Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro

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GLIA 39:193–206 (2002)

Human Cortical Glial Tumors Contain Neural Stem-Like Cells Expressing Astroglial and Neuronal Markers In Vitro TATYANA N. IGNATOVA,1 VALERY G. KUKEKOV,1 ERIC D. LAYWELL,1 OLEG N. SUSLOV,1 FRANK D. VRIONIS,2 AND DENNIS A. STEINDLER1* 1 Departments of Neuroscience and Neurosurgery, McKnight Brain Institute and Shands Cancer Center, University of Florida, Gainesville, Florida 2 NeuroOncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida

KEY WORDS

neural stem cell; glial tumors; neural cell diversity; serum and anchorage withdrawal; pleiotropic growth factors; nestin; Survivin; Delta; Jagged

ABSTRACT Neural stem cells from neurogenic regions of mammalian CNS are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellulose and the pleiotropic growth factors EGF, FGF2, and insulin. The aim of this study was to test whether cortical glial tumors contain stem-like cells capable, under this culture system, of forming clones showing intraclonal heterogeneity in the expression of neural lineage-specific proteins. The high frequencies of clone-forming cells (about 0.1–10 ⫻ 10⫺3) in clinical tumor specimens with mutated p53, and in neurogenic regions of normal human CNS, suggest that the ability to form clones in this culture system is induced epigenetically. RT-PCR analyses of populations of normal brain- and tumorderived sister clones revealed transcripts for nestin, neuron-specific enolase, and glial fibrillary acidic protein (GFAP). However, the tumor-derived clones were different from clones derived from neurogenic regions of normal brain in the expression of transcripts specific for genes associated with neural cell fate determination via the Notch-signaling pathway (Delta and Jagged), and cell survival at G2 or mitotic phases (Survivin). Moreover, the individual glioma-derived clones contain cells immunopositive separately for GFAP or neuronal ␤-III tubulin, as well as single cells coexpressing both glial and neuronal markers. The data suggest that the latent critical stem cell characteristics can be epigenetically induced by growth conditions not only in cells from neurogenic regions of normal CNS but also in cells from cortical glial tumors. Moreover, tumor stem-like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the developmental pathological heterogeneity of glial tumors. GLIA 39:193–206, 2002. ©

2002 Wiley-Liss, Inc.

INTRODUCTION Within the neurogenic subependymal zone (SEZ) and hippocampus throughout life, multipotent glial cells have been found to play a role in persistent neurogenesis (Doetsch et al., 1999; Seri et al., 2001). We have previously demonstrated that neural stem cells (NSCs) from these neurogenic regions are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellylose (MC) and the pleiotropic growth factors epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and insulin (Kukekov et al., 1997, 1999). We have shown also that astrocytes from different brain ©

2002 Wiley-Liss, Inc.

areas during a critical period in development express specific attributes of NSCs (Laywell et al., 2000). Proliferation and differentiation potential of progenitor

T.N. Ignatova and V.G. Kukekov contributed equally to this study. Grant sponsor: NIH/NINDS; Grant number: NS37556; Grant sponsor: the McKnight Brain Institute; Grant sponsor: Department of Neurosurgery and Shands Cancer Center. *Correspondence to: Dr. Dennis A. Steindler, McKnight Brain Institute, Shands Cancer, and Program in Stem Cell Biology, University of Florida, 100 S. Newell Drive, P.O. Box 100244, Gainesville, Fl 32610. E-mail: [email protected] Received 18 December 2001; Accepted 8 April 2002 DOI 10.1002/glia.10094 Published online 11 June 2002 in Wiley InterScience (www.interscience.wiley. com).

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cells and NSCs can be modulated by both endogenous and environmental factors. FGF2, in concert with EGF under serum withdrawal in defined medium, has been shown to promote proliferation/differentiation of different NSCs (Vescovi et al., 1993, 1999; Gritti et al., 1999; Tropepe et al., 1999). FGF2 is able to activate a latent neurogenic program in cells derived from different adult CNS regions, including the cerebral cortex (Palmer et al., 1999). Another pleiotropic growth factor, insulin-like growth factor-1 (IGF-1), can also promote neurogenesis (O’Kusky et al., 2000). NSCs from the normal developing and adult CNS exhibit conserved responses to various endogenous and environmental stimuli, resulting in the induction and activation of proteins responsible for cell proliferation/differentiation. It is not known whether NSC plasticity may also be involved in abnormal cell proliferation/differentiation accompanying CNS hyperplasia (e.g., gliomagenesis). Malignant gliomas are the most common primary tumors of the adult human brain. They include highgrade anaplastic astrocytoma (AA; World Health Organization grade III) and glioblastoma multiforme (GBM; grade IV). Heterogeneity of high-grade glial tumors and their tendency toward fast malignant progression is coupled with the ability of glioma cells to migrate away from a tumor mass into normal brain tissue where they generate multiple new foci and recurrent growth. Glial tumors have been shown to be associated with expression of markers specific for primitive, undifferentiated neural cells (Dahlstrand et al., 1992; Noble and Mayer-Proschel, 1997; Barnett et al., 1998; Toda et al., 2001). The expression of nestin, a cytoskeletal protein associated with NSCs and progenitor cells in the developing CNS (Lendahl et al., 1990), with tumors experimentally induced by PDGF-B viral gene transfer (Uhrbum et al., 1998), and with reactive astrocytes (Lin et al., 1995), argues that nestin-positive cells may be somehow involved in gliomagenesis (Holland et al., 2000). The present study focuses on adult human malignant gliomas from the cerebral cortex in order to determine whether these tumors contain cells capable of forming in vitro neurosphere clones (Reynolds and Weiss, 1992), as do normal adult human NSCs (Kukekov et al., 1999). Our data show that, first, this culture system allows the isolation of clonogenic cells not only from normal adult human brain, but also from cortical gliomas with an abnormal p53 status, and with remarkably similar epigenetic frequencies (about 0,1-10 ⫻ 10⫺3). Second, clonogenic cells derived from gliomas can give rise to cells that express both neuronal and glial cytoskeletal markers. These cells, referred to here as “stem-like,” are therefore similar to NSCs from neurogenic regions of normal human brain when grown in this culture system, though the tumor-derived clones sometimes exhibit ambiguous phenotypy. Third, progeny of clonogenic cells derived from tumors with altered p53 status also reveals an epigenetic modulation in their expression of particular developmentally regulated

genes, including Delta, Jagged, and Survivin, known to be involved in cellular proliferation versus apoptotic and differentiation choices (Li et al., 1998; Miele and Osborne, 1999).

