Oncogene - Isolation = of cancer stem cells from adult glioblastoma multiforme

Isolation of cancer stem cells from adult glioblastoma=20 multiforme

Xiangpeng Yuan¹,=20 James Curtin²,=20 Yizhi Xiong²,=20 Gentao Liu¹,=20 Sebastian Waschsmann-Hogiu²,=20 Daniel L Farkas²,=20 Keith L Black¹=20 and John S Yu¹

¹Maxine Dunitz Neurosurgical Institute, Suite = 800 East,=20 8631 West 3rd Street, Los Angeles, CA 90048, USA
²Department of Medicine and Surgery, = Cedars-Sinai=20 Medical Center, Los Angeles, CA, USA

Correspondence: JS Yu, E-mail: yuj@cshs.org

Received 5 October 2004; Revised=20 21 October 2004; Accepted 21 October 2004; Published = online=20 22 November 2004.

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Abstract

Glioblastoma multiforme (GBM) is the most common = adult=20 primary brain tumor and is comprised of a heterogeneous population of = cells. It=20 is unclear which cells within the tumor mass are responsible for tumor=20 initiation and maintenance. In this study, we report that brain tumor = stem cells=20 can be identified from adult GBMs. These tumor stem cells form = neurospheres,=20 possess the capacity for self-renewal, express genes associated with = neural stem=20 cells (NSCs), generate daughter cells of different phenotypes from one = mother=20 cell, and differentiate into the phenotypically diverse populations of = cells=20 similar to those present in the initial GBM. Having a distinguishing = feature=20 from normal NSCs, these tumor stem cells can reform spheres even after = the=20 induction of differentiation. Furthermore, only these tumor stem cells = were able=20 to form tumors and generate both neurons and glial cells after in = vivo=20 implantation into nude mice. The identification of tumor stem cells = within adult=20 GBM may represent a major step forward in understanding the origin and=20 maintenance of GBM and lead to the identification and testing of new = therapeutic=20 targets.

Keywords:

cancer stem cells, adult brain tumors, glioblastoma=20 multiforme, glioblastoma spheres, in vivo = implantation

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Introduction

Glioblastoma multiforme (GBM) is typically comprised of=20 morphologically diverse cells within the tumor mass. Despite current = advances in=20 therapy, the morbidity and mortality of GBM remain very high (Surawicz=20 et al., 1998). This is due, at least in part, to the focus of = most=20 treatments on the bulk of the tumor. However, the diverse cells within = GBM may=20 play different roles in tumorogenesis. There is increasing evidence that = cancers=20 might contain and arise from stem cells. Studies on leukemia and breast=20 carcinoma have demonstrated that a small group of cells within the tumor = mass is=20 responsible for tumor formation and maintenance. This subpopulation of = stem-like=20 cells plays an important role in the tumorogenic process (Lapidot=20 et al., 1994; Larochelle=20 et al., 1996; Bonnet=20 and Dick, 1997; Al-Hajj=20 et al., 2003; Dick,=20 2003). GBMs contain cells that express neural markers as well as = cells that=20 express glial markers, indicating that there may be multipotent neural = stem=20 cell-like cells (Ignatova=20 et al., 2002; Katsetos=20 et al., 2002). Such mixed glioblastomas may develop from = neural stem=20 cells (NSCs) or from differentiated cell types that acquired = multipotential stem=20 cell-like properties by either reprogramming or de-differentiating in = response=20 to oncogenic mutation (Kondo=20 and Raff, 2000; Bachoo=20 et al., 2002; Zhu=20 and Parada, 2002). This plasticity of central nervous system (CNS) = cells has=20 already been reported as normal oligodendrocyte precursor cells can be = induced=20 to acquire stem cell-like properties (Kondo=20 and Raff, 2000; Belachew=20 et al., 2003; Nunes=20 et al., 2003). Very recent studies show that pediatric brain = tumors=20 and a rat glioma cell line contain cancer stem cells that may be = important for=20 the malignancy (Hemmati=20 et al., 2003; Singh=20 et al., 2003; Kondo=20 et al., 2004). Here, we demonstrate that human adult GBMs = contain a=20 subpopulation of cells that can form neurospheres in defined stem cell = medium=20 with growth factors. The spheres share many characteristics of stem = cells,=20 including self-renewal ability and multipotent differentiation, which = can=20 produce daughter cells of all phenotypes present in the GBM. = Furthermore, we=20 show that these spheres are different from normal neurospheres, being = able to=20 reform new spheres after the induction of differentiation. More = importantly,=20 after in vivo implantation only the isolated tumor stem cells = were able=20 to form tumors that contained both neurons and glial cells. Our findings = suggest=20 that a subpopulation of cells exist within adult GBM, which may = represent a=20 general source of cancer stem cells in adult brain tumor and need to be = targeted=20 for more effective and specific cancer therapy.

