New strategy for the analysis of phenotypic marker antigens in brain tumor–derived neurospheres in mice and humans

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Object

Brain tumor stem cells (TSCs) hypothetically drive the malignant phenotype of glioblastoma multiforme (GBM), and evidence suggests that a better understanding of these TSCs will have profound implications for treating gliomas. When grown in vitro, putative TSCs grow as a solid sphere, making their subsequent characterization, particularly the cells within the center of the sphere, difficult. Therefore, the purpose of this study was to develop a new method to better understand the proteomic profile of the entire population of cells within a sphere.

Methods

Tumor specimens from patients with confirmed GBM and glioma models in mice were mechanically and enzymatically dissociated and grown in traditional stem cell medium to generate neurospheres. The neurospheres were then embedded in freezing medium, cryosectioned, and analyzed with immunofluorescence.

Results

By sectioning neurospheres as thinly as 5 μm, the authors overcame many of the problems associated with immunolabeling whole neurospheres, such as antibody penetration into the core of the sphere and intense background fluorescence that obscures the specificity of immunoreactivity. Moreover, the small quantity of material required and the speed with which this cryosectioning and immunolabeling technique can be performed make it an attractive tool for the rapid assessment of TSC character.

Conclusions

This study is the first to show that cryosectioning of neurospheres derived from glioma models in mice and GBM in humans is a feasible method of better defining the stem cell profile of a glioma.

Abbreviations used in this paper: bFGF = basic fibroblast growth factor; DAPI = 4', 6-diamidino-2-phenylindole dihydrochloride; EGF = epidermal growth factor; GBM = glioblastoma multiforme; GFAP = glial fibrillary acidic protein; IgG = immunoglobulin G; NSC = neural stem cell; PBS = phosphate-buffered saline; PDGF = platelet-derived growth factor; RCAS = replication-competent avian leukosis virus long terminal repeat splice acceptor; TSC = tumor stem cell; WHO = World Health Organization.

Object

Brain tumor stem cells (TSCs) hypothetically drive the malignant phenotype of glioblastoma multiforme (GBM), and evidence suggests that a better understanding of these TSCs will have profound implications for treating gliomas. When grown in vitro, putative TSCs grow as a solid sphere, making their subsequent characterization, particularly the cells within the center of the sphere, difficult. Therefore, the purpose of this study was to develop a new method to better understand the proteomic profile of the entire population of cells within a sphere.

Methods

Tumor specimens from patients with confirmed GBM and glioma models in mice were mechanically and enzymatically dissociated and grown in traditional stem cell medium to generate neurospheres. The neurospheres were then embedded in freezing medium, cryosectioned, and analyzed with immunofluorescence.

Results

By sectioning neurospheres as thinly as 5 μm, the authors overcame many of the problems associated with immunolabeling whole neurospheres, such as antibody penetration into the core of the sphere and intense background fluorescence that obscures the specificity of immunoreactivity. Moreover, the small quantity of material required and the speed with which this cryosectioning and immunolabeling technique can be performed make it an attractive tool for the rapid assessment of TSC character.

Conclusions

This study is the first to show that cryosectioning of neurospheres derived from glioma models in mice and GBM in humans is a feasible method of better defining the stem cell profile of a glioma.

Numerous authors of recent studies have drawn attention to a slowly cycling, but highly tumorigenic, subpopulation of cells in human gliomas with stem cell–like properties. These TSCs, alternatively called “tumor initiating cells,” drive the malignant phenotype of human glial tumors.27,28 The TSC hypothesis has potentially profound implications for both basic biological studies and the development of new therapeutic drugs against GBM. Evidence suggests that studying the TSC population in GBMs is a more relevant system for exploring glioma biology and screening new therapeutic agents than is traditionally used, that is, passaged GBM cell lines whose phenotypic characteristics and genetic aberrations often bear little resemblance to those found within corresponding primary human brain tumors.16