MATERIALS AND METHODS Clinical Specimens of Human Glial Tumors and Normal CNS This study was performed on clinical biopsy samples isolated from the SEZ and hippocampus of patients undergoing temporal lobectomy for the treatment of intractable epilepsy (n ⫽ 3; N1–N3) and clinical specimens of human anaplastic astrocytoma and recurrent malignant GBM (n ⫽ 10; T1–T10), diagnosed histologically. Specimens were obtained in accordance with institutional review board protocols through the University of Tennessee at Memphis, and Methodist and Baptist Memorial Hospitals, Memphis, Tennessee.

Preparation of Single Cell Suspensions and Cloning in Methyl Cellulose The procedures were performed as previously described (Kukekov et al., 1997, 1999), with some additional modifications. Briefly, approximately 10 mm3 of each tissue sample was minced, transferred to a beaker containing 0.25% trypsin in 0.1 mM EDTA (4:1), and slowly stirred on a magnetic plate at room temperature (RT) for 15 min. After trituration through a plastic 5 ml pipette and a fire-polished Pasteur pipette, the dissociate was washed 5– 8 times in 5 ml of medium to eliminate cellular debris. The resultant suspension was filtered through sterile gauze, verified visually to contain only single cells, and counted with a hemocytometer. Finally, cells were resuspended in 0.8% MC-based medium (StemCell Technologies, Vancouver, Canada) and plated at clonal density (105 cells per well) in six-well plates (Corning) coated with a nonadhesive substrate, poly 2-hydroxyethyl methacrylate (Sigma P-3932), as recommended by the manufacturer. Fresh aliquots of EGF and FGF-2 were added to a concentration of 10 ng/ml every 3 days during the 14 –18 days of clone generation. Cells were maintained in a 37°C incubator with 95% air, 5% CO2, and 100% humidity. Experimental Design The general paradigm used in this study is shown schematically in Figure 1. Normal brain and tumor biopsy dissociates were used to generate suspended clones under anchorage and serum withdrawal in semisolid MC (Eaves, 1995) with pleiotropic growth factors. The clones became visible under an inverted phase microscope about 2 weeks after plating. One half of the

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Fig. 1. The experimental paradigm used in this study. Single cell suspensions prepared from tissue specimens of normal brain or glial tumors were cloned in a culture system using MC under anchorage and serum withdrawal, in medium with the addition of pleiotropic growth factors EGF, FGF2, and insulin. A population of clones generated in MC (MC clones), or MC clones transferred to and main-

tained on coverslips coated with the adhesive substrata, poly-L-ornithyne/laminin (MC-PL clones), were used for total RNA extraction and RT-PCR. Individual sister MC-PL clones attached to substrata were processed for immunolabeling with antibodies specific for neuronal (␤-III tubulin) or astroglial (GFAP) differentiation markers.

suspended clones were collected and used for the extraction of total RNA. The other half of suspended clones were individually removed from MC and transferred in a drop of DMEM/F12 medium, with 0.5% serum, to glass coverslips sequentially coated with poly-L-ornithine (1 mg/cm2; Sigma P-3655) and laminin (0.5 mg/cm2; Sigma L-2020). Ten to twenty clones were transferred to each polyornithine/laminin (P/L)coated coverslip, and 12 coverslips were obtained from each of the different specimens. After 24 –36 h, coverslips were flooded with 1 ml of DMEM/F12 medium containing 0.5% fetal bovine serum (FBS) to promote differentiation. Four days later, six coverslips with the attached clones generated from each specimen were processed for immunolabeling as described below. Attached sister clones from the other six coverslips (40 – 60 clones total for each sample) were harvested and mixed together for total RNA extraction, and eventual reverse transcriptase–polymerase chain reaction (RT-PCR) analysis.

RNA Preparation and RT-PCR One hundred to two hundred suspended (MC) clones, and 40 – 69 attached (MC-PL) clones were collected and processed for total RNA extraction and RT-PCR using specific primers in the following protocol (Suslov et al., 2000). Briefly, pellets with 100 –200 MC clones, or with 40 – 60 MC-PL clones, were used for extraction of total RNA using the TRIzol reagent (Gibco-BRL 15596) for first strand of cDNA synthesis with oligo(dT)12–18 primer according to the manufacturer’s instructions (Gibco-BRL 18089-011). The RNA templates were then removed from the cDNA:RNA hybrid mixture by the addition of RNase H (2 U/ml). PCR was performed in a 30 ␮l volume which, in addition to cDNA templates in 1 ⫻ PCR buffer, also contained 0.3– 0.6 ␮M of each primer, 0.2 mM of each dNTP, 1.5 mM of MgCl2, and 1 U of Platinum Taq DNA polymerase (Gibco-BRL 10966-034). Thirty-five to fifty cycles of amplification were performed in a Peltier Thermal Cycler (PTC-200,

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IGNATOVA ET AL. TABLE 1. Primers used for RT-PCR analyses

Transcript

Accession #

nestin

X65964

tenascin

X56160

GFAP

J04569

NSE

X13120

Delta

AF003522

Jagged 1

U73936

Jagged 2

AF029778

survivin

NM001168

Position

Sequence

5⬘-716-736 3⬘-2004-1082 5⬘-5622-5645 3⬘-6125-6103 5⬘-381-403 3⬘-811-790 5⬘-787-809 3⬘-1040-1019 5⬘-530-553 3⬘-1089-1062 5⬘-2170-2194 3⬘-2753-2728 5⬘-2279-2304 3⬘-2764-2739 5⬘-48-67 3⬘-494-475

