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. 2008 Jul 10;19(3):399–408. doi: 10.1111/j.1750-3639.2008.00189.x

Spontaneous In Vitro Transformation of Adult Neural Precursors into Stem‐Like Cancer Cells

Florian A Siebzehnrubl 1,* ,✉, Ina Jeske 1, Dorit Müller 1, Rolf Buslei 1, Roland Coras 1, Eric Hahnen 1,4, Hagen B Huttner 2, Denis Corbeil 5, Johanna Kaesbauer 6, Thomas Appl 3, Stephan Von Hörsten 3, Ingmar Blümcke 1
PMCID: PMC8094744  PMID: 18637011

Abstract

Recent studies have found that cellular self‐renewal capacity in brain cancer is heterogeneous, with only stem‐like cells having this property. A link between adult stem cells and cancer stem cells remains, however, to be shown. Here, we describe the emergence of cancer stem‐like cells from in vitro cultured brain stem cells. Adult rat subventricular zone (SVZ) stem cells transformed into tumorigenic cell lines after expansion in vitro. These cell lines maintained characteristic features of stem‐like cells expressing Nestin, Musashi‐1 and CD133, but continued to proliferate upon differentiation induction. Karyotyping detected multiple acquired chromosomal aberrations, and syngeneic transplantation into the brain of adult rats resulted in malignant tumor formation. Tumors revealed streak necrosis and displayed a neural as well as an undifferentiated phenotype. Deficient downregulation of platelet‐derived growth factor (PDGF) receptor alpha was identified as candidate mechanism for tumor cell proliferation, and its knockdown by siRNA resulted in a reduction of cell growth. Our data point to adult brain precursor cells to be transformed in malignancies. Furthermore, in vitro expansion of adult neural stem cells, which will be mandatory for therapeutic strategies in neurological disorders, also harbors the risk for amplifying precursor cells with acquired genetic abnormalities and induction of malignant tumors after transplantation.

Keywords: adult stem cells, carcinogenesis, CNS, transplantation, tumor formation, tumorigenesis

INTRODUCTION

The cancer stem cell hypothesis is increasingly recognized as the current model to understand tumorigenesis 24, 38. In contrast to the stochastic model of tumor growth and metastasis, where basically each tumor cell is able to drive tumor growth and the likelihood of metastasis is governed by chance and probability, the hierarchical model proposes cancer stem cells as sole culprits for continued growth of tumors and the formation of remote tumor clones (48). Cancer stem cells have self‐renewal capabilities and produce more differentiated tumor cells, which continue to proliferate for a limited time period (though not indefinite) but cannot metastasize upon spreading (1). Cancer stem cells have been identified in a variety of malignancies, for example, leukemia (27), CNS tumors 22, 36, 44 as well as colorectal and prostate carcinomas 8, 33.

Repetitive isolation of stem‐like cells from brain tumors 22, 44 has reentered the intriguing discussion from which cell population these tumors derive. Stem and progenitor cells from the subventricular zone (SVZ) have long been suspected as potential sources of malignant gliomas (41). Current hypotheses favor, therefore, the susceptibility of stem cells to acquire mutations, which lead to cell cycle deregulation or dedifferentiation of more committed progenitor cells, and finally into cancer founding stem cells.

Recent discussions suggest a transforming potential of adult neural stem cells (41), a property that has already been shown for adult mesenchymal stem cells 30, 40. The platelet‐derived growth factor (PDGF) and its alpha receptor are noteworthy candidates for inducing tumorigenic transformation, as PDGF may induce transformation in vitro and in vivo 11, 23, and SVZ stem cells express the PDGF receptor alpha (23).

Here, we used an in vitro expansion culture model to analyze the transforming potential of rodent adult neural precursor cells. SVZ precursors were expanded in culture using standard paradigms. While others have very recently reported that in vitro expanded adult neural stem cells can be maintained stable for extended culture periods (15), we observed a spontaneous transformation of SVZ‐derived precursor cells already after 10 passages in vitro, and these cells were able to form tumors upon orthotopic transplantation into rodent brains.