Tumor stem cells share many of the properties of normal NSCs, including self-renewal, extended proliferation, formation of neurospheres when grown in culture, and potential to differentiate into neurons and glial cells.1,29 They have also been shown to express various progenitor and stem cell proteins such as nestin, Sox2, Oct4, and Musashi.5,7,31 It is likely that tumor-derived neurospheres, once thought to be clonal or derived from a single stem cell, are a heterogeneous population of stem cells, intermediate progenitors, and differentiated cells,19,25 and experiments with video microscopy of genetically labeled cells show that neurospheres are highly motile and prone to fuse.25

In the past, TSCs have been characterized based on functional criteria, such as their proliferation as neurospheres when cultured in the presence of growth factors, which is known as the “neurosphere assay.”20,21 This technique is valuable for the propagation of TSCs but is limited as a characterization assay. Recent publications4,14,16,18,24 have been focused on characterizing the heterogeneous population within neurospheres by immunostaining entire spheres, molecular phenotyping, and flow cytometry. However, the effectiveness of whole-neurosphere immunostaining is diminished because of high background fluorescence and the inability of antibodies to penetrate the sphere to access antigenic sites within the core. In this report, we discuss a new strategy for the analysis of phenotypic markers in both murine and human brain TSC neurospheres through immunolabeling of sphere cryosections. The use of cryosections allows antibody labeling of the sphere core. Using the sections also eliminates the problem of high background fluorescence that occurs with whole spheres. Our technique makes evaluation at the single-cell level possible, and we can simultaneously monitor several target proteins with multiple immunolabelings. Additional major advantages of our method include a reduction in the amount of material required and the speed with which the analysis can be performed.

We are the first to show that the cryosectioning of neurospheres derived from both a GBM model in mice and GBMs in humans is a feasible method to better define the stem cell profile of a glioma in a particular patient. This method undoubtedly will serve as a stepping-stone for more in-depth studies of the neurosphere microenvironment.

Materials and Methods

Specimen Procurement and Stem Cell Culture

Human Tumor Specimen Procurement and Primary Tumor Culture

In accordance with institutional review board approval and after receiving written informed consent, we obtained tumor samples, classified as GBM according to the WHO criteria, from patients undergoing surgical treatment. The GBM tissue was collected in 1× Hanks balanced salt solution containing 0.6% glucose. The tissue was digested in Hanks balanced salt solution containing 12% papain and 10 μg/ml DNase with gentle shaking at 37°C for 1 hour. After 1 hour, fetal bovine serum was added to the solution to abrogate digestion, the tissue was triturated with a 5.0-ml pipette, and the resultant cellular suspension was pipetted through a sterile 70-mm cell filter (BD Biosciences). After spinning for 5 minutes at 1200 rpm, the tumor cells were resuspended in Dulbecco modified Eagle medium/F12 (Gibco) and centrifuged again. The cells were resuspended in stem cell medium consisting of Dulbecco modified Eagle medium/F12 containing B27 (Gibco), 20 μg/ml EGF (Invitrogen), 20 μg/ml bFGF (Invitrogen), 1 μg/ml heparin (Sigma), and 1× antibiotic/ antimycotic (Gibco). Tumor cells were then seeded into tissue culture flasks (Denville Scientific) at a density of 75,000 cells/cm2. Fresh medium was added to the cultures every 48–72 hours, and TSCs were propagated as neurospheres by serial dilution.2,17,29

Murine Tumor Specimen Procurement and Primary Tumor Culture

Platelet-derived growth factor-induced gliomas in mice were generated using the RCAS/tv-a system, which allows in vivo retroviral gene transfer into a specific cell type. Thereafter, DF1 cells were transfected with the RCAS-PDGF construct and intracranially injected into mice expressing tv-a driven by the nestin promotor active in glial progenitors and neuronal precursors.12 As shown in Fig. 1 left, the tumors generated using this model resemble human GBM with the characteristic presence of vascular proliferation and pseudopalisading necrosis.

Fig. 1.
Fig. 1.