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Results

Primary cultures of GBM contain 'neurospheres' forming = cells that=20 can self-renew

Phenotypically diverse cell populations exist in a = tumor=20 mass of GBM. To determine whether there is a population of cells with = distinct=20 proliferative ability characteristic of stem cells distinguishable from = other=20 cells, we studied four GBM primary cultures, GBM1=E2=80=934, grown from = frozen stocks of=20 the first passage of tumor cells, and two freshly resected GBM cultures, = GBM5=20 and 6, directly cultured without freezing. The six GBM primary cultures = were=20 grown as monolayers attached at the bottom of flasks in a medium = containing FBS.=20 We switched the GBM cells into culture conditions known to be permissive = for=20 stem cell proliferation, established previously for isolation of NSC as=20 neurospheres (Reynolds=20 and Weiss, 1992; Kabos=20 et al., 2002). Neurosphere-like colonies appeared in all six = GBM=20 cultures after 1 week and reached 100=E2=80=93200 each after 2 weeks. We = have denoted=20 these colonies as glioblastoma spheres (Figure=20 1a). These glioblastoma spheres were harvested and subjected to=20 subsphere-forming assay by limiting dilution (Kabos=20 et al., 2002). Subspheres formed in the wells seeded with = individual=20 sphere cells with efficiency between 3 and 5% for the six GBMs (Figure=20 1b and c). However, sphere formation was not evident in any wells = seeded=20 with monolayer cells. When compared with normal human NSCs that were = also grown=20 from frozen stocks with the same passage number, there were <1% cells = with=20 subsphere-forming ability (Figure=20 1c). This observation indicates that the glioblastoma spheres have a = self-renewing ability. To confirm this ability, the subspheres were = harvested=20 and a second round subsphere-forming assay was performed as above. = Again, we=20 found that individual cells formed further spheres with an efficiency = similar to=20 that of the previous assay (Figure=20 1c).

Figure=20 1.

Primary cultures of adult GBM form glioblastoma spheres that can = self-renew.=20 (a) Primary neurosphere-like colony, glioblastoma sphere, = developing from=20 monolayer tumor cells. Scale bar=3D50 m.= =20 (b) Subsphere derived from a single cell of primary sphere. Scale = bar=3D50 m.= =20 (c) Two rounds of subsphere-forming assay for glioblastoma = spheres and=20 normal human neurospheres. The data shown represent means and standard=20 deviations of triplicate experiments

Full=20 figure and legend (235K)

One mother-cell-derived glioblastoma spheres express = NSC markers=20 as well as lineage markers

The self-renewal capability of glioblastoma = spheres=20 indicates that sphere cells isolated from GBM primary cultures share = this=20 property with the NSC. We next tested whether the glioblastoma spheres = could=20 express genes characteristic of NSC. Spheres derived from a single = mother cell=20 and isolated by subsphere-forming assay were dissociated and expanded. = Two NSC=20 markers were chosen to characterize the expanded spheres: CD133, a cell = surface=20 marker of normal human NSCs (Miraglia=20 et al., 1997; Uchida=20 et al., 2000), and nestin, a cytoskeletal protein associated = with=20 NSCs and progenitor cells in the developing CNS (Lendahl=20 et al., 1990). For all six GBMs studied, daughter spheres = derived=20 from one mother cell were positive for both CD133 and nestin (Figure=20 2a). However, <0.5% cells of the non-sphere-forming tumor cells = in the=20 monolayer culture stained positive for CD133 and nestin. Some cells = within tumor=20 spheres were also found to be positive for lineage markers = -tubulinIII=20 and myelin/oligodendrocyte, but no GFAP staining was detected within = spheres (Figure=20 2a), although a few cells were positive for GFAP in dissociated = sphere=20 cells. The cells in non-sphere-forming monolayer cultures predominantly=20 expressed the three lineage markers of CNS, = -tubulinIII=20 (30=E2=80=9350%), GFAP (10=E2=80=9325%), and myelin/oligodendrocyte = (20=E2=80=9340%). The pattern was=20 clearly different from that of the glioblastoma sphere staining, it was = 5=E2=80=9315%=20 for = -tubulinIII,=20 1=E2=80=935% for GFAP, and 10=E2=80=9320% for myelin/oligodendrocyte. An = example is shown in Figure=20 2b and c. The observed phenotypic variation of stained cells within = spheres=20 from one mother cell indicates that the mother cell has the potential to = produce=20 the terminal cell types specific to the tumor tissue.

