [8 F9 R  k2 Q

Animal tissue was acquired using an approved protocol in accordance with Institutional Animal Care and Use Committee guidelines. Human pediatric tumor tissue was acquired using an approved protocol in accordance with institutional review board guidelines. Tissues included embryonic day 10.5 (E10.5) mouse cervical spinal cord; 13.5 mouse lateral, medial, and caudal ganglionic eminence (LGE, MGE, and CGE, respectively) and cortex; postnatal day 2 (P2) lateral ventricle wall; and human CNS tumor tissue acquired directly from surgery. Human tumor tissue was gently minced with a scalpel, whereas mouse tissue was left undisturbed before digestion. Initial comparisons were performed with E13.5 mouse LGE, where each LGE pair was resuspended in 1 ml of 1x Hanks' buffered saline solution (HBSS; Ca2 /Mg2 -free, plus Hepes and 1.55 g/l glucose, without bicarbonate, pH 7.2; Invitrogen) containing 200 units/ml DNase I (Roche) and 1 mM MgCl2; the exception was one LGE pair that was resuspended in growth medium (described later herein), one that was resuspended in purified collagenase/neutral protease cocktail in phosphate-buffered saline (PBS)/EDTA (Accutase; Innovative Cell Technologies; used neat, one lot tested) and another LGE pair that was resuspended in a purified trypsin-like replacement in Dulbecco's PBS/EDTA (TrypLE Select; Invitrogen; used neat, two lots tested) along with DNase/MgCl2. To the remaining tubes, we added different concentrations of an high-performance liquid chromatography-purified collagenase/dispase cocktail (Liberase Blendzyme1; Roche; four lots tested, seven vials total) or papain (12 units/ml; Worthington, Lakewood, NJ, http://www.worthington-biochem.com; two lots tested) that was preactivated as directed in 1.1 mM EDTA, 0.067 mM mercaptoethanol, and 5.5 mM cysteine-HCl for 30 minutes before addition. The absence of bicarbonate in the HBSS allowed dissections and incubations to be performed in a room atmosphere.9 C: [. D$ B% z. |
% B$ f" A3 m* q5 ^! S6 S; s
Samples were placed in a 37¡ãC rotator oven during digestion. In some experiments, incubation times were varied for each enzyme preparation (highest concentration only for Liberase-1). Samples were then spun at 200g for 5 minutes, resuspended in fresh HBSS/DNase/MgCl2 without enzyme, and triturated with three rounds of eight passes through an ART 1000E pipette tip (Molecular Bioproducts, Inc., San Diego, http://www.mbpinc.com); a fourth round of trituration was performed with a flamed Pasteur pipette. After each round of trituration, the tissue was allowed to settle for 5 minutes, and the top 800 µl of suspended cells were transferred into a new tube to avoid further disturbance. The combined cell suspensions for each group were spun down again and resuspended in medium appropriate for further use. On the basis of initial comparisons, the standard protocol for tissue digestion for multiple labeling and sorting became 30 minutes (fetal), 60 minutes (neonatal), or 90 minutes (human tumor) incubation with 200 µg/ml Liberase-1, corresponding to 0.62 W¨¹nsch unit (WU)/ml collagenase and 66.7 units/ml dispase. The final step of trituration with a flamed Pasteur pipette was eliminated because it introduced variability in the procedure.

In some instances, human tumor cells were dissociated after acquisition and grafted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice as previously described , using an approved animal protocol. After 4¨C12 weeks to allow tumor growth, tumor tissue was dissected and enzymatically dissociated before further analysis.

Flow Cytometry  ?, }* ~6 c9 i/ m
2 Q; z4 z$ x3 [4 y  B
For flow cytometry, cells were resuspended in flow cytometry buffer, consisting of 1x HBSS, pH 7.2, containing 1.55 g/l glucose and 0.1% fraction V of bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Cells were counted and diluted to a density of 106 cells per milliliter of buffer; all mouse analysis was performed with 100-µl aliquots containing 1 x 105 cells. For viability analysis, we added 300 µl of 1.33x Annexin buffer (diluted from 10x stock) followed by 2 µl of Annexin-V-APC (BD Biosciences, San Diego, http://www.bdbiosciences.com) and incubated at room temperature for 15 minutes; for the final 5 minutes of this incubation, we also added 7-amino-actinomycin-D (7-AAD, Invitrogen) to a final concentration of 50 ng/ml before analysis. Higher concentrations of either dye substantially shifted the fluorescence of the entire population and interfered with detection of other fluorochromes.& D: U; Q8 {+ V( J# d# u% u

For mouse surface marker analysis, we used antibodies against CD24 (phycoerythrin -conjugated mouse IgM; BD Biosciences), CD133 (biotinylated-rat-IgG2b; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), PSA-NCAM (mouse IgM; Chemicon, Temecula, CA, http://www.chemicon.com), A2B5 (mouse IgM; Chemicon), BMPRIA (goat IgG; R&D Systems), and BMPRIB (mouse IgG2a; R&D Systems). Intracellular analysis of BMP activation was performed with a Phospho-Smad1/5/8 antibody (rabbit IgG; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com). For human tumor analysis, we used antibodies against human CD133 (clone AC141-PE, as directed; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) and human CD24 (FITC-conjugated mouse IgG2a; 1:25 dilution; BD Biosciences). Antibodies were titrated over a semi-log scale to determine appropriate dilution and incubated on ice for 30 minutes. Cells were washed in buffer, and then secondary fluorescent-conjugated antibody (if needed) was added at the appropriate dilution and incubated on ice for 15 minutes. Cells were washed once and resuspended in buffer for viability dye staining and analysis.7 H+ V3 d1 L, ?/ D! o
9 t0 [$ \1 X( n: A5 o, l5 }+ J9 z
Cells were analyzed on an FACSCalibur flow cytometer (BD Biosciences). Background fluorescence was measured using unlabeled cells and cells labeled with isotype control or secondary antibody alone; these set gating parameters between positive and negative cell populations. Cell aggregates and small debris were excluded from analysis or isolation on the basis of side scatter (measuring cell granularity) and forward scatter (measuring cell size); dead cells were excluded from analysis on the basis of viability dye fluorescence. Fluorescent intensities for cells in the population were point-plotted on two-axis graphs or histogram using CellQuest software (BD Biosciences).