Photomicrographs demonstrating PDGF-induced glioma in mice and derived NSC culture. Left: Staining of a PDGF-induced tumor reveals the presence of vascular proliferation and pseudopalisading necrosis. H & E, original magnification × 4. Right: The PDGF-driven tumors in mice were dissected, and cells were enzymatically dissociated with papain into a suspension that contained single cells. Neurospheres were obtained by plating the single cell suspension at a density of 5 × 104 cells/ml in neurosphere medium supplemented with EGF and b FGF for a period of 7–10 days. Original magnification × 10.

A protocol similar to that described for processing human GBM specimens was used to isolate cells from PDGF-driven tumors in mice with minor modifications. Briefly, tumors were dissected, and cells were enzymatically dissociated at 37°C for 15 minutes using papain and DNase, with subsequent inactivation via ovomucoid (Worthington). The cell suspension was consecutively washed and resuspended to break apart the remaining cellular aggregates into single cells. The cells were seeded at 5 × 104 cells/ml and grown in neurosphere medium (NeuroCult, StemCell Technologies, Inc.), according to the manufacturer's instructions. The medium contained NSC basal medium for mice, NSC proliferation supplements,20 μg/ml EGF, 20 μg/ml bFGF, and 1 μg/ml heparin.

Sectioning Neurospheres

Neurospheres, grown in suspension, were collected by centrifugation at 1200 rpm at room temperature for 5 minutes and were subsequently resuspended in 1× PBS. The PBS was removed by centrifugation (Galaxy mini microcentrifuge, VWR) at 6000 rpm for 30 seconds or until a visible pellet had formed. The neurospheres were then fixed in 4% paraformaldehyde for 5 minutes. The paraformaldehyde was removed by centrifugation at 6000 rpm, followed by a single wash with 1× PBS. After removal of the PBS, the neurospheres were resuspended in a mixture of 1× PBS and trypan blue (ratio of 50:1) for subsequent visualization of the neurospheres during cryosectioning. The spheres were frozen in a mold with cryosectioning embedding medium (Sakura Finetek) by submerging the mold in 100% ethanol chilled in solid CO2 and stored at –80°C. Neurospheres were serially sectioned on a standard cryostat (Microm HM 505 E, Global Medical Instrumentation) at –21°C at thicknesses of 5 to 15 μm and were electrostatically adhered to microscope slides (VWR). Specimens were allowed to dry at room temperature for 1 hour after sectioning and then were stored at –80°C until they were processed for immunofluorescence.

Immunocytochemical Studies

To label cytosolic antigens, sectioned neurospheres were blocked in 1× PBS containing 0.1% Tween and 5% normal goat serum for 90 minutes and subsequently were incubated in primary antibody diluted in blocking solution (Table 1). To label transcription factors, the spheres were permeabilized through incubation in 0.1% Triton X-100 in 1× PBS for 1 hour at room temperature, and primary antibodies were diluted in blocking solution containing 10% horse serum or normal goat serum, 1% bovine serum albumin, and 0.25% Triton X-100. Sections were incubated in primary antibody overnight at 4°C to detect both cytoplasmic and nuclear antigens. Alexa Fluor 488 (green) or 594 (red) chicken anti–mouse IgG, chicken anti–rabbit IgG, and donkey anti–goat IgG secondary antibodies (Molecular Probes) revealed immunoreactivity. Coverslips were applied to the sections with mounting medium containing DAPI (Molecular Probes) to identify cell nuclei. Proper negative controls were processed by omitting the primary antibody.

TABLE 1

Primary antibodies used in this study

AntibodyHostDilutionManufacturer
anti-GFAPrabbit1:300Dako, Carpinteria, CA
anti-nestinmouse1:100BD Bioscience, Franklin Lakes, NJ
anti-vimentinmouse1:100Dako, Carpinteria, CA
anti–Ki 67mouse1:100Dako, Carpinteria, CA
anti-Musashirabbit1:300Chemicon, Temecula, CA
anti-nanoggoat1:100R&D Systems, Minneapolis, MN
anti-Oct4mouse1:300Abcam, Cambridge, MA
anti-Sox2goat1:100Santa Cruz Biotechnology, Santa Cruz, CA

Sectioned neurospheres were viewed with a conventional fluorescence microscope (DMI6000 B) and were imaged using a monochrome digital camera (DFC350 FX) and Leica FW4000 (version 1.2.1, all Leica Microsystems) imaging software.