MATERIALS AND METHODS

Isolation and expansion of rodent stem cells

Postnatal Wistar rat SVZ stem and progenitor cells were isolated and maintained as described (43). All animals were handled according to the “Principles of Laboratory Animal Care” (NIH publication No. 86‐23, revised 1985) and the German Law on the Protection of Animals. Briefly, brains were dissected and SVZs dissociated using PPD solution, consisting of Dispase II (1 mg/mL, Sigma, Schnelldorf, Germany), DNase I (0.1 mg/mL, Sigma) and papain (0.1 mg/mL, Sigma) in Hank's balanced salt solution (Invitrogen, Karlsruhe, Germany) without Ca2+. After 30 minutes incubation, tissue was mechanically dissociated by pipetting. The isolated cells were washed twice in PBS (Biochrom, Berlin, Germany) and were pelleted by centrifugation at 120 g for 10 minutes. For each obtained cell line, primary cells from five animals were pooled and plated into six‐well culture plates with DMEM/F‐12 medium (1:1, Invitrogen) containing N2 supplements and 20 ng/mL EGF and FGF2 (R&D Systems, Minneapolis, MN, USA). Neurospheres were kept at densities of 2–5 × 104 cells/cm2 and were passaged every 3–5 days as described (43), depending on sphere size and density. Growth factors were added to the cultures every third day. Every 10 passages, aliquots were cryo‐preserved and stored in liquid nitrogen.

Transplantation studies

Fifty thousand cells were transplanted into the brains of 300‐g Wistar rats at the coordinates Bregma +1.20 mm, lateral −2.5 mm, vertical −4.5 mm (34) in a total volume of 5 µL. Neurological deficits were scored every day [grade 0: normal, grade 1: tail weakness or tail paralysis, grade 2: hind leg paraparesis or hemiparesis, grade 3: hind leg paralysis or hemiparalysis, grade 4: complete paralysis (tetraplegia), moribund stage or death]. Tetraplegic animals were killed. Animals were deeply anesthetized, perfused using 4% paraformaldehyde, the brains removed and embedded in paraffin for immunohistochemistry.

Side‐population analysis

For side population analysis, cells were incubated with 2.5 µg/mL Hoechst 33342 (Sigma) for 90 minutes at 37°C. Afterwards, cells were collected by centrifugation and dissociated using Accutase (PAA, Coelbe, Germany) for 15 minutes at 37°C. The resulting single cell suspension was washed once in PBS and resuspended in ice‐cold PBS containing 2 mM EDTA. Controls consisted of Hoechst 33342 staining in the presence of 100 µM Verapamil (Sigma). Cells were analyzed on a MoFlo cell sorter (Dako Cytomation, Glostrup, Denmark).

Immunocytochemistry

Immunostaining of fixed cells or tissues was performed as described (43). The following primary antibodies and dilutions were used: mouse anti‐p21 (1:30, Dako, Glostrup, Denmark), rabbit anti‐Ki‐67 (1:300, Chemicon, Chandlers Ford, UK), mouse anti‐βIII tubulin (1:2000, Promega, Mannheim, Germany), rabbit antiglial fibrillary acidic protein (1:1000, GFAP, Dako), rabbit anti‐CD133 (prominin‐1; 1:2000, clone K8) 9, 10, mouse anti‐Sox2 (1:600, Chemicon), rabbit anti‐Musashi‐1 (1:200, Chemicon), mouse anti‐Nestin (1:400, Chemicon) and mouse anti‐platelet‐derived growth factor (PDGF) receptor (1:50, Chemicon).

Secondary antibodies were used at 1:200 dilutions (goat antimouse Alexa488 and goat anti‐rabbit Alexa555, Invitrogen). For nuclear counterstaining, Hoechst 33342 (500 ng/mL, Sigma) was used. Coverslips were mounted using Gel Mount (Sigma). No fluorescence signal was detectable when omitting primary antibodies.

Immunohistochemistry

Immunostaining of 4% paraformaldehyde‐fixed, paraffin‐embedded tissue was performed as described elsewhere (7). Immunohistochemical reactions were performed using an automated staining apparatus and the streptavidin‐biotin method (Ventana, Strasbourg, France) using 3,3′‐diaminobenzidine as chromogen and hematoxylin counterstaining. The following antibodies were used: mouse anti‐Map2 (1:25, clone C, kind gift of Dr B. Riederer, Lausanne, Switzerland), mouse anti‐βIII tubulin (1:2000), mouse anti‐GFAP (1:2000, Dako), mouse anti‐Nestin (1:400, Chemicon), mouse anti‐p21 (1:30, Dako) and rabbit anti‐Ki67 (1:300, Chemicon).