Cell Sorting
9 ]* _3 ~! M+ h
Cells were filtered through a 70-µm nylon mesh before final centrifugation, although filtration was not required after fetal tissue dissociation with Liberase-1, then resuspended in flow cytometry buffer. All sorts were performed on cells dissociated with 200 µg/ml Liberase-1 (0.62 WU/ml collagenase and 66.7 units/ml dispase). Mouse cells were sorted on fluorescence-activated cell sorter (FACS), either a FACSAria (BD Biosciences) or an Influx (Cytopeia, Seattle, WA, http://www.cytopeia.com) sorter. Single viable cells were gated on the basis of Annexin-V exclusion and pulse width, and then physically sorted into collection tubes for limiting dilution plating. Postsort purity analysis was performed on aliquots from each sort group. FACSDiva (BD Biosciences) or FlowJo (http://www.flowjo.com) software was used for analysis. Human tumor cells were sorted by magnetic cell separation (MACS; Miltenyi Biotec) using both selection and detection antibodies against human CD133 (clone AC133/1-microbead and clone AC141-PE, as directed; Miltenyi Biotec).3 n; @" P; Y' p* _9 L

Cell Culture and Clonal Analysis, V% ?2 k/ e7 E; s, M, ~- g, k* X
' y0 c' Q/ L- K% z# k! k3 m1 T
Dissociated mouse cells were assayed for viability by trypan blue exclusion, plated onto fibronectin-coated tissue culture dishes, and cultured in 5% O2, 5% CO2 in the presence of 20 ng/ml basic fibroblast growth factor (bFGF; R&D Systems) either acutely or added daily as previously described . Immunofluorescence was performed using primary antibodies against MAP2a b (mouse, 1:500, Sigma-Aldrich), glial fibrillary acidic protein (GFAP; rabbit, 1:800; Dako, Fort Collins, CO, http://www.dako.com) and O4 (mouse, 1:100, Sigma-Aldrich), followed by fluorescent secondary antibodies (Alexa dyes; Invitrogen). Cells were counterstained with 4'-6-diamidino-2-phenylindole (DAPI) to measure total cell number. Staining was visualized by epifluorescence (BX60 upright microscope; Olympus, Tokyo, http://www.olympus-global.com), and images were compiled for figures using Photoshop 7.0 (Adobe, San Jose, CA, http://www.adobe.com).9 c8 Z6 a% m; l# x3 [8 z$ Z5 o

RESULTS

Comparison of Mouse CNS Tissue Dissociation Methods/ l& _6 b; A  e
+ X* C6 i# r* _1 g7 G$ i" M  D) q
We compared several methods of dissociation using nonenzymatic buffers or increasing incubation times of four enzyme preparations: papain, purified trypsin (TrypLE), and two purified collagenase/neutral protease cocktails (Liberase-1 or Accutase). Initial comparisons were done with E13.5 forebrain tissue and extended to E10.5 mouse cervical spinal cord, which contains substantial numbers of proliferative precursors . To assess viability, we incubated the dissociated cells with Annexin-V to identify early apoptotic cells and 7-AAD to identify late apoptotic and necrotic cells (Fig. 1; Table 1). By flow cytometric analysis, we found that the poorest results came from dissociating the cells with growth medium or HBSS alone. In contrast, each of the four enzyme preparations greatly improved dissociation and viability, both in terms of percentage of the main population (intact cells) and the total population (all detected events, including debris). Cell aggregates, as measured by high forward scatter, were common in nonenzyme dissociated cells but minimal in the enzyme-treated preparations. Pulse-width analysis of Liberase-1-treated tissue also verified a high efficiency of single-cell dissociation (supplemental online Fig. 1A, 1B).2 D( I/ t2 h6 i0 \2 H2 ~/ j1 A3 o# q/ X2 n
4 q: Y+ [2 J2 V' H, s  ~
Figure 1. Flow cytometric analysis of mouse fetal central nervous system cell viability. E13.5 mouse lateral ganglionic eminence was dissociated with HBSS alone (A), 400 µg/ml Liberase-1 in HBSS (B), 12 units/ml preactivated papain in HBSS (C), TrypLE purified trypsin replacement (D), or Accutase (E). The top panel shows physical characteristics of entire unfiltered population measured by object size (forward scatter) and granularity (side scatter). A polygon delimits the main population of intact cells, whereas events outside of main population consist of pyknotic cells and cellular debris; number indicates main population as a percentage of total events. The bottom panel shows viability of cells within the main population; high Annexin-V staining indicates early apoptotic cells, whereas 7-AAD staining indicates late apoptotic and necrotic cells. Numbers indicate percentages of main population events within each quadrant for this experiment only; see Table 1 and supplemental online Table 1 for complete results. Abbreviations: 7-AAD, 7-amino-actinomycin-D; HBSS, Hanks' buffered saline solution.
" c. A; P! L  ~
Table 1. Viability assays after dissociation
! n4 |+ m& ]1 j" f2 F7 d1 K' R
Although papain and TrypLE were usually highly effective in dissociating tissue, they gave more variable results. On two occasions with papain and three occasions with TrypLE, we saw free-DNA aggregation even in the presence of DNase I, indicating substantial cell lysis. We could not definitively determine the cause of this variability. Two additional papain and one TrypLE sample could not be properly assayed by hemocytometer because of DNA aggregation and so were discarded. Accutase and Liberase-1 never generated free DNA aggregates. Flow cytometric viability agreed with trypan blue exclusion counts shortly after dissociation (Table 1). Viability of cells isolated with Liberase-1 stayed consistent after sequential primary and secondary antibody labeling, washing and up to 8 hours on ice postdissociation, which are typical durations when cell sorting (supplemental online Table 1).