5⬘-CAGCGTTGGAACAGAGGTTGG-3⬘ 5⬘-TGGCACAGGTGTCTCAAGGGTAG-3⬘ 5⬘-CGAGCCTGCCACGGAATACACAC-3⬘ 5⬘-ACAATCCATCCACCCCCATCAGA-3⬘ 5⬘-GCTCGATCAACTCACCGCCAACA-3⬘ 5⬘-GACCTGACAGACGCTGCTGCCC-3⬘ 5⬘-CCCACTGATCCTTCCGATACAT-3⬘ 5⬘-TGCTCAGGTCAACCAGATCGG-3⬘ 5⬘-TGCTGGGCGTCGACTCCTTCAGT-3⬘ 5⬘-GCCTGGATAGCGGATACACTCGTCACA-3⬘ 5⬘-ACACACCTGAAGGGGTGCGGTATA-3⬘ 5⬘-AGGGCTGCAGTCATTGGTATTCTGA-3⬘ 5⬘-CAGTGGCTTTACTGGCACCTACTGC-3⬘ 5⬘-GGGTTGCAGTCGTTGGTATTGTGAG-3⬘ 5⬘-GCATGGGTGCCCCGACGTTG-3⬘ 5⬘-GCTCCGGCCAGAGGCCTCAA-3⬘

MJ Research) in 0.5 ml thin-wall tubes (MJ Research). PCR products were analyzed electrophoretically in 1.5%–2% gels of SeaKem/GTG agarose (FMC BioProducts, 50070), prepared with TAE buffer, supplemented with ethidium bromide for visualization. Tubes containing the PCR cocktail mixture, but no cDNA template, were used as negative control samples to monitor possible contamination of the reagents. PCR product size was verified using standard 50 bp (Gibco-BRL 10416-014) or 100 bp DNA markers (Gibco-BRL 10380012). Photographs were prepared using a UV transilluminator with a Kodak Digital Science DC120 Zoom Digital Camera and Kodak Digital Science 1D Image Analysis software. The program Oligo 5.0 was used to design all primers (Table 1) and to calculate the values of annealing temperatures. The primers for the Survivin gene were published previously (Mahotka et al., 1999). DNA sequence data for the corresponding genes were taken from GeneBank in accordance with the indicated positions and accession numbers shown in Table 1.

Immunostaining Clones derived from glioma and normal adult brain dissociates attached to P/L-coated glass coverslips (10 –20 clones per coverslip) were washed three times with Dulbecco’s PBS (DPBS) at RT, then fixed with ice-cold 4% paraformaldehyde in a DPBS solution for 15 min. The clones were then permeabilized with cold 0.5% Triton-X-100 in DPBS for another 10 min at RT, and washed with cold DPBS. DPBS supplemented with 10% FBS and 5% nonfat dry milk was used as a blocking solution (30 min, RT). After washing with DPBS 3 times, the clones were incubated at 4°C overnight in primary rabbit polyclonal antibodies against GFAP (Immunon, 460740) diluted 1:20, and/or with a primary mouse monoclonal IgG2b antibody raised against a synthetic peptide corresponding to the carboxy-terminal sequence of the human ␤-III tubulin, a neuronspecific isotype (Sigma T8660) diluted 1:1,000. After washing, the cultures were incubated at RT for 1 h with fluorescent-labeled conjugates of affinity-purified

goat antirabbit IgG (Vector) or horse antimouse IgG conjugates (Vector) diluted 1:50. Some clones were counterstained with PI or DAPI (1 ␮g/ml) in secondary antibody solution to show cell nuclei and were then examined under a Leitz fluorescence microscope.

Polymerase Chain Reaction–Single-Strand Conformation Polymorphism (PCR-SSCP) Analyses of p53 Genomic Mutations To test the p53 gene for mutations, high-molecularweight DNA was isolated from primary cultures of normal human brain (specimens N1–N3) and tumor specimens (T1–T10) with standard phenol extraction procedure and ethanol precipitation. The first 20 cycles of a conventional amplification by PCR, with primers homologous to sequences in the introns adjacent to exons 5, 6, 7, and 8 of the p53 gene (Gaidano et al., 1991), were followed by 5 additional cycles with 32PdCTP (Amersham). The labeled PCR products were denatured by heat in the presence of formamide. The single-stranded structures that emerged after cooling were resolved in gels prepared with a 2 ⫻ concentrate of an MDE Gel Solution (FMC 50620) according to the manufacturer’s instructions. The three-dimensional structures, unique to the primary normal sequences and aberrant structures specific for mutated sequences, were run with 0.6 or 1 ⫻ TBE in nondenaturing MDE gel. The sensitivity of mutation detection is high because single base changes radically alter the migration of the DNA conformomers. After drying on 3MM filter paper, the gels were exposed to X-ray films and developed using standard techniques.