Figure=20 2.

Single-mother-cell-derived glioblastoma spheres express NSC markers = as well=20 as lineage markers, but with different staining profile from non-sphere = tumor=20 cells. (a) Glioblastoma spheres derived from a single mother cell = were=20 stained by NSC markers CD133 (red) and Nestin (green). Lineage markers = for=20 neuron, = -tubulinIII=20 (red) and oligodendrocyte, myelin/oligodendrocyte (green) were stained, = but=20 staining for the astrocyte marker, GFAP, was not observed in the = glioblastoma=20 sphere. Cells were located by counterstaining with DAPI (blue). Scale=20 bar=3D50 m.= =20 (b) Dissociated glioblastoma sphere cells show immunostaining for = various=20 markers. (c) Non-sphere monolayer cells show different staining = profile=20 from sphere cells. The data shown in (b) and (c) represent = means=20 and standard deviations of triplicate experiments

Full=20 figure and legend (273K)

One mother-cell-derived glioblastoma sphere is = multipotent

To test whether glioblastoma spheres derived from = one mother=20 cell have multipotent ability and produce progenies of different = lineages, the=20 spheres were subjected to a differentiation assay. Single-cell = suspensions of=20 glioblastoma spheres were differentiated and stained for various lineage = markers. The results demonstrate that cells differentiated from tumor = sphere=20 were positive for = -tubulinIII,=20 GFAP, and myelin/oligodendrocyte. Furthermore, a few cells were nestin = or CD133=20 positive (Figure=20 3a). For all GBM samples, the staining pattern of the differentiated = progeny=20 was similar in profile to that of primary cultured tumor cells from = which the=20 glioblastoma spheres had originally been isolated (Figure=20 3b and c). However, the phenotype pattern was different from that of = normal=20 human NSC, which was also grown from frozen stock, and cultured for the = same=20 period of time as that of glioblastoma spheres. As shown in Figure=20 3d, the differentiated progeny of normal NSCs was characterized with = 50=E2=80=9360%=20 GFAP-positive cells, 20=E2=80=9330% = -tubulinIII-positive=20 cells, myelin/oligodendrocyte-stained cells accounting for 10%, and less = than 5%=20 cells positive for CD133 or nestin, respectively. In contrast, = glioblastoma=20 spheres predominantly differentiated into = -tubulinIII=20 (80%) and myelin/oligodendrocyte (25%)-positive cells that recapitulated = the=20 parental tumor phenotype (Figure=20 3b and c). Furthermore, we found that there were approximately 5% = = -tubulinIII=20 and GFAP double-positive-stained cells only in glioblastoma=20 sphere-differentiated progenies. These results reveal that glioblastoma = spheres=20 are multipotent for the three neural cell types, but different from = normal human=20 neurospheres regarding the phenotype of their progenies.

Figure=20 3.

Glioblastoma sphere-differentiated progenies show a phenotype similar = to the=20 parental tumor, but different from that of normal human neurospheres. = (a)=20 Differentiated progenies of glioblastoma spheres were stained for the = NSC=20 markers, CD133 (red) and Nestin (green). The cells were also stained for = lineage-specific markers of neuron (red), astrocyte (green) and = oligodendrocyte=20 (green). Scale bar=3D50 m.= =20 (b) The phenotype of primary cultured parental tumor cells. = (c)=20 The phenotype of differentiated progenies from glioblastoma spheres. = (d)=20 The phenotype of differentiated progenies from human normal = neurospheres. The=20 data shown in (b=E2=80=93d) represent means and standard = deviations of=20 triplicate experiments

Full=20 figure and legend (242K)

The mRNA transcriptional levels are different between=20 glioblastoma spheres and their differentiated progeny