Because CNS tissue dissociation is typically followed by in vitro culturing, we plated cells in monolayer culture to determine how plating efficiency was affected by dissociation method (supplemental online Fig. 1C¨C1F). Nearly all cells adhered within 1 hour of plating, although papain and TrypLE-treated cells remained phase bright longer, suggesting weaker adhesion. Cells were counted 16 hours after plating to determine viability (supplemental online Fig. 1E) and the number of clusters of three or more cells (unlikely to result from cell division; supplemental online Fig. 1F). Treatment for 30 minutes with Liberase-1 or 30¨C60 minutes with papain yielded the highest cell viability after plating. Although the majority (64%) of surviving HBSS-dissociated cells remained in clusters, all four enzyme treatments strongly reduced the number of clusters. Of these, Accutase left the most cell aggregates after the digestion and trituration process, even after 60 minutes of treatment. Although both Liberase-1 and Accutase are cocktails of collagenase and neutral protease, it is possible that differences in protease type or proprietary formulation could account for the different dissociation efficiencies. On the basis of these results, we conclude that all four enzymatic preparations yield efficient single-cell dissociation and viability compared with HBSS alone, with a rank order of papain > Liberase-1 > TrypLE > Accutase >>> HBSS.) L; D' R) p! A# o( @
1 H& E5 E& F" W# N' z4 Q
Titration of Antibodies to Mouse CNS Cell Surface Antigens  d. C& o4 s! I2 M
2 x. ^5 d/ u& I. Z
For assaying surface marker expression, we first used 30 minutes of Liberase-1 treatment to acutely isolate cells from E13.5 cortex and postnatal day 2 (P2) mouse lateral ventricle wall, which is enriched in multipotent stem cells and neuronal progenitors . For each marker, we titrated the concentration of the antibody to optimally distinguish positive from negative cell populations. We identified distinct populations of mouse cells on the basis of the expression of CD133, CD15, CD24, A2B5, and PSA-NCAM (Fig. 2).% p5 l/ z0 }  x) m! _/ L8 [
2 b" t: U: l: z' C& M- y# e4 E6 N9 W
Figure 2. Titration of antibodies for CD133, CD15, CD24, A2B5, and PSA-NCAM against mouse central nervous system cells. E13.5 mouse cortex (A¨CC) or postnatal day 2 mouse lateral ventricle tissue (D, E) was dissociated with 200 µg/ml Liberase-1 in Hanks' buffered saline solution. Cells numbering 1 x 105 in 100 µl of flow cytometry buffer were incubated with serial dilutions of antibodies against CD24 (A), CD15 (B), CD133 (C), PSA-NCAM (D), or A2B5 (E), followed by secondary antibodies where appropriate, then analyzed by flow cytometry. Each column shows isotype control or secondary-only antibody (nonspecific staining) at top followed by increasing concentrations of each antibody. Exact antibody concentration is listed when known; fold dilution is listed for PSA-NCAM and A2B5. Frequency histogram for each antibody concentration is shown by black filled distribution; nonspecific staining is overlaid in a gray line for comparison. Concentrations used for subsequent experiments (per 100 µl of suspension): 0.01 µg of CD24; 0.6 µg of CD15; 1.0 µg of CD133; 1/300,000 PSA-NCAM; 1/30,000 A2B5. Abbreviation: K, x1,000.8 }0 S  e( t2 q, g
& I) o1 ~. S) @/ ~
The importance of high viability to proper titration was underscored by the observation that dead cells preferentially bound anti-CD133, thereby saturating out the specific binding capacity of the antibody (Fig. 3A). Tissue dissociation with Liberase-1 solved this problem by increasing cell viability (Fig. 3B). Although all four enzymes preserved equivalent antibody recognition of CD133 and CD15 (not shown), papain completely eliminated staining for CD24, whereas Liberase-1, TrypLE, and Accutase preserved similar CD24 staining (Fig. 3C¨C3F). Additionally, staining for BMP receptors IA and IB was lost after Liberase-1 or papain digestion (Fig. 3G¨C3J); staining was not tested after TrypLE or Accutase treatment. Loss of BMP receptors was verified by the reduction of SMAD activation (measured by phospo-Smad1, -5, and -8 recognition) in cultured precursor cells after treatment with BMP2, which is known to efficiently induce neural crest-like fates in these cells .5 x1 d: Z5 P  |+ a7 Y+ T
) [: d$ i1 S2 a5 _& {2 @( M" e/ U) ?
Figure 3. Effect of dissociation method on antigen recognition. (A, B): Nonenzymatic dissociation interferes with discrimination of CD133  cells. Mouse fetal cortex was dissociated with HBSS alone (A) or with 200 µg/ml Liberase-1 (B), followed by labeling with anti-CD133. Viability analysis with 7-AAD showed that CD133 antibody preferentially bound dead (7-AAD ) cells, thus limiting availability for binding live cells. Increased cell viability after Liberase-1 dissociation of tissue allowed a distinct CD133  population to be identified. Numbers indicate percentage of CD133-stained cells that are living and dead. (C¨CF): CD24 antigenicity is lost after papain treatment but preserved equally after Liberase-1, TrypLE, or Accutase treatment. CD133 and CD15 antigenicity is preserved equally after all four methods (not shown). Numbers show percentage of CD24hi cells. (G¨CJ): Liberase-1 and papain treatment reduce or eliminate BMP responsiveness of central nervous system precursors. Expanded cultures from E13.5 cortex were passaged with HBSS and then incubated for 15 minutes in suspension with HBSS alone (G¨CJ), 200 µg/ml Liberase-1 (G¨CI), or 12 units/ml papain (J). Cells were then treated with 10 ng/ml BMP2 for 15 minutes before labeling with an antibody against phospho-Smad-1/5/8. Cells passaged with HBSS alone (gray line distribution) show a shifted peak and shoulder indicating BMPR-IA and BMPR-IB staining and a separate peak indicating SMAD phosphorylation, whereas cells treated with Liberase-1 or papain (dark filled distribution) show lower BMP receptor immunoreactivity and/or BMP responsiveness as measured by reduced SMAD phosphorylation. Abbreviations: 7-AAD, 7-amino-actinomycin-D; HBSS, Hanks' buffered saline solution.