RNA extraction and real‐time PCR

Extraction of RNA was performed as described (6). For cDNA synthesis, the SuperScript II first‐strand synthesis kit (Invitrogen) was used according to the manufacturer's instructions. PCR reactions were performed in a total volume of 10 µL using 20 ng of total cDNA in a PCR buffer containing 1.5 mM MgCl2 (Qiagen, Hilden, Germany), 5 pmol of each primer, 200 µM of each dNTP and 0.25 U Taq polymerase (Qiagen). The cycling conditions were 40 cycles of denaturation at 94°C for 35 s, annealing at 55°C for 40 s and extension at 72°C for 40 s. Quantitative real‐time PCR reactions were performed using the Power SYBR green PCR master mix (Applera, Darmstadt, Germany) according to the manufacturer's instructions. Cycling conditions for real‐time PCR were 40 cycles of denaturation at 95°C for 30 s, followed by annealing at 60°C for 60 s and extension at 72°C for 30 s. Primer sequences will be provided upon request. The comparative method of relative quantification (2−ddCt) was used to calculate the relative expression levels of each target gene (normalized to GAPDH). RT‐PCR specificity of each reaction was verified by melting curve analysis.

Chromosome preparation

Chromosome preparation was performed as described (49). Briefly, cells were grown as monolayer in 5 mL of N2/10% fetal calf serum (FCS) (see above), treated with 100 µL Colcemid (10 µg/mL) (Roche, Mannheim, Germany) for 120 minutes at 37°C. The cells were detached by treatment with trypsin–EDTA (Biochrom) and centrifuged. The cell pellet was suspended in hypotonic solution (75 mM KCl) for 20 minutes at 37°C, centrifuged, and fixed in methanol and acetic acid. Metaphase spreads were prepared on slides, dried and Giemsa stained after trypsin pretreatment.

siRNA transfection and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenoltetrazoliumbromide (MTT) assay

siRNA molecules targeted against the rat PDGFRα sequence (XM_001067574) were obtained from Qiagen (sequences available upon request). To test cell growth reduction, R2303 cells were seeded at 5000 cells/well in 96‐well plates and were transfected with 5 nM of PDGFRα siRNAs using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Controls consisted of cells incubated with Lipofectamine only and cells mock transfected with oligonucleotides bearing no sequence similarity to any known rodent gene. Cell viability was analyzed 7 days after transfection using the MTT assay as described (21).

The PDGFR‐specific receptor tyrosine kinase inhibitor (RTKI) 4‐(6,7‐dimethoxy‐4‐quinazolinyl)‐N‐(4‐phenoxyphenyl)‐1‐piperazinecarboxamide was obtained from Calbiochem (Darmstadt, Germany). RTKI‐induced cell growth reduction was assayed by MTT. Data were analyzed using GraphPad Prizm Version 4.00 (GraphPad Software, San Diego, CA, USA). Data were fitted to a four‐parameter logistic equation comprising the top plateau, bottom plateau, inflection point IC50 and curve slope n H. The parameters “bottom,” IC50 and n H were set as variables; “top” was the control value of cell viability and was set constant = 100%.

Western blotting

R2303 cells were withdrawn from serum and growth factors for 48 h. Following treatment with PDGF‐AA (R&D Systems), cells were lysed on ice using RIPA buffer. Protein concentrations were determined and equal amounts of proteins were loaded on a Bis‐Tris 4%–12% gradient gel for SDS‐PAGE (Invitrogen) and were separated at 200 V for 45 minutes, followed by transfer on a 0.2 µm nitrocellulose membrane (Invitrogen). The membrane was blocked with 5% milk in tris‐buffered saline (TBS) containing Triton X‐100 for 1 h at room temperature. The following primary antibodies were incubated overnight at 4°C: rabbit anti‐PDGFRa (1:300, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and goat anti‐PDGFRa‐phospho Y720 (1:300, Santa Cruz). Secondary antibodies were horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit (1:5000, Invitrogen) and bovine antigoat (1:5000, Santa Cruz). For detection, enhanced chemoluminescence (ECL) reagents were used (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