To better define the multipotent ability, a single = glioblastoma sphere derived from one mother cell was scaled up for=20 semiquantitative RT=E2=80=93PCR to assess the mRNA levels of lineage = marker and NSC=20 genes. Half of the spheres were directly subjected to RNA extraction. = The rest=20 was differentiated first for 2 weeks prior to RNA extraction. As shown = in Figure=20 4a as a representative, nestin mRNA was only detected in the = glioblastoma=20 sphere and not in the differentiated progeny. Conversely, the PCR = product of=20 astrocyte marker GFAP was amplified in the differentiated progeny but = not in the=20 sphere. Transcripts of neuronal and oligodendrocyte-specific genes were = elevated=20 in the differentiated progenies. These results are consistent with the = above=20 findings that glioblastoma sphere derived from one mother cell was able = to=20 generate multiple-lineage daughter cells.

Figure=20 4.

Semiquantitative RT=E2=80=93PCR and new spheres reformed from = differentiated cells.=20 (a) Semiquantitative RT=E2=80=93PCR showing mRNA transcription = levels within=20 undifferentiated glioblastoma sphere and differentiated monolayer cells. = Both=20 were derived from the same single mother cell. Lane 1: 1 kb ladder = (L);=20 lanes 2 and 6: NSC marker gene, nestin (N); lanes 3 and 7: neuron marker = gene,=20 = -tubulin=20 (T); lanes 4 and 8: oligodendrocyte marker gene, MOBP, myelin-associated = oligodendrocyte basic protein (M); lanes 5 and 9: astrocyte marker gene, = GFAP,=20 glial fibrillary acidic protein (G). A representative of three = experiments is=20 shown. (b) New spheres developed again after the previous = glioblastoma=20 spheres differentiation process. The spheres were stained for CD133 = (upper=20 panel, red) and Nestin (lower panel, green). Cells were located by=20 counterstaining with DAPI (blue). Scale bar=3D50 m<= /P>Full=20 figure and legend (150K)

Glioblastoma spheres are different from normal = neurospheres in=20 their ability to regain or maintain NSC features after = differentiation

The above results demonstrate that glioblastoma = spheres=20 possess a phenotype different from that of normal human neurospheres = upon=20 differentiation (Figure=20 3c and d). To further determine that glioblastoma spheres are cancer = stem=20 cells and not derived from contaminating normal NSC, we conducted a=20 differentiation experiment for both glioblastoma spheres and normal = human=20 neurospheres by dissociating individual spheres into a single-cell = suspension=20 and subjecting the cells to differentiation conditions. After 2 weeks of = differentiation, all cells were attached and growing as a monolayer. = Cells were=20 then switched into NSC growth medium. New spheres started to form in the = GBM-differentiated monolayers after 1 week. No spheres were observed in = the=20 normal human neurosphere-differentiated monolayer even after 4 weeks. = The newly=20 formed glioblastoma spheres stained positive for nestin and CD133 (Figure=20 4b). We also karyotyped the glioblastoma sphere cells and identified = chromosome loss, gain, and translocation events. Taken together, both = cellular=20 analysis and genetic data support our hypothesis that glioblastoma = spheres=20 isolated from primary GBM cultures are not normal NSCs migrated into the = tumor=20 mass. Rather, these glioblastoma spheres possess abnormal = characteristics=20 including enhanced self-renewal and altered differentiation abilites, = which may=20 be responsible for maintaining the tumor stem cell pool and also for = generating=20 the differentiated progeny.

Glioblastoma spheres represent the tumor stem cells and = are=20 capable of forming tumors in vivo

To address whether glioblastoma spheres and=20 non-sphere-forming monolayer cells differ in their abilities to form = tumor in=20 vivo, we implanted the isolated glioblastoma spheres or = non-sphere-forming=20 monolayer cells into the brain of nude mice. There was no tumor detected = 6 weeks=20 after 50 000 cells per mouse implantation of non-sphere-forming = monolayer=20 cells (n=3D6; Figure=20 5c). However, all the mice developed brain tumors after the same = number of=20 glioblastoma sphere cell implantation (n=3D6) (Figure=20 5a and c). We next increased the non-sphere-forming monolayer cells = to=20 250 000 and decreased the glioblastoma sphere cells to 5000 per = mouse for=20 the intracranial implantation. After 6 weeks, there was still no tumor = detected=20 in the non-sphere-forming monolayer cell-implanted mice (n=3D6), = but brain=20 tumors were detected in all mice (n=3D6) implanted with = glioblastoma sphere=20 cells, although the tumor size was smaller than that of the above (data = not=20 shown).