Comparison of Human CNS Tumor Dissociation Methods; v8 l0 d- U( g1 ~4 q. b  E* E

We also tested the ability of these enzymes to dissociate human neural tissue. A solid high-grade glioma was divided with a scalpel and dissociated with Liberase-1, papain, TrypLE, or Accutase (Fig. 4A¨C4O). Viability was high for all four treatment groups (Table 1). Analysis of CD24 expression (Fig. 4F¨C4J) showed that TrypLE and Accutase yielded the highest number of CD24  cells (approximately 90%), whereas Liberase-1 yielded less (44%) and papain yielded greatly reduced CD24  numbers (14%). All four methods yielded CD133  cells (Fig. 4K¨C4O), although the highest percentage came after digestion with Liberase-1 (62%) and the lowest came from papain (37%) and Accutase (35%). Liberase-1 effectively dissociated a wide variety of resected tumors (supplemental online Table 2) with minimal undissociated tissue, except when tumors contained necrotic tissue. In these cases, the necrotic portions failed to digest and thus did not affect the viability counts. Additionally, we were able to more effectively passage cultured tumor cells with Liberase-1 compared with Cell Dissociation Buffer, an HBSS-based buffer containing a calcium chelator (Table 1).1 I; y! J& {* Z4 t6 S2 j/ n8 V0 U
/ S* }! ~6 C2 o; J
Figure 4. Dissociation, CD24 and CD133 labeling, analysis, and sorting of human tumor cells. (A¨CO): Solid high-grade glioma recovered from nonobese diabetic/severe combined immunodeficiency mouse graft was divided four ways and dissociated with different enzymes before antibody labeling. (A): Light scatter profile after Liberase-1 digestion shows that whole cells (main) makes up about 60% of total events. Background fluorescence (B) and viability staining of main population cells with Annexin-V and 7-AAD (C) after Liberase-1; see Table 1 for other enzymes. (D¨CO): Profile of viable cells based on main, Annexin-V¨C gating. (D, E): Background fluorescence in detection channels for CD24 and CD133; same cells with different overlaid gates. (F¨CJ): CD24 antigenicity was retained at highest levels after TrypLE or Accutase digestion, and was reduced by half after Liberase-1 digestion and nearly eliminated after papain digestion; (F) shows isotype control. (K¨CO): CD133 antigenicity was retained with all four dissociation methods but was highest after Liberase-1 digestion; (K) shows isotype control. (P¨CT): Surgically resected pediatric atypical teratoid/rhabdoid tumor was dissociated with 200 µg/ml Liberase-1 for 60 minutes and immediately incubated with anti-CD133 (AC133/1-microbeads plus AC133/2-PE) for magnetic selection. Light scatter profile (P) shows that whole central nervous system (CNS) cells (main, partially off scale) make up 4.1% of total events; concentrated low side scatter events are putative erythrocytes (93% of viable cells by trypan blue exclusion). (Q¨CT): Profile of viable CNS cells based on main, Annexin-V¨C gating. (Q): Nonspecific staining. (R): Before selection, CD133  cells make up 7.1% (polygon) of all intact CNS tumor cells. (S): After magnetic selection, unbound (flow-through) cells contain 4.7% CD133  cells. (T): Bound and eluted fraction is 97.5% CD133  cells. No recovery step to permit CD133 re-expression is necessary. Differences in physical profile in (A) and (P) are due in part to forward scatter voltage settings and side scatter scale (log in , linear in [P[). Abbreviation: 7-AAD, 7-amino-actinomycin-D." R; m. [, O% t+ ^8 R; g2 V& N

Because CD133 expression is predictive of tumor reinitiating activity in some CNS tumors .$ s* N( E7 f$ T; W, A% J0 u5 m7 l& g9 {

CD133 and CD15 Coexpress in a Similar Manner in Mouse Forebrain( N* |0 }: V) a6 c

Previous studies have used high expression of CD133 or CD15 to enrich for multipotent mouse stem cells . To address this, we dissociated E13.5 LGE and P2 subventricular zone (SVZ), and then performed triple labeling with CD24, CD133, and either CD15, A2B5, or PSA-NCAM (Fig. 5; supplemental online Fig. 2).