Results

Immunolabeling of Cryosectioned Neurospheres From Induced Murine GBMs

As previously described,23 PDGF-induced mouse gliomas were histopathologically similar to human GBM. As seen in Fig. 1 left, these densely cellular tumors contain pleomorphic nuclei, areas of pseudopalisading necrosis, and new vessel formation and thereby meet the WHO criteria for GBM. Tumor stem cells were successfully isolated from mouse specimens and formed neurospheres when cultured in stem cell medium (Fig. 1 right). Immediately following tumor dissection, the dissociated cells grew as a monolayer. After 7–10 days in culture, collections of cells that had grown together began to grow as a “polypoid” structure out of the plane of the culture flask or dish. Soon thereafter, the cells detached from the monolayer to yield free-floating neurospheres. Once formed, the neurospheres were mechanically or enzymatically dissociated and kept for numerous passages, which confirmed their ability to self-renew.

Cells on the surface of and within the tumor-derived neurospheres were immunolabeled with antibodies for various stem cell antigens after cryosectioning. Consistent with previous reports, the neurospheres stained positive for the intermediate filament protein, nestin, and the cytoskeletal protein GFAP (Fig. 2, green staining).16,7 Neurospheres also strongly expressed transcription factors Oct4 and Sox2 and the neural RNA-binding protein Musashi-1, all of which are known to be expressed in developmental and adult mouse and human NSCs.7,31 Oct4 and Musashi-1 each colabeled neurospheres with GFAP and nestin.

Fig. 2.
Fig. 2.

Immunofluorescence images obtained during immunocytochemical analysis of neurospheres derived from PDGF-driven tumors in mice. Cryosectioned neuropsheres were fixed in paraformaldehyde and stained overnight with specific stem cell marker antibodies. A: Single staining of nestin, GFAP, Musashi-1, Sox2, and Oct4 revealing expression patterns in mouse TSCs. B: Double staining showing the coexpression of stem cell markers: Oct4 with either GFAP or nestin, and Musashi with GFAP or nestin. Nuclei were counterstained with DAPI. Bar = 50 μm.

Detection of Stem Cell Antigens in Cryosected Neurospheres Derived From Human GBM Specimens

We routinely isolate TSCs from patients with GBM. We also harvest and culture parental glioma cells and the non-TSC populations, which serve as controls. Thirty consecutive tumor samples classified as GBM, based on WHO criteria, were obtained from patients undergoing surgical treatment at Weill Cornell Medical College in accordance with institutional review board approval. Within 1–3 hours after surgical removal, tumors were washed and enzymatically dissociated into single cells. Human brain TSCs were cultured in conditions optimal for propagation and the prevention of differentiation of normal NSCs. Tumor cells cultured in this medium readily proliferated as nonadherent, multicellular neurospheres in uncoated plates (Fig. 3 right), as in the mice (Fig. 1 right). After crysosectioning, neurospheres were immunolabeled for Sox2, nanog, vimentin, nestin, and GFAP, whose expression overlapped within a given sphere (Fig. 4B–G). Moreover, relatively few cells within a neurosphere were dividing, as shown by probing for the proliferation marker Ki 67, which would be expected from slowly cycling progenitors.

Fig. 3.
Fig. 3.

Left: Photomicrograph demonstrating staining of neurospheres grown from specimens from a patient with confirmed Grade IV astrocytoma. Note the hyperdensity and pleomorphism of the nuclei as well as the pseudopalisading necrosis (asterisks) and angiogenesis (arrows). H & E, original magnification ×10. Right: Brightfield microscopic image showing a neurosphere grown from the surgical specimen featured in panel A. Bar = 50 mm.