RESULTS Cells From Normal Adult Brain and Cortical Glial Tumors Generate Clones Under Serum and Anchorage Withdrawal With Pleiotropic Growth Factors Normal cells in vitro require contact with an adhesive substratum (anchorage) and serum growth factors

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MULTIPOTENT GLIOMA CELLS TABLE 2. Frequency of clonogenic cells from clinical specimens of normal adult temporal lobe and cortical glial tumors Clinical specimensa N1 N2 N3 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 a b

Clones per wellb

Mean

Standard Error

Frequency (⫻ 10⫺3)

16, 20, 21, 15, 18, 19 28, 22, 21, 25, 24, 24 6, 10, 11, 5, 4, 10 70, 79, 55, 73, 92, 85 3, 3, 0, 1, 4, 8 26, 25, 19, 17, 11, 23 7, 9, 5, 10, 14, 19 8, 11, 16, 12, 6, 13 10, 18, 13, 17, 15, 14 30, 24, 36, 33, 26, 25 no clones no clones less than 50 cells/clone

18.2 23.8 7.7 75.7 3.2 20.2 10.7 11.0 14.5 29.0

0.94 0.87 1.23 5.26 1.14 2.31 2.07 1.46 1.17 1.97

3.0 3.9 1.3 12.6 0.5 3.4 1.8 1.8 2.4 4.8

N-normal brain; T-tumor plating density 6 ⫻ 104 cells/well

to survive and proliferate. Therefore, it was surprising when our earlier studies (Kukekov et al., 1997, 1999) demonstrated that the neurogenic regions of normal adult human brain contain cells capable of generating suspended clones under serum and anchorage withdrawal in MC in the presence of insulin, EGF, and FGF2. In the present study, we analyzed three clinical biopsy specimens of SEZ (from the ventricular wall of the inferior horn of the lateral ventricle) and hippocampus from adult human brain for the frequency of clonogenic cells following plating as single-cell suspensions in our culture system. We found that neurogenic regions of normal human brain obtained from three different donors (specimens N1, N2, and N3) contained clonogenic cells in our system, with frequencies of 1.3, 3.0, and 3.9 per 103 cells, respectively (Table 2). The morphology of the suspended spherical clones under the phase microscope resembled neurospheres, which consist of different numbers of round cells tightly embedded within an apparent extracellular matrix (Fig. 2, inset) (Kukekov et al., 1997, 1999). The approximate number of cells within such clones varied between 50 and 1,000. Ten clinical specimens of cortical glial tumors from adult human brain also were analyzed for the presence of cells capable of clone formation under the same culture conditions. Even though neoplastic transformation eliminates or significantly decreases cellular requirements for anchorage and serum, as a rule, not every transformed cell can survive and form clones without anchorage in MC and under serum withdrawal. The results of culturing clinical specimens of cortical glioma from adult patients also confirm the presence of cells capable of generating clones under serum and anchorage withdrawal in the presence of pleiotropic growth factors (Fig. 3, specimens T1–T7 in Table 2). The frequency of clone-forming cells retrieved from tumors varied between 0.5–12.6 ⫻ 10⫺3. The number of cells within the clones also varied between 50 and more than 2,000 cells. It is interesting that some tumors have cells that form small clones (less than 50 cells; T10), and some tumors appear to have no clonogenic cells at all (T8, T9).

The morphological features of both suspended and attached clones from normal brain, observed under the phase microscope (Fig. 2), were indistinguishable from the clones derived from tumors (compare Fig. 2 inset with Fig. 3). Attached clones derived from tumor specimens T1, T2, T3, T6, and T7 exhibited multiple outgrowing cells with processes that extended outside the spherical core of the clone, which consisted of cells without discernible processes (see representative microphotographs of Fig. 3). These results suggest that our culture system allows for the isolation of clonogenic cells from normal brain and cortical glial tumors, with a similar clonogenic frequency of 0.5–10.0 ⫻ 10⫺3. It was then important to test whether the clones derived from cortical glial tumors are similar to those derived from neurogenic regions of normal brain, with respect to intraclonal neural cell lineage diversity.

Individual Normal Brain- and Tumor-Derived Clones Have Cells That Separately Express Neuronal or Glial Markers Our previous studies showed that parental clonogenic cells from neurogenic regions of normal brain specimens were capable of forming clones in this in vitro system, and separate progeny within these clones expressed neural markers specific for either neurons or glia. By these criteria, each individual parental cloneforming cell could be retrospectively identified as an NSC, or “stem/progenitor” cell [see Kukekov et al. (1999) and Scheffler et al. (1999) for additional information]. Immunolabeling of attached tumor-derived clones demonstrate that the parental clone-forming cells are capable of generating intraclonal cellular diversity, as individual progenies are either immunopositive for neuronal beta-III tubulin and negative for GFAP (␤-III tubulin⫹, GFAP⫺), or ␤-III tubulin⫺ and GFAP⫹ (Fig. 4A–E). Virtually identical results were seen in clones derived from cases T1, T2, T3, T6, and T7.

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Fig. 2. An individual, representative clone derived from normal adult brain (specimen N1) is shown under phase microscopy (inset) and after double immunolabeling with antibodies against GFAP (A) and ␤-III tubulin (B). GFAP and ␤-III tubulin labeling shows immunopositive cells within the clone as well as in cells growing out from the clone. These phenotypic markers label different cells and are not colocalized. It documents the intraclonal neural cell fate diversity and allows the suggestion that the parental clone-forming cells from neurogenic regions (hippocampus, SEZ) of normal brain are competent to divide asymmetrically and activate a neurodevelopmental program for cell fate diversification during a number of divisions in our in vitro system. Scale bar in B is 100 ␮m; in inset, 150 ␮m.

Fig. 3. The morphological features of representative clones derived from a cortical glial tumor (specimen T1), grown in MC (inset) or after transfer to P/L-coated coverslips. Cells appear to migrate away from the attached clones where they develop multiple processes. This photomicrograph documents that the T1 tumor sample contained cells competent to generate clones in this culture system and produce progeny with a specific differentiated phenotype when plated on P/L-coated coverslips. Scale bar is 150 ␮m.