Figure 5. Coexpression of CD24, CD133, CD15, PSA-NCAMs, and A2B5 on fetal mouse forebrain cells. (A¨CO): E13.5 mouse lateral ganglionic eminence was freshly dissociated with 0.2 mg/ml of Liberase-1. Light scatter profile (A) shows that whole cells (main) make up 90% of total events. (B): 7-amino-actinomycin-D (7-AAD) staining (shown without main gate) shows that live cells are 98% of main population, 88% of total events. (C¨CO): Profile of live cells based on main, 7-AAD¨C gating. (C): Example of isotype control (for CD24) on two-dimensional plot. (D¨CO): Frequency histogram for CD133 (D) and CD24 (E), and dot plot of coexpression (F); CD15 histogram (G) and CD15 coexpression with CD133 (H) and CD24 (I); A2B5 histogram (J) and A2B5 coexpression with CD133 (K) and CD24 (L); PSA-NCAM histogram (M) and PSA-NCAM coexpression with CD133 (N) and CD24 (O). Green overlay indicates controls for nonspecific staining. CD133 and CD15 are coexpressed similarly among cortical cells, with highest expression in cells also expressing high levels of CD24 and A2B5. PSA-NCAM is expressed only in cells expressing high levels of CD24 and is rarely coexpressed with CD133. (P¨CR): Separate analysis of CD133 and CD24 staining in E13.5 mouse cortex (P) medial ganglionic eminence (Q) and caudal ganglionic eminence (R); profile of live cells based on main 7-AAD¨C gating. Profile is similar when gating is performed on 7-AAD¨C or Annexin-V¨C, cells.0 X3 h% j% N5 _! q9 }# w/ s
# Q' p. ?; V' J0 M+ g2 r
From E13.5 LGE, we found one distinct population (approximately two thirds of total) that expressed high levels of CD24 and no/low levels of CD15 or CD133 (i.e., CD15lo/¨CCD133lo/¨C CD24hi). A second distinct population centered on the high expression of CD15 and/or CD133 with low expression of CD24 (i.e., CD15hiCD133hiCD24lo). Within this second population, some cells expressed lower levels of CD15/CD133 but were not negative. Comparison of CD15 and CD133 expression indicated that most cells expressed similar levels of both antigens (Fig. 5H; supplemental online Fig. 3A¨C3E), suggesting that they identify similar cell types. However, there was a small but distinct CD15hiCD133loCD24hi population in both E13.5 LGE (Fig. 5F, 5I) and P2 SVZ (supplemental online Fig. 2F, 2I) that appeared repeatedly. We also triple labeled cells with CD133, CD24, and either A2B5, a glial progenitor marker . Triple labeling with CD133, CD24, and A2B5 showed that A2B5  cells (approximately 20% in both tissues) were predominantly CD133hi (Fig. 5J¨C5L; supplemental online Fig. 3F¨C3J). Nearly all PSA-NCAM  cells were CD133lo and CD24hi (Fig. 5M¨C5O; supplemental online Fig. 3K¨C3O). Analysis of CD24 against increasing concentrations of CD133 and PSA-NCAM (not shown) showed that this distribution remained the same.

We then compared coexpression of CD133 and CD24 from other mouse E13.5 forebrain regions, including the cortex, MGE, and CGE (Fig. 5P¨C5R). The LGE, MGE, and CGE are known to generate distinct interneuron derivatives and varying contributions of oligodendrocytes , had comparatively few of these cells but much larger numbers of CD133¨CCD24hi cells (Fig. 5F, 5Q, 5R). Interestingly, the LGE, MGE, and cortex all contained approximately 20% CD133loCD24lo cells.

CD133/CD24 and CD15/CD24 Coexpression Similarly Distinguish Fetal Mouse Forebrain Stem Cells from Neuronal Progenitors and Neurons( \4 o) n; h4 Z3 Q, u
) Q' c: o) _$ p) ?; m
In E13.5 LGE, the most highly CD133-/CD15-expressing cells often expressed high levels of CD24 (Fig. 5F, 5I). This was even more pronounced in E13.5 cortex (Figs. 5P, 6A), which has a high rate of new neuronal birth at this stage . To investigate differences in potency among these cells, we freshly dissociated E13.5 mouse cortex with Liberase-1, prospectively isolated cells based on CD133 (or CD15) versus CD24 expression (Fig. 6B¨C6D; supplemental online Fig. 4), and performed in vitro clonal analysis.
# i5 t- v& j0 f
Figure 6. Prospective isolation and clonal analysis of fetal mouse cortical cells. (A): Colors denote sort gates overlaid on CD133/CD24 expression profile from E13.5 mouse cortex after Annexin-V  (nonviable) cells were gated out; (B¨CD): example of purity analysis showing cell enrichment after gating (rectangles) for CD133loCD24lo (B), CD133hiCD24lo (C), and CD133hiCD24hi (D); each plot shows mean relative fluorescent intensity of CD24 (x-mean) and CD133 (y-mean) for enriched population, along with percentage of cells within gate area before (top) and after (bottom) sorting. (E): Example of two-cell clone, 16 hours postclonal density plating. (F, G): Cell survival (F) and percentage of cells dividing (G) at 16 hours postplating. (H¨CM): examples of clone types generated after sorting, 3 days of expansion, 6 days of differentiation, and staining for MAP2a b (neurons), GFAP (astrocytes), O4 (oligodendrocytes) and DAPI (all nuclei). Type 1 is a single neuron and is not shown. (N¨CO): Quantitation of each clone type generated from CD133-/CD24-sorted (N) or CD15-/CD24-sorted (O) cells. Mean ¡À SEM, n = 3 for (F, G, N, O). Abbreviations: AO, astrocyte-oligodendrocyte clone; DAPI, 4'-6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; N, neuron-only clone; NAO, neuron-astrocyte-oligodendrocyte clone; NO, neuron-oligodendrocyte clone. Scale bars = 50 (D) and 100 µm (C¨CF).3 \) M+ X$ G: H" m4 c
% W6 ^) j0 q1 h/ A
Cell division in the presence of bFGF could easily be visualized within the first 16 hours because of the "mirroring" behavior of daughter cells (Fig. 6E), which allowed extrapolation of how many initially plated cells survived and divided (Fig. 6F¨C6G). Notably, CD133¨C/loCD24hi cells did not divide; these cells had extensive fine filopodia that distinguished them from the short process-bearing dividing cells. Cells sorted on the basis of CD15/CD24 expression showed similar plating and clonal efficiencies (supplemental online Fig. 4G, 4H) to those seen after CD133/CD24 sorting, further supporting the contention that CD133 and CD15 expression predicts similar mouse cell populations.