RESULTS

Generation of SVZ precursor cell lines

Using a neurosphere culture model in which cells were seeded at a density of 20 000–50 000 cells/cm2 into anti‐adhesive cell culture plates, we were able to expand adult rodent SVZ precursor cells with two passages per week. During the first eight passages, all spheres showed characteristics of neural precursor cell cultures, that is, multipotent differentiation [data not shown; see Siebzehnrubl et al (43)]. Between passages 8 and 10, cultured cells changed their morphological appearance and formed spheroids resembling cellular aggregates rather than genuine neurospheres, which are a more compact mass of cells (Figure 1A). This was accompanied by their ability to expand also under differentiation conditions, that is, growth factor (EGF, FGF2) withdrawal in the presence of serum (see Figure S1). However, the expressions of neuronal (βIII tubulin, Map2) and glial markers (GFAP) were unaffected under differentiation conditions (Figure 1A). We continued passaging the derived cell line (designated “R2303” henceforth) for more than 110 passages without a decline in the doubling rate of approximately 3 days. Repeating the above‐specified experimental approach in an independent setting generated another cell line (termed “R2902”).

Figure 1.

Figure 1

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Generation of cell lines. A. Expansion of subventricular zone (SVZ) precursor cells over 10 passages in vitro resulted in morphologically homogenous cultures (termed R2303 and R2902) that presented as spheroid aggregates in suspension cultures. Scale bar: 50 µm. Plating R2303 and R2902 spheres onto Laminin/poly‐ornithine coated surface and growth factor withdrawal resulted in differentiated phenotypes that are immunopositive for neuronal (βIII tubulin) and glial (GFAP) markers. While most cells expressed either neuronal or glial markers, some cells were positive for both (arrowhead), indicating a possible transition phenotype (28). Furthermore, R2303 cells express stem cell markers Sox2 and CD133 in culture. Scale bar: 50 µm. B. RT‐PCR for stem cell‐associated genes revealed that R2303 and R2902 cells express Nestin and Musashi‐1 (MSI1H) mRNA in vitro. Expression of both genes can be detected under expansion (ie, sphere culture, Exp) and differentiation (ie, growth factor withdrawal and serum addition, Diff) conditions. C. Side‐population analysis. R2303 cells were stained with Hoechst 33342 and were analyzed on a MoFlo Cell Sorter. A significant side population consisting of 0.3% of all cells was found (100 000 events analyzed). The side population could be suppressed by Verapamil treatment.

R2303 cells were more extensively characterized, but both cell lines can be expanded indefinitely and express neural stem cell markers (ie, Sox2, Musashi‐1, CD133/prominin‐1 and Nestin) under expansion conditions and neuronal and glial markers (ie, βIII tubulin, Map2, GFAP) under differentiation conditions (Figure 1A). Moreover, RT‐PCR revealed expression of neural stem cell‐associated genes in both cell lines (ie, Nestin and Musashi‐1). However, expression of these stem cell markers continued under differentiation conditions (Figure 1B). Immunostaining for Nestin and Musashi‐1 showed that only a subpopulation of morphologically different cells continue to express Musashi under differentiation conditions (Figure S2).

The existence of a “side population” has been confirmed in both adult and cancer stem cells 20, 25. Its higher drug efflux ability is caused by ABCG2 drug transporter expression (20), resulting in low staining capacity for the cell‐permeable DNA binding dye Hoechst 33342. Fluorescence‐activated cell sorting (FACS) analysis of R2303 cells detected a significant side population, which could be blocked using Verapamil, an ABCG2 inhibitor (Figure 1C).

Chromosomal analysis

We performed chromosomal analyses for both cell lines at different passages. These analyses revealed multiple numeric and structural chromosomal aberrations and overall genomic instability (Figure 2). Eighty percent (R2303) and 52% (R2902) of analyzed metaphases had 38 chromosomes compared to 42 for normal rats. While a high‐grade chromosomal mosaicism was found, some chromosomal aberrations were present in all mitoses analyzed. These included derivative chromosomes 6, 11 and 14 as well as an isochromosome 2. R2902 cells also showed a trisomy 7, which was not found in R2303 cells. Of note, both cell lines showed one derivative chromosome 14 (inset, Figure 2).