Figure=20 5.

Evidence of in vivo tumor-forming ability of glioblastoma = spheres. The=20 isolated glioblastoma spheres were able to form brain tumors in nude = mice after=20 intracranial implantations (a, b), but the = non-sphere-forming=20 tumor cells did not, even when implanted cells number reached 50 times = of the=20 glioblastoma sphere cells (c) by hematoxylin & eosin = staining. The=20 tumors developed in the mice brain could be stained by human-specific = antibodies=20 against NSC markers, nestin (d, e) and CD133 = (g=E2=80=93i).=20 The sections were labeled also with DAPI (blue) to identify nuclei = (f,=20 h). The merged images were also shown (i). Scale=20 bar=3D1000 m = (a, d); Scale bar=3D250 m = (b, c, e=E2=80=93i)

Full=20 figure and legend (421K)

The tumors, which developed in mice brains after = implantation of=20 human glioblastoma spheres, could be stained by human-specific = antibodies=20 against nestin (Figure=20 5d and e) and CD133 (Figure=20 5g=E2=80=93i), the NSC-related markers. Also, the tumor cells were = stained positive=20 for anti-GFAP, which was not stained in the glioblastoma spheres in = vitro=20 (Figure=20 6a=E2=80=93c), anti-myelin/oligodendrocyte (Figure=20 6d=E2=80=93f), and anti-= -tubulinIII=20 (Figure=20 6g=E2=80=93i), suggesting that the implanted glioblastoma spheres = can generate both=20 glial cells and neurons in vivo. Thus, the malignance of the = human adult=20 GBM apparently depends on the insolated glioblastoma spheres, the cancer = stem=20 cells.

Figure=20 6.

Glioblastoma spheres can generate both neurons and glial cells after=20 intracranial implantations. The tumor mass developed by intracranial=20 implantation of glioblastoma spheres contained positive stained cells = for glial=20 cell marker, GFAP (a=E2=80=93c), oligodendrocyte = (d-f), and=20 neuron marker, = -tubulinIII=20 (g=E2=80=93i). The sections were labeled also with DAPI = (blue) to identify=20 nuclei (b, e, h). The merged images were also shown = (c, f, i). Scale bar=3D250 m<= /P>Full=20 figure and legend (614K)

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Discussion

The application of principles used for studying the NSCs = to tumor=20 biology indicates a link between normal neurogenesis and brain = tumorigenesis (Holland=20 et al., 1998; 2000;=20 Holland,=20 2001; Kabos=20 et al., 2002; Ehtesham=20 et al., 2004). NSCs have the ability to generate three cell = lineages:=20 neuron, astrocyte, and oligodendrocyte. Human adult GBM also contains = all the=20 three cell types within the tumor mass. Until now, there has been no = convincing=20 evidence to show which cell type in GBM is the root for tumor growth. Is = there a=20 stem cell population present within the tumor that is responsible for = the tumor=20 growth and maintenance in GBM? Our data at least partially answered this = question. Currently, there is increasing evidence that some malignant = tumors,=20 both soluble and solid, contain cancer stem cells that are responsible = for the=20 initiation and growth of malignancy (Lapidot=20 et al., 1994; Larochelle=20 et al., 1996; Bonnet=20 and Dick, 1997; Al-Hajj=20 et al., 2003; Dick,=20 2003). Cancer stem cells are potentially important because it is = likely that=20 the stem cell population within tumor mass plays a key role for the = recurrences=20 that occur after current treatments. Thus, it is critical for cancer = therapy=20 that treatments must target and eliminate this special population of = cancer=20 cells. Consequently, the need to identify and study cancer stem cells = becomes=20 significant.

Our data indicate that human adult GBMs contain a = subpopulation of=20 cells that can self-renew and differentiate into mature cell types,=20 recapitulating the diverse complexity of primary GBMs. Several lines of = evidence=20 support that we have isolated cancer stem cells from adult human GBMs: = (1) the=20 isolated cells only account for a small fraction in tumor mass, form = spheres=20 that are morphologically indistinguishable from normal neurospheres, and = express=20 known NSC markers; (2) the cells can self-renew and proliferate to = generate=20 subspheres and different progenies; (3) the isolated single mother cell = can=20 differentiate into multi-lineage progenies; (4) spheres derived from a = single=20 mother cell possess a different phenotype compared with normal NSCs upon = differentiation, can reform spheres after the induction of = differentiation, and=20 display genetic aberrations commonly found in brain tumor cells; (5) the = isolated neurosphere-forming cells can produce brain tumors in nude = mice,=20 whereas the non-sphere-forming monolayer cells do not.