After differentiation by bFGF withdrawal, we performed quadruple staining of cells for MAP2a b (neurons), GFAP (astrocytes), O4 (oligodendrocytes), and DAPI (total nuclei) and classified clones into seven types (Fig. 6H¨C6N). The CD133¨C/lo CD24hi fraction that did not divide yielded only single complex neurons (referred to as type 1). The CD133hiCD24hi fraction generated predominantly neuron-only clones of 4¨C16 cells with long processes (type 2) or 50¨C200 cells with short bipolar morphologies (type 3). CD133loCD24lo and CD133hiCD24lo cells generated mostly large three-fate clones with faint glial staining (type 4) or strong glial staining (type 5), indicating that they are multipotent. Additionally, we found three-fate clones that contained nearly all astro-oligo fates (type 6) or neuron-oligo fates (type 7). Although both CD24lo quadrants generated three-fate clones, CD133loCD24lo and CD15loCD24lo cells generated a higher proportion of type 4 clones, whereas CD133hiCD24lo cells generated higher numbers of type 5 and 7 clones. Similar results were found after CD15/CD24 selection, although type 7 clones also were generated from the CD15hiCD24hi population (Fig. 6O). On the basis of these results, we conclude that CD133/CD24 and CD15/CD24 expression can be used in a similar manner to predict mouse CNS precursor potency. Interestingly, the cell fraction enriched in clone-forming neuronal progenitors expresses higher levels of CD133 and CD15 than does the cell fraction enriched in multipotent stem cells. CD24 expression distinguished mouse fetal stem cells from postmitotic neurons and their progenitors; however, treatments that degrade CD24 expression (such as papain; Fig. 3) prevent such functional characterization.2 f% x% d/ F9 @* M

DISCUSSION

Methods of neural tissue dissociation vary widely and lead to differences in dissociation efficiency and viability as measured by flow cytometry . Interestingly, a neuronal progenitor population expresses higher levels of CD15 and CD133 than multipotent stem cells of the fetal mouse forebrain. Coexpression with CD24 enhances the discrimination of these two populations, indicating that Liberase-1, TrypLE, and Accutase have an advantage over papain for this type of analysis in mouse.
6 b7 e$ E6 S1 X" `" D& c8 \
In human CNS tissue studies, cruder preparations of trypsin can cleave important cell surface antigens such as CD133, requiring a recovery period for antigen re-expression . Suspended aggregate (neurosphere) cultures are often difficult to dissociate into single-cell suspensions without killing a substantial proportion of the cells. We found that viability and efficiency of passaging tumor cultures, in monolayer or in suspended aggregates, worked well with Liberase-1 (Table 1). TrypLE also worked well for this purpose, whereas papain yielded poor viability (not shown). Mouse cell passaging from fibronectin-coated dishes was not facilitated by Liberase-1 when compared with nonenzymatic passaging (not shown). We did not compare the effectiveness of these enzymes in passaging mouse neurospheres.2 w$ `3 n1 o, f7 ~/ N
7 i7 z8 ]4 P5 F& K1 `
The high viability of cell dissociation also allowed us to perform multiantigen analysis with high confidence that results are representative of the cell types in the tissue. Our results show that CD133 and CD15 can be used almost interchangeably along with CD24 to distinguish mouse multipotent stem cells from neuronal progenitors and postmitotic neurons. Interestingly, we find that the cells expressing the highest levels of CD133 and CD15 are more likely to be neuronal progenitors than multipotent stem cells. In contrast, we find that P2 SVZ has fewer CD133hiCD15hiCD24hi cells, which may be why selection on the basis of CD15 alone works for enriching multipotent stem cells from postnatal SVZ .7 Z5 V% J. m7 p4 S9 f! E# ]2 u
6 m2 H) X- f% g0 ?# m6 n
Our results show that multipotent fetal cells are enriched in the CD24lo fraction, similar to results in adult SVZ  these cell subgroups. Our future studies will test these possibilities.# u+ w  y! }7 ]0 s

In summary, we show that the flow cytometric analysis of the important neural antigens CD133, CD15, and CD24 are affected by the manner of dissociation. Although nonenzymatic dissociation does not digest antigens, this method is not useful for flow cytometric analysis since poor viability prevents proper titration of antibodies like CD133. We show that CD24 antigenicity, which is retained during Liberase-1, TrypLE, or Accutase treatment but lost during papain treatment, provides a useful discrimination between mouse fetal multipotent stem cells, neuronal-committed progenitors and neurons when paired with CD133 or CD15. These results reinforce the importance of comparing multiple methods of dissociation when characterizing novel selectable markers in future neural lineage studies.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST8 n7 i" u: Q/ m& r+ U

The authors indicate no potential conflicts of interest.
6 I( P+ j3 r' R  b* W1 q" x
ACKNOWLEDGMENTS. L2 d0 S% t4 T3 Z2 B
4 D9 p3 Q* v( A& V( }7 g  V3 V
This study was supported by funds from the NIH Mental Retardation and Developmental Disabilities Research Center Grant P30HD40677, the Frank and Nancy Parsons Fund, the Georgia Derrico and Rod Porter Fund for the Children's National Medical Center (CNMC)/University of Padova Sister Program, the CNMC Research Advisory Council, and two CNMC Board of Visitor Grants. We thank William King and Bhargavi Rajan for flow cytometry support; Dr. Brian Rood and Hui-Zhen Zhang for assistance in acquiring human tumors; and Dr. Vittorio Gallo and Dr. Giuseppe Basso for advice and support.
      【参考文献】/ |7 |% c0 V% W6 D

Panchision DM, McKay RD. The control of neural stem cells by morphogenic signals. Curr Opin Genet Dev 2002;12:478¨C487.5 Z$ {$ Z# \( M  W- k  T' ]
6 q" H( p5 S7 u  f) L3 p
Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RD. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev 1996;10:3129¨C3140.9 k: T" p! G  h" s; _9 ]

Rajan P, Panchision DM, Newell LF, McKay RD. BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells. J Cell Biol 2003;161:911¨C921.: M0 Z5 w1 c# v" Z2 h. K+ Y
& Z( M& n& Z) P' v8 G( T4 w
Dupin E, Ziller C, Le Douarin NM. The avian embryo as a model in developmental studies: Chimeras and in vitro clonal analysis. Curr Top Dev Biol 1998;36:1¨C35.
: h2 f( r0 T7 N2 M% P: @( L
Lindvall O, Kokaia Z, Martinez-Serrano A. Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat Med 2004;10 (suppl):S42¨C50.