Figure 2.

Figure 2

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Karyotyping of cell lines. Karyotyping of R2303 and R2903 cells revealed multiple structural and numeric chromosomal aberrations. All metaphases analyzed in both cell lines had an isochromosome 2 and derivative chromosomes 6, 11 and 14. Several metaphases showed further derivative chromosomes 8, 10, 13 and 15 compatible with a high‐grade mosaicism. Some chromosomal derivatives could not be identified. Inset: Pairwise comparison of chromosome 14 (harboring the gene locus of PDGFRα) from different metaphases. In each sample, derivation in the short arm of one chromosome was recognized.

Transplantation of R2303 cell line into rat brain

We transplanted 50 000 R2303 cells into the right striatum of syngeneic and immunocompetent male adult rats. Extensive tumor masses were recognized in four out of five transplanted animals. Histopathological examination of these tumors revealed streak necrosis, invasive growth (Figure 3A) and expression of neuronal (Map2ab, βIII tubulin) as well as undifferentiated (Nestin) markers (Figure 3B). No GFAP immunoreactivity was observed in these cases. These features are similar to other experimentally induced gliomas 16, 42, 47.

Figure 3.

Figure 3

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Tumor formation after R2303 transplantation. A. Transplantation of 50 000 R2303 cells into the right striatum of adult male Wistar rats resulted in the formation of solid tumors (27 days post transplantation of a representative tumor depicted). (a) higher magnification (12.5×) of the upper inset reveals invasion into surrounding brain parenchyma (arrows), and (b) higher magnification (40×) of the lower inset reveals streak necrosis (asterisk). B. Immunohistochemistry shows the expression of neuronal (Map2, 200× and βIII tubulin, 100×) and undifferentiated markers (Nestin, 12.5×).

Further immunohistochemical analysis identified two distinct populations of tumor cells, one characterized by nuclear accumulation of p21, the other expressing the proliferation marker Ki‐67 (Figure 4A,B). Double fluorescence staining confirmed that both populations are mutually exclusive (Figure 4C). We detected no nuclear accumulation of p53 or Olig2 (data not shown).

Figure 4.

Figure 4

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Proliferation and cell cycle arrest within R2303‐derived tumors. A. Staining for the proliferation marker Ki‐67 revealed a high proliferative index of R2303‐derived tumors, compatible with high malignancy (200×). B. A population with nuclear accumulation of the cell‐cycle regulator p21Waf1/cip was revealed by immunohistochemistry. Nuclear p21 accumulation normally results in cell cycle arrest (200×). C. Fluorescence immunostaining for p21 (green) and Ki67 (red) showed both populations to be mutually exclusive, pointing to a proliferative and quiescent subpopulation of tumor cells (400×). Blue: nuclear counterstaining (Hoechst).

Gene expression analysis

To further characterize cell lines R2303 and R2902, we studied gene expression of oncogenes and tumor suppressor genes frequently associated with malignant brain tumors, including EGFR, Olig2, p21, p53, PDGFRα and PTEN. The rationale was based on previous chromosomal analysis identifying prominent structural and numeric aberrations (see above). In addition, EGFR and PFGFRα are located on rat chromosome 14, which was found aberrant in all studied metaphases. All target genes listed above were expressed in R2303 and R2902 cell lines (data not shown). Quantitative real‐time PCR identified significantly different expression levels only for p21 and PDGFRα when comparing R2303 and R2902 cell lines to syngeneic primary SVZ precursors. Significant upregulation of p21 was detected for R2303 and R2902 cells under differentiation conditions (P < 0.001, two‐way ANOVA, data not shown). Furthermore, we observed PDGFRα downregulation during differentiation of normal rodent precursor cells, while expression levels remained unchanged in R2303 and R2902 cell lines under expansion or differentiation conditions (P < 0.001, two‐way ANOVA; Figure 5A). Prominent PDGFR staining can also be detected immunohistochemically in R2303 transplanted tumors (Figure 5B). RT‐PCR revealed that both cell lines and normal precursor cells express the growth factor subunits PDGF‐A and ‐B in vitro (Figure 5C). PDGFRα ligands include PDGF‐AA, ‐AB and ‐BB (4).

Figure 5.