In addition to providing further evidence for the = existence of=20 cancer stem cell in solid tumor, as the presence of stem-like precursors = in=20 adult human glioblastomas has been independently reported after our work = was=20 completed (Galli=20 et al., 2004), our finding that cancer stem cells can reform = new=20 spheres after the induction of differentiation has an important = implication. It=20 may, for example, provide a basic strategy for identifying cancer stem = cells=20 from normal NSC. Although flow cytometric isolation of subpopulations of = cells=20 from tumors has been used (Al-Hajj=20 et al., 2003; Singh=20 et al., 2003; Kondo=20 et al., 2004), there is no appropriate marker for isolating = cancer=20 stem cells, especially for distinguishing cancer stem cells from NSCs. = It is a=20 fundamental step to identify and isolate cancer stem cells, so that = elucidating=20 the pathways that account for tumorigenic potential and developing = effective and=20 specific anticancer strategy may be realized. We found that glioblastoma = spheres=20 derived from a single mother cell can differentiate into the three CNS = cell=20 lineages and recapitulate the phenotypically complex property of the = parental=20 tumor. These data suggest that the presence of varied cell types within = the=20 tumor is not simply a consequence of different types of cells growing = together=20 to form tumor mass, but rather is an intrinsic property of cancer stem = cells.=20 The isolated brain tumor stem cells, not only giving rise to further = stem cells=20 but also to a diverse population of other phenotypes, may serve as a = useful=20 object for understanding the fundamental similarities and differences = between=20 normal neurogenesis and tumorigenesis in CNS.

An interesting implication arises from the ability of = one=20 glioblastoma cell to generate neurons as well as astrocytes and=20 oligodendrocytes. These cells were isolated from adults, suggesting that = there=20 are two possibilities. On the one hand, these might be converted from = normal NSC=20 by oncogenic stimulation. They were stem cells before acquiring the = tumorigenic=20 property. On the other hand, these cells might be somehow either = reprogrammed,=20 or de-differentiated from a more differentiated cell type in response to = some=20 signal transduction cascades (Kondo=20 and Raff, 2000; Bachoo=20 et al., 2002; Zhu=20 and Parada, 2002). Studying the molecular basis involved in the = oncogenic=20 conversion of stem cells or the conversion from more differentiated = cells into a=20 stem cell-like phenotype might shed light on the biology of both NSCs = and cancer=20 cells.

In conclusion, the present study highlights the = importance of=20 cancer stem cells in brain tumor research and suggests a basic strategy = to=20 identify brain cancer stem cells from normal NSCs. The study also = indicates that=20 the cancer stem cell of adult brain tumor may serve as a tool for = studying the=20 basic biology of adult stem cells.

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Materials and methods

Culture of primary glioblastoma cells and spheres

Tumor specimens were obtained within half an hour = of=20 surgical resection from six adult GBM patients, as approved by the = Institutional=20 Review Boards at the Cedars-Sinai Medical Center. Tumor tissue was = washed,=20 minced, and enzymatically dissociated (Reynolds=20 and Weiss, 1992). Tumor cells were resuspended in DMEM/F12 medium = containing=20 10% fetal bovine serum (FBS) as growth medium and plated at a density of = 2 =20 10⁶ live cells per 75 cm² flask. The cells = attached=20 and grew as a monolayer in flasks. The frozen stock tumor cells, which = were also=20 subjected to the same procedure as above and cryopreserved from = monolayer=20 cultures, were recovered into the growth medium. All the six = monolayer-growing=20 adult glioblastoma cells were switched into a defined serum-free NSC = medium (Reynolds=20 and Weiss, 1992) containing 20 ng/ml of basic fibroblast growth = factor=20 (bFGF, Peprotech, Rocky Hill, NJ, USA), 20 ng/ml of epidermal = growth factor=20 (EGF, Peprotech) and 20 ng/ml leukemia inhibitory factor (LIF,=20 Chemicon).