Fig. 4.
Fig. 4.

Immunofluorescence images revealing neurospheres derived from human GBM specimens expressing stem cell markers. As expected of slowly cycling stem cells, relatively few nuclei stained positively for Ki 67, a marker of proliferation. All Ki 67+ cells were colabeled with GFAP (A). Vimentin and GFAP were colocalized within human GBM–derived neurospheres (B), as were nestin and GFAP (C). The transcription factor Sox2 labeled the majority of nuclei within the sphere and labeled cells that were also vimentin+ (D) and nestin+ (E). Nanog+ cells were also vimentin+ (F) and nestin+ (G). Bar = 50 μm.

Discussion

In this report, we describe a novel technique of characterizing neurospheres derived from human and mouse models of glioma. These models involved cryosectioning of embedded spheres followed by immunocytochemical analysis with antibodies against known stem cell proteins.

Recent data have suggested that cancers may develop from a small subset of cells with self-renewal properties analogous to organ stem cells.6 In the 1990s, studies of acute myelogenous leukemia provided compelling evidence for the existence of a cancer stem cell subpopulation. Stem cells in patients with leukemia possess proliferative and self-renewal capacities absent in the majority of leukemic cells. The ability to fractionate and functionally analyze leukemic stem cells has led to the determination that these cells are necessary and sufficient to maintain the disease.15

A growing body of evidence indicates that stem cells drive tumorigenesis in solid tumors. Stem cell–like cells that have the capacity for self-renewal and can produce tumors when xenografted have been isolated from human breast and prostate cancers.6 As with other solid tumors, authors have verified the presence of a slowly cycling, but highly tumorigenic, subpopulation of cells with stem cell–like properties in adult and pediatric human glial malignancies.1,10,11,24,27,28 Tumor stem cells, derived from resected GBM tissue, have shown similarities both to the relatively quiescent population of adult NSCs embedded within the subventricular zone and dentate gyrus and to the TSCs from other malignancies.30 Among these commonalities is the ability to form neurospheres that self-renew and generate astrocytes, neurons, or oligodendrocytes under permissive conditions.13,26,28

These findings have confirmed that brain tumors contain undifferentiated neural precursors reminiscent of NSCs and that these cells can be isolated from a tumor mass.9,13 We as well as others have shown that, when transplanted into immunodeficient animals, TSCs generate tumors with the cardinal features of the GBM from which they derive, including an infiltrative phenotype and histopathological features such as hypercellularity, pseudopalisading necrosis, and angiogenesis.26,29 In contrast, traditionally passaged cells lines from human GBM, such as U87MG and others, or cells derived directly from surgical specimens grown in medium containing serum do not generate tumors with the aforementioned characteristics.16 This evidence suggests that studying the TSC population of GBMs is a more biologically relevant model system for exploring glioma biology.

Therefore, accurate selection of TSCs is of the utmost importance to most closely study the parental tumor in vitro. This selection becomes technically challenging for 4 practical reasons: 1) a minority of cells within the tumor are stem cells; 2) to date, no single antigen has been shown to reliably segregate tumorigenic stem cells from the rest of the tumor specimen; 3) tumor-derived neurospheres, once thought to be clonal or derived from a single stem cell, are a heterogeneous population containing stem and non-stem cells;19,25 and 4) until this report, there has been no description of the makeup of entire tumor-derived neurospheres, including the core, which is important to characterize the constituent cells of the sphere.

Recently, sorting for membrane-bound epitopes through fluorescence-activated cell sorting, magnetic bead sorting, or immunopanning has received much attention because of the ease with which these techniques might separate stem cells from the rest of the tumor. Results have been equivocal, however,4,18,28 and are best characterized by the controversy surrounding the separation of stem cells by CD133, a 5 transmembrane domain glycoprotein of unknown function.