Figure 5

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Functional analysis of transformed cell lines. A. Quantitative real‐time PCR of normal neurospheres [non‐transformed precursor cells (NPC)] as well as R2303 and R2902 cells under expansion (Exp, sphere culture) and differentiation (Diff, growth factor withdrawal and serum addition) conditions revealed differences in PDGFRα expression levels (normalized to expansion conditions). Normal subventricular zone (SVZ) precursor cells downregulate the PDGFRα upon induction of differentiation (P < 0.001, two‐way ANOVA), while both R2303 and R2902 cells show no significant differences in expression of PDGFRα. Target gene expression was normalized to the housekeeping gene GAPDH. B. Fluorescence immunostaining for PDGFR (green) within R2303‐induced tumors revealed prominent expression of this receptor (400×). C. RT‐PCR for PDGF‐A and ‐B revealed that both subunits are expressed by R2303 and R2902 cells as well as normal precursor cultures (NPC), but PDGF‐B is downregulated during expansion conditions (Exp). D. siRNA knockdown of the PDGFRαin vitro as well as treatment with a PDGFRα‐specific receptor tyrosine kinase inhibitor (RTKI) resulted in growth inhibition of R2303 cells. Data are from a cell viability assay [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), n = 8]. Control: R2303 cells incubated with lipofectamine. Mock: R2303 cells transfected with nonsense oligonucleotides. RNA1 and RNA2: R2303 cells transfected with siRNA directed against different parts of the PDGFRα sequence. Asterisks indicate statistical significance (Student's t‐test, P < 0.01). A PDGFRα RTKI reduced cell growth dose‐dependently. Depicted is a best fit curve for the induction of growth inhibition in R2303 cells. Ordinate: cell viability normalized to untreated controls (100%). Abscissa: RTKI log concentration (mol/L). E. Immunofluorescence staining for PDGFR (100×). R2303 cells were either mock or PDGFRα‐siRNA transfected and immunostained for PDGFR 96 h after transfection. F. Western blot for phosphorylation (Tyr 720) of PDGFRα. R2303 cells were starved from serum and growth factors for 48 h, followed by treatment with PDGF‐AA for the indicated time. A PDGFR antibody demonstrates equal loading, while the phosphorylation of Tyr 720 increases after exposure to PDGF‐AA. G. PDGF‐AA increases the proliferation rate of R2303 cells. Five hundred thousand cells were plated in a medium deprived of serum and growth factors and treated with vehicle (control), PDGF‐AA or a specific inhibitor against the PDGFRα (RTKI). Although R2303 cells continued proliferating in serum and growth factor‐free medium, PDGF‐AA significantly increased cell proliferation (one‐way ANOVA, P < 0.005), while a PDGFR inhibitor reduced proliferation.

Functional analysis of PDGFRα signaling pathway

To analyze whether the continued expression of PDGFRα is required for the proliferation of cancer stem‐like cells in culture, we performed a knockdown experiment for the PDGFRα in R2303 cells. Five thousand cells were seeded into 96‐well plates and transfected with 5 nM of two different siRNA molecules designed against rat PDGFRα. R2303 cells transfected with oligonucleotides with no sequence similarity to any known gene were used as controls. Cell growth was quantified using the MTT assay (31) 7 days after transfection. Cell viability compared to mock transfected controls was decreased by 50% and 85%, respectively, in siRNA‐transfected cells (P < 0.01, t‐Test; Figure 5D). This was accompanied by a reduction of protein expression, as revealed by immunofluorescence staining (Figure 5E). Furthermore, a specific inhibitor of the PDGFα receptor tyrosine kinase was able to inhibit growth of R2303 cells in low micromolar concentrations (IC50 = 1.4 µM, Figure 5D). Western blotting revealed active phosphorylation of the PDGFRα after stimulation with exogenous PDGF‐AA (Figure 5F). When R2303 cells were cultured with exogenous PDGF‐AA in the absence of serum or other growth factors, we found that cell numbers increased over time compared to untreated control cells. The numbers of cells treated with a specific PDGFRα inhibitor were decreased in contrast to the untreated controls (Figure 5G).