Subsphere-forming assay

After primary spheres formed and reached = 100=E2=80=93200 cells each=20 in the monolayer culture, the sphere cells were harvested, dissociated = into=20 single cells and plated into a 96-well plate for the subsphere-forming = assay by=20 limiting dilution as described previously (Kabos=20 et al., 2002). In brief, the cells in single-cell suspension = were=20 diluted and plated at 1=E2=80=932 cells/well. In parallel, the cell = monolayer remaining=20 in the flasks after harvesting of the spheres was also plated. Cells = were fed by=20 changing half of the medium every 2 days. After plating, the cells were = observed=20 and only wells containing a single cell were considered. The wells were = scored=20 for sphere formation after 14 days.

Differentiation assay of spheres derived from one = mother=20 cell

Glioblastoma sphere derived from one mother cell = in the=20 subsphere-forming assay was trypsinized into single-cell suspension and = seeded=20 into chamber slides (Lab-TekII, Nalge Nunc International) for = differentiation=20 assay. The cells were grown in medium devoid of growth factors but = permissive=20 for differentiation for 14 days, and processed for immunocytochemistry = (Kabos=20 et al., 2002). For the new sphere-reforming assay, the cells = were=20 switched back into NSC culture medium containing growth factors after=20 differentiation process.

Immunocytochemistry staining

To examine the expression of NSC markers and = lineage=20 markers, immunostaining was performed as described previously (Kabos=20 et al., 2002). For staining the primary cultured tumor cells = and=20 differentiated glioblastoma sphere or normal neurosphere, the cells = growing in=20 precoated chamber slides were fixed with 2% paraformaldehyde for = 15 min at=20 room temperature, treated with 10% donkey serum, and then stained with = the=20 following antibodies: anti-CD133/1 (mouse monoclonal IgG1; = 1 : 10;=20 Milteny Biotec), anti-nestin (mouse monoclonal IgG1; 1 : 200;=20 Chemicon), anti-= -tubulinIII=20 (mouse monoclonal IgG1; 1 : 200; Chemicon), anti-GFAP (rabbit=20 polyclonal; 1 : 1000; Chemicon), and = anti-myelin/oligodendrocyte=20 (mouse monoclonal; 1 : 1000; Chemicon). The primary antibodies = were=20 detected with Tex-Red or FITC-conjugated donkey anti-mouse IgG and=20 FITC-conjugated donkey anti-rabbit IgG (1 : 200; Jackson=20 ImmunoRescearch). The cells were counterstained with=20 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories) to identify = all=20 nuclei. For immunostaining of glioblastoma spheres, the spheres or = dissociated=20 sphere cells were freely floated in 'U'-bottom wells of a 96-well plate = during=20 the staining process. Then, the cells were resuspended into the mounting = medium=20 containing DAPI and mounted on microslides. Quantification of cells = positive for=20 a specific marker was carried out by counting all the stained cells = within 20=20 randomly selected microscope fields per specimen, and the percentage was = calculated based on the total number of nuclei counted.

RNA extraction and RT=E2=80=93PCR assay

Glioblastoma spheres derived from a single mother = cell were=20 expanded and differentiated as outlined previously. Spheres and = differentiated=20 progeny were respectively subjected to total RNA extraction with RNeasy = kit=20 (GIAGEN) and reverse transcripted by using a Bioscript kit (Bioline) and = Olig(dT)_{12=E2=80=9318} primer (Invitrogen). The PCR was = carried out in a=20 20 l = reaction mixture that contained 1 l = cDNA as template, specific oligonucleotide primer pairs (Table=20 1), and Accuzyme (Bioline). Each specific gene was concurrently = amplified=20 with internal control = -actin in=20 the same reaction tube. The final primer concentrations were 400 and=20 200 nM for specific gene and = -actin,=20 respectively. Cycle parameters for nestin, = -tubulinIII,=20 GFAP, myelin-associated oligodendrocyte basic protein (MOBP), and = = -actin=20 amplification were 30 s at 94=C2=B0C, 30 s at 61=C2=B0C, and = 60 s at 72=C2=B0C=20 for 25 cycles. The amplified products were identified by agarose gel=20 electrophoresis and ethidium bromide staining.

Table=20 1 - Oligonucleotides for PCR.