Reportedly, CD133 is present on the membrane of stem cells and progenitor cells in various tissues.6,28 The CD133+ cells purified from human gliomas are reported to grow as neurospheres, and the transplantation of as few as 100 CD133+ human glioma cells into the brains of immunodeficient mice initiates the development of glioma.28 However, authors of a recent paper have indicated that CD133 is a marker of embryonic, not adult, NSCs (meaning that CD133+ stem cells are not the stem cell of origin in glioma) or that CD133 is aberrantly upregulated in brain TSCs.18 Moreover, Beier et al.4 recently have shown that CD133– cells derived from GBM specimens fulfill all stem cell criteria and are equally as tumorigenic as CD133+ cells when xenografted into immunodeficient mice.

Many groups have performed immunocytochemical studies of whole spheres to characterize their constitution. There are several issues that challenge the effectiveness of whole-neurosphere immunostaining, including resistance to penetration by antibodies, availability of antigenic sites within the specimen, and high background fluorescence. By cryosectioning embedded neurospheres, we eradicated the problems hindering immunocytochemical analysis of the entire neurosphere. For example, antibody penetration is not a problem using our method, and we can monitor several target proteins simultaneously by multiple immunolabeling to discriminate segregated expression from colocalization. Additionally, our method requires small quantities of material and can be performed quickly. By sectioning the spheres as thinly as 5 μm, background fluorescence is eliminated from cells stained within the sphere but outside the focal plane.

With our cryosectioning method and immunolabeling, we showed that intact GBM-derived human and PDGF-induced mouse glioma neurospheres are similar in their patterns of stem cell protein expression. They express various stem cell markers, such as nestin, GFAP, nanog, Oct4, Sox2, vimentin, and Musashi-1, previously shown to be present in neurospheres derived from normal, mammalian NSCs.7,31 This technique allows comparison between human glioma– and mouse model–derived intact neurospheres, and, more importantly, may provide the first step toward remedying the aforementioned problems with harvesting pure TSCs.

Given that TSCs are the cells putatively driving tumorigenesis in vivo, they are obvious therapeutic targets. As one might predict from their slow cycling, however, TSCs have been shown to be resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol), and etoposide (VP-16)17 as well as radiotherapy.2,3 In addition, CD133 expression was shown to be significantly higher in recurrent GBM tissue obtained from 5 patients compared with their respective newly diagnosed tumors, suggesting that CD133+ cancer stem cells strongly affect a tumor's resistance to chemotherapy.17,22 These data suggest that TSCs isolated from human GBM may represent novel targets for therapeutics.

Furthermore, cancer cell lines, such as U87MG, have been the historical standard both for exploring the biology of human brain tumors and as preclinical models for screening potential therapeutic agents.16 However, the phenotypic and genotypic aberrations found within these cell lines often bear little resemblance to those in the corresponding primary GBM. This factor probably explains why cancer cell line–based preclinical models have been poorly predictive for identifying clinically useful therapeutic agents in GBM.

In fact, a GBM-specific neurosphere assay has recently been proposed to test the drug sensitivity of TSCs.8 The neurospheres were exposed to various drugs, and treatment induced dispersion of the spheres leading to cell death. The utilization of our staining technique in concert with the neurosphere assay could become a useful tool for investigating the effects of various drugs on important signaling molecules involved in regulating the cell cycle or cell death in TSCs, such as caspase-3. Cryosectioning and immunofluorescence could also be used as tools to analyze the presence of proteins that may sensitize or promote the resistance of GBM neurospheres to radiation treatment. Applying radiation to GBM neurospheres has been shown to increase the percentage of stem cell–like cells, suggesting that radiotherapy has a greater effect on differentiated cells and that cancer stem cell–like cells are the resistant population.3 Ultimately, treatments that change the stem cell phenotype of glioma-derived neurospheres may yield better outcomes in patients with GBM.