DISCUSSION

The origin of brain tumors remains enigmatic, although current hypotheses favor stem cells as source for these malignancies 22, 36, 41, 44, 45. Here, we generated two cancer stem‐like cell lines from adult neural precursors following expansion in a culture assay adopted for SVZ stem and progenitor cells. Irrespective of the artificial culture environment, these findings indicate the risk and potential of SVZ precursors to transform into malignant brain tumor‐initiating cells.

In a low‐density culture assay, adult stem cells did not transform after prolonged expansion in vitro (15). In contrast to the neurosphere assay 18, 39, however, culture paradigms employed in the present study were designed to achieve rapid expansion of stem and progenitor cells, allowing passaging every 3–5 days. This will be mandatory if ex vivo cell expansion is required within a reasonable therapeutic time schedule, that is, stem cell transplantation in neurological disorders. It is likely, therefore, that our culture assay particularly recruits transit‐amplifying cells (type C cells), which are the fastest proliferating cells in the SVZ (12). Interestingly, it has been reported that type C cells of the SVZ can be reverted into multipotent cells in culture and can become invasive as a result of prolonged EGF exposure (13). Moreover, we found no spontaneous transformation of rodent olfactory bulb derived precursor cells in our culture model despite several attempts to passage these cells beyond passage eight (F. Siebzehnrubl, unpub. obs.), indicating that only cells with self‐renewing capacities are sensitive to genomic instability in our culture model.

We are aware that rodent cells are more likely to transform spontaneously in culture than in human cells. Nonetheless, precursor cells in our assays transformed already after 10 passages in vitro in two independent settings. This does not exclude the possibility of human precursor cell transformation, which may need more genetic alterations (37).

Neoplastic transformation during cell culture periods may result from accidental exposure to chemical drugs and/or viruses. However, we have successfully cultured and differentiated adult rodent brain stem cells and performed functional culture assays in our lab 6, 43 without any indication for aberrant behavior of other cells. The rate for spontaneous brain tumor formation over the lifetime of Wistar rats is below 1.5% (5); thus, contamination of the initial cultures from five juvenile animals with pre‐existing tumor cells is extremely unlikely.

Detailed histopathological analysis of transplanted tumors detected distinct cell populations, that is, those with nuclear accumulation of p21. Double immunofluorescence for p21 and Ki‐67 separated this population from a Ki‐67 immunoreactive proliferating cell pool (Figure 4). Others described prominent expression of Olig2 as inducer of proliferation in gliomas as well as in adult stem cells (29), which we could not confirm in our cell lines. p21 expression has been associated with both cell cycle arrest/senescence and stem cell quiescence (26). We cannot detect the senescence‐associated marker β‐galactosidase (17) in R2303 cells in vitro or in vivo (data not shown). Therefore, the issue of p21 accumulating senescent vs. quiescent tumor cells will need further clarification.

Proof of stemness in cancer cells remains challenging, and several reports postulate CD133 (prominin‐1) expression (48) or the detection of a side population as a reliable experimental paradigm 20, 25, but a controversial report has been published (32). Here, we identified CD133 expression in R2303 cells as well as a significant side population. In addition, both R2303 and R2902 cells expressed the stem cell markers Sox2, Musashi‐1 and Nestin (Figure 1). We conclude from these experiments that both cell lines retain stem‐like cell properties even after prolonged passages in vitro (>100).

A recent report described CD133/prominin‐1 expression in adult brain and cancer stem cells (35). However, isolation of CD133‐ cancer stem cells in glioblastomas (3) argues against CD133+ cells as tumor founder population. With respect to the latter study (3), the impact of CD133 expression remains controversial and may not be an imperative event during malignant transformation.