Full=20 table

Spectral karyotype analysis of glioblastoma sphere = cells

The spheres were cultured and dissociated as = described=20 above. The cultured cells were harvested within 5 days with Colemid = (Invitrogen)=20 for 2=E2=80=933 h, KCL treated, and fixed with = methanol : acetic acid=20 (3 : 1). The spectral karyotype analysis was performed on = fixed=20 metaphase cells according to the manufacturer's instruction (ACI, = Carlsbad, CA,=20 USA). Spectral images were acquired and analysed.

Implantation into nude mice

Athymic nude mice (nu/nu; 6=E2=80=938 weeks = old; Charles=20 River Laboratories, Wilmington, MA, USA) were anesthetized with i.p. = ketamine=20 and xylazine, and stereotactically implanted with either isolated = glioblastoma=20 sphere cells (5000; 50 000 per mouse) or non-sphere-forming = monolayer cells=20 (50 000; 250 000 per mouse) in 2.5 l = of=20 1.2% methylcellulose/PBS in the right striatum. The experiment was = repeated once=20 with identical situations. The implanted mice were killed after 6 weeks = by=20 intracordic perfusion=E2=80=93fixation with 4% paraformaldehyde and = examined for tumors=20 on the brain sections as described below. All of the animals used were=20 experimented in strict accordance with the Institutional Animal Care and = Use=20 Committee guidelines in force at the Cedars-Sinai Medical Center.

Hematoxylin=E2=80=93eosin and immunohistochemistry = staining of brain=20 sections

The tumor-cell-implanted mice brains were = postfixed after=20 the perfusion=E2=80=93fixation with 4% paraformaldehyde, and cut with a = vibratome into=20 40 m = coral sections. For hematoxylin=E2=80=93eosin staining, brain sections = were mounted on=20 slide and stained with Harris hematoxylin for 2 min first, and then = counterstained with alcoholic eosin. To characterize the brain tissue by = immunohistochemistry, free-floating sections were treated with 10% = donkey serum=20 (sigma) for 30 min at room temperature and then stained with = primary=20 antibodies for human-specific anti-nestin (mouse monoclonal IgG1;=20 1 : 200; Chemicon), anti-CD133/1 (mouse monoclonal IgG1;=20 1 : 10; Milteny Biotec), and both human and mice reactive = anti-= -tubulinIII=20 (mouse monoclonal IgG1; 1 : 200; Chemicon), anti-GFAP (rabbit=20 polyclonal; 1 : 1000; Chemicon), and = anti-myelin/oligodendrocyte=20 (mouse monoclonal; 1 : 1000; Chemicon) antibodies. The primary = antibodies were detected as described above.

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References

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Acknowledgements

This work was supported in part by NIH grants 1K23 = NS02232 and=20 1R01 NS048959 to JS Yu. We thank Dr Phillip H Koeffler for the critical = review=20 of the manuscript and helpful comments.

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nature jobs

Postdoctoral=20 Position
- Fox Chase Cancer Center
- Philadelphia, Pennsylvania, United States, 19111
Senior=20 DMPK scientist
- Cancer Research Technology (CRT)
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Original Paper

Isolation of cancer stem cells from adult glioblastoma=20 multiforme

Abstract

Keywords:

Introduction

Results

Primary cultures of GBM contain 'neurospheres' forming = cells that=20 can self-renew

Figure=20 1.

One mother-cell-derived glioblastoma spheres express = NSC markers=20 as well as lineage markers

Figure=20 2.

One mother-cell-derived glioblastoma sphere is = multipotent

Figure=20 3.

The mRNA transcriptional levels are different between=20 glioblastoma spheres and their differentiated progeny

Figure=20 4.

Glioblastoma spheres are different from normal = neurospheres in=20 their ability to regain or maintain NSC features after = differentiation

Glioblastoma spheres represent the tumor stem cells and = are=20 capable of forming tumors in vivo

Figure=20 5.

Figure=20 6.

Discussion

Materials and methods

Culture of primary glioblastoma cells and spheres

Subsphere-forming assay

Differentiation assay of spheres derived from one = mother=20 cell

Immunocytochemistry staining

RNA extraction and RT=E2=80=93PCR assay

Table=20 1 - Oligonucleotides for PCR.

Spectral karyotype analysis of glioblastoma sphere = cells

Implantation into nude mice

Hematoxylin=E2=80=93eosin and immunohistochemistry = staining of brain=20 sections

References

Acknowledgements

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