At the same time, within a representative clone derived from tumor specimen T3, it is possible to recognize, in addition to cells expressing ␤-III tubulin and GFAP separately (Fig. 4C–E, short arrows and arrowhead in F and G), individual cells positive for both of these markers (e.g., Fig. 4F and G, long arrows). Such

“neurogliaform” cells (Bordey and Sontheimer, 1998; Gritti et al., 2000) have so far not been observed in our normal brain-derived clones. Unlike clones derived from normal brain, glioma-derived clones also contain cells with atypical morphologies and patterns of immunolabeling (e.g., there are large cells immunopositive

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Fig. 4. Immunofluorescence photomicrographs of representative MC-PL clones derived from different cortical glial tumor specimens (T1–T3, T6, and T7) processed for double (A, B, C, E, F) or single (B inset, D) labeling for different neural markers. The cells positive for the neuronal marker ␤-III tubulin are stained green (FITC), and the cells that expressed the astroglial marker GFAP are stained blue

(AMCA). The arrowhead in F points out a cell that is not labeled for GFAP, but is immunopositive for ␤- III tubulin (arrowhead in G). The short arrow in F points out a cell that is immunolabeled for GFAP, but immunonegative for ␤-III tubulin (short arrow in G). The long arrow in F and G points out a cell that is labeled with both immunomarkers. Scale bars are 10 ␮m (F and G) and 100 ␮m (A–E).

for neuronal ␤-III tubulin with protracted processes found in specimen T1 and T3; Fig. 4D–G). These results suggest that the parental clone-forming cell from the tumor specimens, although being competent to activate some separate latent attributes of neural developmental program in our culture system, was also

somehow aberrant comparing to normal brain-derived clones shown in Figure 2. In principle, these data show that tumor-derived cells, retrieved by their ability to survive and generate clones in our culture system, have some similarities to clonogenic cells from normal adult brain with respect

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to their ability to activate the expression of latent separate neural lineage-specific markers within individual clones, irrespective of culture condition. In other words, tumor-derived cells clonogenic in this culture system demonstrate similarities to NSCs from neurogenic regions of normal brain. At the same time, the clones derived from these stem-like tumor cells show phenotypic abnormalities that are not seen in clones derived from NSCs from normal specimens. It was therefore important to test whether normal brain-derived clones and tumor-derived clones express RNA transcripts specific for the genes that are expressed in self-renewing cells (e.g., nestin), as well as transcripts for differentiated neurons (␤-III tubulin, neuron-specific enolase) and glia (GFAP).

Populations of Normal Brain-Derived Clones and Clones Derived From Gliomas Express Nestin as Well as Phenotypic Markers of Neuronal and Glial Lineages The cytoskeletal intermediate filament protein nestin has been reported to be a developmental marker of neural cells that are capable of self-renewal, with implications toward possible roles for nestin-positive cells in brain tumorigenesis (Brustle and McKay, 1995). Nestin expression is downregulated in terminally differentiated astrocytes, but along with other developmentally regulated proteins (McKeon et al., 1991; Laywell, et al., 1999) it is upregulated in reactive astrocytes under pathological conditions, including traumatic injury (Lin et al., 1995). We previously reported (Kukekov et al., 1999) that neurosphere clones derived from normal adult CNS, and generated in this culture system, express RNA transcripts coding for nestin. In the present study, RT-PCR was performed with primers specific for nestin (Table 1) on RNAs extracted from mixtures of multiple sister clones derived from normal brain (specimen N1) and glioma (specimens T1, T7), either in suspension or attached to coverslips. The results demonstrate that all groups of clones, irrespective of their origin or anchorage, express nestin (Table 3, Fig. 5A). This finding indicates that the parental clone-forming cells from tumor specimens, clonogenic in our culture system, are similar to clone-forming cells from a representative specimen (N1) prepared from neurogenic regions of normal adult brain (e.g., temporal lobe SEZ and hippocampus) not only with regard to clone-forming ability and competence to activate neural cell lineage diversification, but also with regard to their self-renewing ability associated with the expression of nestin, a feature of NSCs and a response to altered growth conditions. We next asked whether nestin expression is correlated with the expression of genes specific for different neural lineages (astroglial and neuronal) and whether culture conditions might influence the expression of these genes in tumor- and normal brain-derived clones. RNAs extracted from a population of tumor- and normal adult CNS-derived clones were probed for the pres-

ence of transcripts for neuron- specific enolase (NSE) (Schmechel et al., 1987), and the astrocyte-specific intermediate filament protein marker, GFAP. The results show (Table 3) that the presence of nestin transcripts in RNA prepared from more than 100 clones correlates with the expression of genes specific for two different neural lineages: astroglial GFAP and neuronal NSE (in addition to ␤-III tubulin, as demonstrated with immunolabeling; Fig. 4). This analysis showed that all clones, again irrespective of their origin and growth condition, expressed transcripts associated with both astroglial and neuronal lineages. Given that the expression of genes associated with self-renewal (e.g., nestin), and with differentiation along neural lineages, may be activated in the progeny of clonogenic cells derived from glial tumors, we next addressed the question if there might be differential expression, between normal- and tumor-derived clones, of those genes known to be developmentally regulated and/or associated with cell survival and asymmetric division.

Tumor-Derived Clones, Unlike Normal BrainDerived Clones, Do Not Express Jagged-2, and They Differentially Express Delta and Survivin We focused on four developmentally regulated genes: Delta, Jagged-1-2, Survivin, and Tenascin-C (a prominent extracellular matrix protein that is expressed in normal human brain-derived clones) (Kukekov et al., 1999). The expression of Notch and its ligands Delta and Jagged-1 and -2 in neurogenic regions, including the ventricular germinal matrix of both the developing vertebrate and invertebrate brain (Weinmaster, 2000), is consistent with their involvement in asymmetrical cell division that could contribute to the activation of specific sets of genes necessary for stem/progenitor cell fate choice (Lindsell et al., 1996). The focus here on the genes Delta, Jagged-1-2, Survivin, and Tenascin-C in glioma- versus normal brain-derived clones is based on the supposition that tumor cells may possess abnormalities in the coupling of cell division with differentiation. Delta expression in tumor-derived clones, but not in normal brain-derived clones, reveals an epigenetic modulation that is dependent on culture conditions following clone formation. Tumor-derived clones in suspension were Delta-negative, whereas attached tumor clones expressed Delta as normal brain-derived clones (Table 3). All of the tumor-derived clones studied here were positive for Jagged-1 expression, similar to that seen for the normal brain-derived clones. However, tumor-derived clones were negative for Jagged-2 expression, while normal brain-derived clones were Jagged-2–positive (Table 3). As for Tenascin-C (Fig. 5B, Table 3), all clones tested here (from tumors or normal brain, suspended or attached) expressed Tenascin-C. This was not particularly surprising, since this matrix protein is prominently expressed in a variety of tumors, including gliomas (Brodkey et al., 1995; Kurpad et al., 1995; Thomas and Steindler, 1995; Bouterfa et al., 1999), developing brain and the adult SEZ