Singh SK, Hawkins C, Clarke ID et al. Identification of human brain tumour initiating cells. Nature 2004;432:396¨C401., ?9 [/ s4 o7 J9 G% Z- {
, A  }6 S& i# {, j* k
Mujtaba T, Piper DR, Kalyani A et al. Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells. Dev Biol 1999;214:113¨C127.

Morrison SJ, White PM, Zock C et al. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell 1999;96:737¨C749.
# p. a' o8 R1 W9 [
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.% r. s8 V* M; J  I+ h
+ T* ~8 {! T; v; \* X
Maric D, Maric I, Chang YH et al. Prospective cell sorting of embryonic rat neural stem cells and neuronal and glial progenitors reveals selective effects of basic fibroblast growth factor and epidermal growth factor on self-renewal and differentiation. J Neurosci 2003;23:240¨C251.
, W6 s7 u) f1 i4 D$ C5 h
Belachew S, Chittajallu R, Aguirre AA et al. Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons. J Cell Biol 2003;161:169¨C186.6 b2 M8 F+ D/ M  ?

Uchida N, Buck DW, He D et al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA 2000;97:14720¨C14725.
/ g! @0 n: E/ B1 Q  |9 E
Barraud P, Stott S, Mollgard K et al. In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133. J Neurosci Res 2007;85:250¨C259.

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.

Fargeas CA, Florek M, Huttner WB et al. Characterization of prominin-2, a new member of the prominin family of pentaspan membrane glycoproteins. J Biol Chem 2003;278:8586¨C8596.  w( c1 Q2 \; W
4 x; W' L# P; K
Weigmann A, Corbeil D, Hellwig A et al. Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells. Proc Natl Acad Sci U S A 1997;94:12425¨C12430., r  {+ {; Y5 I( _

Lee A, Kessler JD, Read TA et al. Isolation of neural stem cells from the postnatal cerebellum. Nat Neurosci 2005;8:723¨C729.0 T5 |0 `: [/ D- o) i

Doetsch F, Petreanu L, Caille I et al. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 2002;36:1021¨C1034.

Calaora V, Chazal G, Nielsen PJ et al. mCD24 expression in the developing mouse brain and in zones of secondary neurogenesis in the adult. Neuroscience 1996;73:581¨C594.2 |1 J0 U7 Q) i4 \( n( N; g7 v

Nieoullon V, Belvindrah R, Rougon G et al. mCD24 regulates proliferation of neuronal committed precursors in the subventricular zone. Mol Cell Neurosci 2005;28:462¨C474.% X! Z/ ?# Q+ F6 E  ^
9 L8 _: U* k) N' j8 t
Capela A, Temple S. LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal. Neuron 2002;35:865¨C875.
" V/ e0 W& L. X
Sun Y, Goderie SK, Temple S. Asymmetric distribution of EGFR receptor during mitosis generates diverse CNS progenitor cells. Neuron 2005;45:873¨C886.8 Z( P3 S( Z3 H+ T
1 |# h" t! n3 [+ c9 g  ?
Capela A, Temple S. LeX is expressed by principle progenitor cells in the embryonic nervous system, is secreted into their environment and binds Wnt-1. Dev Biol 2006;291:300¨C313.

Mukouyama YS, Deneen B, Lukaszewicz A et al. Olig2  neuroepithelial motoneuron progenitors are not multipotent stem cells in vivo. Proc Natl Acad Sci U S A 2006;103:1551¨C1556.+ K5 g' M6 g; f
2 \4 @6 y: c  _% |  d- M; n  S/ I! a
Kalyani AJ, Piper D, Mujtaba T et al. Spinal cord neuronal precursors generate multiple neuronal phenotypes in culture. J Neurosci 1998;18:7856¨C7868.$ F: V0 y" n6 @$ n) H/ n( Y) [
' z0 C0 w4 Y" Q
Pennartz S, Belvindrah R, Tomiuk S et al. Purification of neuronal precursors from the adult mouse brain: Comprehensive gene expression analysis provides new insights into the control of cell migration, differentiation, and homeostasis. Mol Cell Neurosci 2004;25:692¨C706.7 d# N- F% J5 Z3 s& U* p- Y

Cai J, Wu Y, Mirua T et al. Properties of a fetal multipotent neural stem cell (NEP cell). Dev Biol 2002;251:221¨C240.8 f; _7 T* K6 \# `7 Z$ U8 `. j

Mayer-Proschel M, Kalyani AJ, Mujtaba T et al. Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron 1997;19:773¨C785.
7 L! T. n* k- Q6 d% D5 P
Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A. Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 1997;17:5046¨C5061.
" A% y; `" T$ a/ C1 x/ Q
Rao MS, Mayer-Proschel M. Glial-restricted precursors are derived from multipotent neuroepithelial stem cells. Dev Biol 1997;188:48¨C63.
$ C' G2 h; R. p" x! U7 h! h
Rao MS, Noble M, Mayer-Proschel M. A tripotential glial precursor cell is present in the developing spinal cord. Proc Natl Acad Sci U S A 1998;95:3996¨C4001.
+ G6 z# V8 r, _& h7 T
Maric D, Barker JL. Neural stem cells redefined: A FACS perspective. Mol Neurobiol 2004;30:49¨C76.* r# O8 J" e9 N3 s3 B

Hulspas R, Quesenberry PJ. Characterization of neurosphere cell phenotypes by flow cytometry. Cytometry 2000;40:245¨C250.+ y! p" T- b, @) h: T

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.

Maric D, Maric I, Barker JL. Buoyant density gradient fractionation and flow cytometric analysis of embryonic rat cortical neurons and progenitor cells. Methods 1998;16:247¨C259.