The very characteristics of TSCs—extensive proliferation and self renewal—make the concept of patient-specific stem cell lines attractive and eminently feasible. The limitless number of brain TSCs that can be created during neurosphere culturing provides what amounts to a personalized cell line upon which individualized patient-specific chemotherapeutic regimens theoretically can be designed.

Conclusions

Accumulating evidence suggests that a better understanding of TSCs will have profound implications for treating cancers, particularly GBM. Tumor stem cells are immediately available for expansion and use in basic biological assays, animal implantation studies, and comparative analyses and have the propensity to form neurospheres. A variety of methods have been developed to facilitate their study. Large-scale expansion of these spheres can be used to study the unique molecular profile of a tumor in a particular patient, which may prove essential to effectively treat patients suffering from glial neoplasms. However, the effectiveness of whole-neurosphere characterization by immunocytochemistry is diminished because of reduced penetration of antibodies into the sphere core, the concealment of antigenic sites within the specimen, and high background fluorescence. In this report, we discussed a new strategy for the analysis of phenotypic marker antigens in both murine and human brain TSC neurospheres by using cryosectioning and immunocytochemistry. The use of this technique avoids problems with antibody penetration and allows immunophenotyping of TSCs to better profile the GBM in each particular patient.

Disclaimer

None of the authors has a financial interest in the subject matter presented herein or any of the companies mentioned.

Acknowledgments

Anne-Marie Bleau, Brian M. Howard, and Lauren A. Taylor contributed equally to this work.

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This work was supported by a grant from the American Brain Tumor Association (J.A.B.) and funds from The Starr Foundation (J.A.B.)

Article Information

Address correspondence to: John A. Boockvar, M.D, Laboratory of Translational Stem Cell Research, Weill Cornell Brain Tumor Center, Department of Neurological Surgery, Weill Cornell Medical College, 525 East 68th Street, Box 99, New York, New York 10021. email: jab2029@med.cornell.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Photomicrographs demonstrating PDGF-induced glioma in mice and derived NSC culture. Left: Staining of a PDGF-induced tumor reveals the presence of vascular proliferation and pseudopalisading necrosis. H & E, original magnification × 4. Right: The PDGF-driven tumors in mice were dissected, and cells were enzymatically dissociated with papain into a suspension that contained single cells. Neurospheres were obtained by plating the single cell suspension at a density of 5 × 104 cells/ml in neurosphere medium supplemented with EGF and b FGF for a period of 7–10 days. Original magnification × 10.

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    Immunofluorescence images obtained during immunocytochemical analysis of neurospheres derived from PDGF-driven tumors in mice. Cryosectioned neuropsheres were fixed in paraformaldehyde and stained overnight with specific stem cell marker antibodies. A: Single staining of nestin, GFAP, Musashi-1, Sox2, and Oct4 revealing expression patterns in mouse TSCs. B: Double staining showing the coexpression of stem cell markers: Oct4 with either GFAP or nestin, and Musashi with GFAP or nestin. Nuclei were counterstained with DAPI. Bar = 50 μm.

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    Left: Photomicrograph demonstrating staining of neurospheres grown from specimens from a patient with confirmed Grade IV astrocytoma. Note the hyperdensity and pleomorphism of the nuclei as well as the pseudopalisading necrosis (asterisks) and angiogenesis (arrows). H & E, original magnification ×10. Right: Brightfield microscopic image showing a neurosphere grown from the surgical specimen featured in panel A. Bar = 50 mm.

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    Immunofluorescence images revealing neurospheres derived from human GBM specimens expressing stem cell markers. As expected of slowly cycling stem cells, relatively few nuclei stained positively for Ki 67, a marker of proliferation. All Ki 67+ cells were colabeled with GFAP (A). Vimentin and GFAP were colocalized within human GBM–derived neurospheres (B), as were nestin and GFAP (C). The transcription factor Sox2 labeled the majority of nuclei within the sphere and labeled cells that were also vimentin+ (D) and nestin+ (E). Nanog+ cells were also vimentin+ (F) and nestin+ (G). Bar = 50 μm.

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