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Fig. 5. Representative results of RT-PCR analyses of clones derived from glial tumors (T7, AA; T1, GBM) and normal brain specimen N1. The transcripts specific for nestin (A), tenascin-C (B), Delta (C), Jagged-1-2 (D, E), and survivin (F) are found in RNAs extracted from

a population of sister MC clones (lanes 1, 3, and 5) or from a population of sister MC-PL clones (lines 2, 4, and 6) derived from the same specimens.

(Gates et al., 1995), as well as in normal adult human brain neurosphere clones (Kukekov et al., 1999). We chose to examine Survivin because its expression downregulated in terminally differentiated adult, or nondividing embryo cells (Adida et al., 1998), and is upregulated in cells at the G2 phase, or in mitotic cells (Ambrosini et al., 1997; Li et al., 1998; Kobayashi et al., 1999). Survivin was found to be expressed in both suspended

and attached clones derived from neurogenic regions of normal adult CNS (Fig. 5F, Table 3). In contrast, in clones derived from tumors T1 and T7, Survivin was detected only in suspended clones. At the same time, the analyses of the expression pattern of several other genes associated with apoptosis, such as the members of the Bcl-2 and IAP families (Bax, Bcl-x(L), and XIAP), showed that tumor-derived sister clones generated under serum

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IGNATOVA ET AL. TABLE 3. Expression of differentiation markers and developmentally regulated genes in clones generated from normal brain and glial tumors AA (T7)-derived clones

Nestin GFAP NSE Tenascin Delta Jagged 1 Jagged 2 Survivin

GBM (T1)-derived clones

Human Normal Brain (N1)

MC-clones (mixture of more than 100 clones)

MC-PL-clones (mixture of about 50 clones)

MC-clones (mixture of more than 100 clones)

MC-PL-clones (mixture of about 50 clones)

MC-clones (mixture of more than 100 clones)

MC-PL-clones (mixture of about 50 clones)

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Fig. 6. SSCP analyses of PCR products using primers specific for different exons of the p53 gene of DNA samples extracted from primary cultures of clinical specimens of neurogenic regions of normal adult human brain (N1–N3) and cortical glial tumors (T1–T7). A: p53 status in clinical samples of normal adult brain and glial tumors (WT, wild type; M, mutation). B: The representative aberrant conformomers, or bands, produced with the primers specific for exons 5, 6, and 7 of the p53 gene in tumor DNA samples versus the normal conformomers in normal brain specimens are shown.

and anchorage withdrawal do not exhibit the dependence on culture condition in the expression of interclonal heterogeneity. All of these genes were expressed in all of the samples studied (data not shown). To test normal brain- and tumor-derived clones for epigenetic differences in the response to growth condition, RT-PCR analyses of the Notch ligands Delta and Jagged-1-2 was performed (Figs. 5C–E). The results of these analyses indicate that tumor-derived clonogenic cells modulate epigenetic responses to growth condition that is different from the response of normal adult brain-derived cells, suggesting that normal and tumor cells may be distinguished by their capacity to couple cell division with cell fate determinations. They also suggest that tumor cells may have genetic or epigenetic alterations in the molecular mechanisms that stringently control the events at the interface of cell division, survival, and differentiation. Such a suggestion prompted analysis of p53 status in tumor specimens, since this gene is not only a well known guard of genomic stability at critical cell cycle checkpoints, but also a regulator of transcription and translation of the FGF2 gene (Sidransky at al., 1992; Galy et al., 2001).

PCR-SSCP Analyses of Status of the p53 Gene in Genomic DNA From Clinical Specimens of Normal Neurogenic Regions and Cortical Glial Tumors p53 status was examined using SSCP analysis of PCR products prepared from DNAs extracted from primary-cultured cells derived from three specimens (N1– N3) of normal human adult brain, and from seven tumor specimens (T1–T7) with the primers for separate exons (Gaidano et al., 1988). All tumor specimens tested had alterations in different exons of the p53 gene (Fig. 6). One can see that T7 (anaplastic astrocytoma) had alterations in exon 6, whereas GBM tumors T1–T6 (Fig. 6B) possessed alterations in exons 5 and 7 as well as in exon 8 (this case is not shown). Normal adult brain-derived clones do not have any alterations within these exons. These results suggest no correlation between p53 status and the observed capacity of brain- or tumor-derived clones to demonstrate intraclonal heterogeneity in the expression of neural lineage-specific markers.