Finkbeiner S, Stevens CF. Applications of quantitative measurements for assessing glutamate neurotoxicity. Proc Natl Acad Sci U S A 1988;85:4071¨C4074.$ n$ r& j% Y+ p# ^
9 k/ R' ?1 N5 W
Huettner JE, Baughman RW. Primary culture of identified neurons from the visual cortex of postnatal rats. J Neurosci 1986;6:3044¨C3060." V& @. B; Y1 S
  V( K7 W" m, _) K
Sawamoto K, Yamamoto A, Kawaguchi A et al. Direct isolation of committed neuronal progenitor cells from transgenic mice coexpressing spectrally distinct fluorescent proteins regulated by stage-specific neural promoters. J Neurosci Res 2001;65:220¨C227.- |/ v* f# G3 p7 {
) m3 q" Z% t9 B
Kim M, Morshead CM. Distinct populations of forebrain neural stem and progenitor cells can be isolated using side-population analysis. J Neurosci 2003;23:10703¨C10709./ T: A# J  _, t7 B# R

Rao MS, Landis SC. Characterization of a target-derived neuronal cholinergic differentiation factor. Neuron 1990;5:899¨C910.

Thompson LH, Andersson E, Jensen JB et al. Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon. Exp Neurol 2006;198:183¨C198.+ f2 ]' c. y% K! d% |
7 ~& f( R; i. o8 C9 D5 \
Kim J-H, Panchision DM, Kittappa R et al. Generating CNS neurons from embryonic, fetal and adult stem cells. In: Wassarman PM, Keller GM, eds. Differentiation of Embryonic Stem Cells.New York: Academic Press,2003;Vol 365.

Alvarez-Buylla A, Lim DA. For the long run: Maintaining germinal niches in the adult brain. Neuron 2004;41:683¨C686.

Panchision DM, Pickel JM, Studer L et al. Sequential actions of BMP receptors control neural precursor cell production and fate. Genes Dev 2001;15:2094¨C2110.* k: p. \7 e' U2 F
6 b! h$ d, v! \
Sailer MH, Hazel TG, Panchision DM et al. BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells. J Cell Sci 2005;118:5849¨C5860.
. g2 E+ E; \9 X" n
Taylor MD, Poppleton H, Fuller C et al. Radial glia cells are candidate stem cells of ependymoma. Cancer Cell 2005;8:323¨C335.* r/ V# k+ ]( l

Piccirillo SG, Reynolds BA, Zanetti N et al. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 2006;444:761¨C765.

Corbin JG, Nery S, Fishell G. Telencephalic cells take a tangent: Non-radial migration in the mammalian forebrain. Nat Neurosci 2001;4 (suppl):1177¨C1182.
  L; @0 L/ y1 {) i8 Y$ t7 K
Richardson WD, Kessaris N, Pringle N. Oligodendrocyte wars. Nat Rev Neurosci 2006;7:11¨C18.

Nery S, Fishell G, Corbin JG. The caudal ganglionic eminence is a source of distinct cortical and subcortical cell populations. Nat Neurosci 2002;5:1279¨C1287.. {5 _$ @; H0 J( S9 Z9 n
! Y& U- E0 I6 F- H9 ?
Caviness VS Jr., Goto T, Tarui T et al. Cell output, cell cycle duration and neuronal specification: A model of integrated mechanisms of the neocortical proliferative process. Cereb Cortex 2003;13:592¨C598.% Y% o8 V! |% T/ r. |+ x) M& Y, M; u
" W# c& N( o! ?5 H" n
Smart IH, McSherry GM. Growth patterns in the lateral wall of the mouse telencephalon. II. Histological changes during and subsequent to the period of isocortical neuron production. J Anat 1982;134:415¨C442./ g$ f4 q4 E* B8 a
4 h. x8 S( g: k' P. c* H
Bhide PG. Cell cycle kinetics in the embryonic mouse corpus striatum. J Comp Neurol 1996;374:506¨C522.

Sheth AN, Bhide PG. Concurrent cellular output from two proliferative populations in the early embryonic mouse corpus striatum. J Comp Neurol 1997;383:220¨C230.

Corver WE, Cornelisse CJ, Hermans J et al. Limited loss of nine tumor-associated surface antigenic determinants after tryptic cell dissociation. Cytometry 1995;19:267¨C272., R5 b2 N! o$ X' Z' b
* Y2 n7 ~3 F- q* w0 {  ?
Jakob M, Demarteau O, Schafer D et al. Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells. Connect Tissue Res 2003;44:173¨C180.5 G/ Q9 _( n( H2 b+ Y) m

Ziegler BL, Thoma SJ, Lamping CP et al. Surface antigen expression on CD34  cord blood cells: Comparative analysis by flow cytometry and limiting dilution (LD) RT-PCR of chymopapain-treated or untreated cells. Cytometry 1996;25:46¨C57.

Ravindranath MH, Bauer PM, Cornillez-Ty C et al. Quantitation of the density of cell surface carbohydrate antigens on cancer cells with a sensitive cell-suspension ELISA. J Immunol Methods 1996;197:51¨C67.
+ A4 E- v7 E, _/ ^! p3 }3 q: `% @
Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281¨C292.
0 {# }9 s1 F, W1 O
Xu RH, Peck RM, Li DS et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2005;2:185¨C190.

Kalyani A, Hobson K, Rao MS. Neuroepithelial stem cells from the embryonic spinal cord: Isolation, characterization, and clonal analysis. Dev Biol 1997;186:202¨C223., P# Z# I* _1 h" A$ y0 [

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¨C188.7 Y% N9 t0 [+ k
% G( h& U9 Z, s6 f* X
Martens DJ, Tropepe V, van Der K. Separate proliferation kinetics of fibroblast growth factor-responsive and epidermal growth factor-responsive neural stem cells within the embryonic forebrain germinal zone. J Neurosci 2000;20:1085¨C1095.% B3 b5 _0 w6 v2 H% c

Kuhn HG, Winkler J, Kempermann G et al. Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J Neurosci 1997;17:5820¨C5829.

Burrows RC, Wancio D, Levitt P et al. Response diversity and the timing of progenitor cell maturation are regulated by developmental changes in EGFR expression in the cortex. Neuron 1997;19:251¨C267.

收藏0 分享0 顶0 踩0