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DISCUSSION In our earlier reports, the culture system exploiting serum and anchorage withdrawal in medium supplemented with MC, and the pleiotropic growth factors EGF, FGF2, and insulin, allowed us to isolate clonogenic cells from adult mouse (Kukekov et al., 1997) and human CNS (Kukekov et al., 1999). These cells proliferated to form clones, or neurospheres, which showed intraclonal heterogeneity in the expression of neural lineage-specific marker proteins, the critical feature of NSCs. The routine isolation of clonogenic NSCs from neurogenic regions of normal adult brain, or so-called brain marrow regions (Steindler et al., 1996; Scheffler et al., 1999; Suslov et al., 2000), by using this system is different from the previous approaches, which relied on monolayer cultures to generate neurospheres suspended in growth factor-containing medium, or on surfaces coated with different adhesive substances (Temple and Raff, 1985; Reynolds and Weiss, 1992; Vescovi et al., 1993; Tsai and McKay, 2000). Our in vitro system exploits the ability of stem/progenitor cells to survive and form clones under serum and anchorage withdrawal, as well as to generate neural lineage-specific diversity during successive rounds of cell division of clone formation. It is important that, in order to survive under growth conditions of this culture system, the cells must be responsive to supplements of the pleiotropic growth factors, insulin, FGF2, and EGF. This system eliminates the cells that are dependent on serum growth factors and anchorage for survival, and thus selects the cells with critical characteristics of stem/progenitor cells. The aim of this study was to test several clinical specimens of adult cortical glial tumors for the presence of cells resembling NSCs, based on the ability to form clones and activate the latent neural lineagespecific attributes in this culture system. The results demonstrate that this system allows for the retrieval of clonogenic cells not only from normal adult CNS, but also from clinical specimens of adult cortical glial tumors, as has been suggested from previous studies attempting to isolate malignant glioma cells in soft agarose (Silbergeld and Chicoine, 1997). Seven out of ten tested clinical tumor samples contain stem-like cells capable of forming clones under the growth conditions of this system, and with a frequency similar to NSCs isolated from normal samples. Furthermore, it is also important that clonal populations, which include a mixture of about 50 or 100 clones, demonstrate the presence of different transcripts specific for undifferentiated neural cells and reactive astrocytes (e.g., nestin), neurons (e.g., NSE and ␤-III tubulin), and astroglia (e.g., GFAP) revealed by RT-PCR analyses (Fig. 5, Table 3). Although these data did not allow us to evaluate the rate of interclonal heterogeneity in the expression of different neural lineage-specific transcripts, the complementary results obtained here after investigation of the individual tumor-derived sister clones immunolabeled for neural lineage-specific protein markers dem-

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onstrate that neural lineage diversification can occur within the individual clones irrespective of their origin from normal CNS or cortical tumors (Figs. 3 and 4). In addition, the data also suggest that separate, latent neuronal and astroglial attributes can be activated in clone-forming cells irrespective of origin. Thus, tumorderived cells are similar to NSCs isolated from normal temporal lobe (even though these biopsy samples are removed to treat intractable epilepsy, we regard the tissue as normal in the sense that it displays no signs of hyperplastic tendencies and appears to behave similarly to normal postmortem samples of the same areas) of adult CNS, both in this study and in our earlier reports (Kukekov et al., 1997, 1999). Altogether, the results allow us to conclude that cortical glial tumors contain cells clonogenic in this culture system, which express separate attributes of neuronal and astroglial programs, and define them as NSC-like cells. What is the genetic nature of these tumor stem-like cells? These cells might result from mutational events induced within tumor tissue in vivo, or by growth conditions in vitro. Or they may be induced by epigenetic stimulation of growth conditions and, consequently, represent epigenetic modulations of cell responses to growth conditions. In order to distinguish between these two possibilities (mutational versus epigenetic mechanisms) underlying the features of tumor stemlike cells, we investigated the frequency of clone formation and found this frequency is similar (1 ⫻ 10⫺4– 10⫺2; see Table 2). Such frequency is too high for mutational events that might have occurred in vivo or in vitro under pathological growth conditions within the glial tumor, or within neurogenic regions of brain tissue under normal physiological conditions. On the other hand, such cells could be uniquely generated under the conditions of our culture system. Mutant cells capable of survival and clone formation under the two selective conditions, withdrawal of serum and anchorage, must occur with a frequency of double mutational events, i.e., 10⫺14–10⫺12 (the frequency of a single mutational event has to be about 1 ⫻ 10⫺8–10⫺6). Therefore, the observed frequency of 10⫺4–10⫺2 favors epigenetic rather than a mutational origin of cloneforming cells. We conclude that the clonogenic cells detected under these growth conditions have been induced de novo, under instructive influences of in vivo tumor growth conditions or in an in vitro system. This in vitro culture system has recently been shown to activate transient attributes, including nestin expression and de novo appearance of clonogenic stemlike cells among mouse astrocytes during a critical period in CNS postnatal development (Laywell et al., 2000). Moreover, neural cells growing in vitro at different densities or on different surfaces in the presence of growth factors in different combinations have been shown to induce de novo appearance of cells expressing an entire myogenic differentiation program of smooth (Tsai and McKay, 2000) or skeletal (Galli et al., 2000) muscle.

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The data presented here imply that unknown molecular mechanisms can de-differentiate, or a-differentiate (i.e., expunge), the cellular differentiation program toward a more primitive status. In this respect, the previous study (Laywell et al., 2000) reinforces a notion that potential abnormal astrocytic developmental multipotency can take place under different pathological conditions, whereas induction of a myogenic differentiation program mentioned above can result from transdifferentiation events induced by in vitro growth conditions. Recent studies support the impressive epigenetic plasticity of different tissue-specific stem and stem-like cells, as well as their competence for reciprocal transitions between different embryo layers (Gussoni et al., 1999; Jackson et al., 1999; Kopen et al., 1999; Petersen et al., 1999; Brazelton et al., 2000; Lagasse et al., 2000; Mezey et al., 2000; SanchezRamos et al., 2000; Woodbury et al., 2000). One important question about tumor-derived NSClike cells is whether they have an altered stringent genetic control that couples cell fate determination with cell survival and proliferative potential, as compared to normal NSCs. We studied the expression of Delta and Jagged in order to address this question. It is well known that Notch activation by its ligands Delta and Jagged might act as a master switch controlling cell cycling, programmed apoptotic cell death, fate determination, and differentiation (Miele and Osborne, 1999). Notch signaling can vary in different cells and under different conditions. In the CNS, Notch has been suggested specifically to direct stem cells toward the glial lineage rather than simply inhibiting their differentiation and thus preserving stemness (Morrison et al., 1999; Furukawa et al., 2000). Therefore, we assumed that the regulation of Notch expression by its ligands in clonogenic tumor-derived cells might be altered and can therefore be differentially regulated by different culture conditions. Our results show that Delta and Jagged expression in glioma-derived clones is different compared to normal brain-derived clones and is dependent on particular in vitro growth conditions (Table 3, Fig. 5). Suppression of Delta expression in tumor-derived MC clones, in the presence FGF2, is consistent with the recently described expression of Delta in neuroepithelial cells under the influence of this growth factor (Faux et al., 2001). In addition to Delta and Jagged-1-2, we also explored the expression of the survivin gene, a crucial participant at the G2/M checkpoint. Survivin is a wide-spectrum antiapoptotic inhibitor, overexpressed at the Sand G2/M phases of cell cycle, and is necessary for survival and fidelity of cell division (Li et al., 1998). In addition to data that Survivin is developmentally regulated and suppressed in differentiated cells (Adida et al., 1998; Ambrosini et al., 1999), it has been recently proposed that Survivin may also contribute to the stem cell origin of colon cancer (Zhang et al., 2001). Here we show Survivin expression in suspended, tumor-derived clones is suppressed following clone attachment to the substrate (unlike that seen for attached clones from the

normal brain; Table 3, Fig. 5F). These results are consistent with an epigenetic cell cycle– dependent regulation of Survivin expression, and the possible molecular mechanisms of epigenetic control of Survivin expression, such as methylation of its promoter region, has been recently discussed (Hattori et al., 2001). Studies of Delta, Jagged-1-2, and Survivin expression suggest that the presence of NSC-like cells in glial tumors does not result from contamination of tumor sample by real NSCs from normal surrounding brain tissue, since normal NSC-derived clones have different patterns of expression of the above genes. At the same time, these NSC-like cells can be recruited from surrounding normal brain tissue and activate their latent attributes under the influence of tumors. Our previous studies showing a paucity of clonogenic cells from the normal adult human cerebral cortex (Kukekov et al., 1999) support this conclusion. We assume that an altered regulation of the expression of Delta, Jagged, and Survivin is indicative of an altered mechanism that integrates cell fate determination, survival, and cell cycle control in stem-like cells derived from glial tumors. Such alterations in stringent control events might lead to a deregulation of an entire neurodevelopmental program and contribute to the generation of developmentally abnormal heterogeneous progeny. Some progeny might exhibit ambiguous cellular phenotypes and simultaneously express markers specific for two separate neural lineages, as well as initiate anaplastic, metaplastic, and neoplastic growth. In general, phenotypes of stem-like cells derived from tumors might reflect the stages of neurodevelopment, or socalled neuropoietic hierarchy, at which alterations in cell cycle and fate choice mechanisms might have occurred. Alternatively, a genetic alteration itself might determine the proliferation and differentiation potential of the clonal progeny derived from an affected NSC-like cell. Clonal expansion of glial tumor cells has been associated with p53 status (Sidransky et al., 1992; Morse et al., 1994). It has been shown that the p53 gene plays a pivotal role at the interface between cell cycle transition, cell survival, differentiation, and genomic stability of neural cells (Sidransky et al., 1992), as well as in the clonal expansion of glial tumors (Sidransky et al., 1992). However, there is a paucity of information on the possible contribution of p53 to the stringent control of asymmetrical division of stem/progenitor cells. The wild-type p53 has been shown to inhibit cellular FGF2 production at the posttranslational (Galy et al., 2001) and transcriptional (Ueba et al., 1994) levels, whereas mutated p53 might activate the transcription of this growth factor that is known to be involved in proliferation and differentiation of NSCs. Given the instructive role of FGF2 for neurodevelopmental programs in vivo and in vitro (Vescovi et al., 1993, 1999; Gritti et al., 1999; Tropepe et al., 1999; Palmer et al., 2000), we asked whether the status of p53 in tumor specimens might correlate with the ability to form clones and generate intraclonal cell lineage diversity. We hypoth-

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esized that mutations in the p53 gene might correlate with abnormal proliferation, fate choices, and differentiation of real NSCs and tumor NSC-like cells. Mutant p53 could drive tumor progression, increasing survival rates via an escape from cell cycle arrest or apoptosis, or by promoting the clonal expansion of cells with mutated p53. The activation of FGF2 expression in cells with mutated p53 (Ueba et al., 1994), or activation of the entire FGF2 pathway in regulation for cell cycle control (e.g., via chromatin remodeling), provides a possible link between p53 status in the tumors studied here and the presence of NSC-like cells capable of forming clones and activating latent elements of neurodevelopmental lineages. It may appear to be a contradiction that both normal brain-derived cells with wild-type p53 status and glioma-derived cells with an altered p53 status are both capable of generating intraclonal neural cell lineage diversity. However, we suggest that the presence of FGF2 in our culture system, by itself, could epigenetically activate this program in normal brain-derived cells with wild-type p53 status. In conclusion, it is possible that abnormal NSC-like cells, such as pathologically damaged astroglial cells, contribute to neurodevelopmental heterogeneity and thus contribute to hyperplastic and neoplstic growth of human brain glial tumors. This conclusion is consistent with our previous studies (Laywell et al., 2000) showing the existence of normal, clonogenic, multipotent astrocytes throughout the neuraxis during CNS development, and their persistence within the adult SEZ and hippocampus shown by others (Seri et al., 2001). These data suggest that such cells can be the targets for cues prompting de-differentiation, chromosomal imbalance, and mutations that could result in abnormal proliferation and differentiation of NSC-like cells. We consider the in vitro culture system used here as a approach capable of activating latent elements of a neurodevelopmental program in competent NSCs from neurogenic regions of the normal adult CNS and in tumor NSC-like cells. In addition, this culture system affords the generation of clones that can be exploited for studying mechanisms that control normal and pathological neurogenesis. In this regard, the clones generated under this culture system might be considered as an in vitro model of normal neurogenesis and pathological gliomagenesis.

ACKNOWLEDGMENT This study is dedicated to the memory of Dr. Diane Daly Ralston.

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