Several mechanisms of malignant transformation have been recognized in brain tumors, and the molecular alteration is likely to associate with specific tumor entities. In this respect, constitutive activation of the PDGFRα pathway is common in several brain malignancies, such as malignant gliomas and primitive neuroectodermal tumors 19, 46. Our gene expression analysis in R2303 cells revealed continued expression of PDGFRα after onset of differentiation (Figure 5A). Notably, PDGF is not a component of either expansion or differentiation media in our culture assay, but is expressed by normal precursor cultures and both cell lines (Figure 5C). PDGFR expression was confirmed by immunofluorescence microscopy in tumors derived from R2303 grafts. Of note, PDGFRα is located on chromosome 14p, which was found structurally aberrant in one chromosome 14 in R2303 and R2902 cells. Thus, it is most likely that lasting expression of PDGFRα in these cell lines results in growth advantage. While PDGFRα is downregulated upon differentiation in normal SVZ precursor cells, its maintained expression in R2303 and R2902 cell lines may account for their continued proliferation and tumorigenicity (Figure 5). This is substantiated by the fact that normal precursor cells as well as transformed cells express PDGF in vitro. Furthermore, a knockdown of PDGFRα results in a reduction of protein levels accompanied by reduced cell growth in R2303 cells (Figure 5D,E). A highly specific PDGFRα RTKI also inhibits cell growth. Cell proliferation can be stimulated by exogenous PDGF‐AA (Figure 5G), which also results in phosphorylation of the PDGFRα (Figure 5F). Taken together, the PDGF pathway is of major importance for the continued proliferation of these transformed cell lines. Importantly, PDGFRα is expressed on SVZ stem cells, and cerebroventricular infusion of PDGF as well as overexpression of PDGF in astrocytes and neural progenitors result in malignant glioma formation 11, 23.

CONCLUSION

Our findings are compatible with a tumor progression model in the CNS, which is adopted from the current concept of adult brain stem cell development (2). Type B stem cells reside lifelong within their SVZ niche. These cells are characterized by GFAP and PDGFRα expression (23) and progress into transit‐amplifying type C cells, downregulating both GFAP and PDGFRα. We propose transformed type C cells as cancer stem‐like cell immortalized in our cell lines R2303 and R2902. Lack of GFAP and constitutive PDGFRα expression support this notion, and the latter is likely to result from acquired genetic aberrations. Hence, the risk of graft carcinogenesis, which is well documented for embryonic stem cells 14, 50, also applies to therapeutic adult stem cell transplantation strategies. In light of the report of Foroni et al (15) and our present data, particular efforts have to be dedicated to expansion of adult precursor cells in vitro, as paradigms not as restrictive as the neurosphere assay may lead to favored growth of transformed cells. Careful examinations for aberrant growth patterns are therefore mandatory in any (pre‐) clinical reconstructive therapy regimen.

Continuous expansion of precursor cells in culture harbors the risk of spontaneous transformation. Transformation of rodent cells in vitro has been repetitively proven in many reports, although adult brain stem cells have been thought to be resistant to transforming events. As precursor cell cultures always contain an aggregation of stem and progenitor cell populations, transformation of one subpopulation is likely to result in tumor formation in graft recipients. This notion underlines the need for a systematic and careful examination of stem cell expansion for therapeutic applications.

Supporting information

Figure S1. Proliferation and viability of cancer stem‐like cells. A. Cell viability assay [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay]. Cell viability during expansion and differentiation was analyzed for non‐transformed precursor cells (NPC) as well as cell lines R2303 and R2902 (n = 6). Shown is the relative viability of R2303 and R2902 compared to NPCs (set = 100%). Both cell lines show increased viability under expansion as well as differentiation conditions, which may be explained by their higher proliferative activity. B. Fluorescence immunostaining for the proliferative marker Ki‐67 in NPCs and R2303 cells shows increased proliferation of R2303 cells under differentiation conditions. Nuclei are counterstained with Hoechst. Magnification 200×. C. Cell numbers of NPCs, R2303 and R2902 cultures under expansion and differentiation conditions. In each condition, 150 000 cells were plated in 5 mL culture medium, cultured for 7 days and counted in a hemocytometer. Cell counts after 7 days in culture (DIC) are normalized to initial cell numbers (0 DIC). Photomicrographs of representative visual fields of the cultures at 7 DIC exemplify differences in cell numbers as well. Magnification 100×.

Figure S2. Expression of Nestin and Musashi‐1 in non‐transformed precursor cells (NPCs) and transformed cell lines. Both NPCs and transformed cell lines R2303 and R2902 continue to express Nestin (red) after 7 days of differentiation. NPCs also show frequent expression of Musashi‐1 (green), while a subpopulation of Musashi‐1 positive cells exists in R2303 and R2902 cell lines. The Musashi‐1 expressing cells differ in morphology from the Musashi‐negative cells. Nuclei are counterstained with Hoechst (blue). Scale bar 50 µm.

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ACKNOWLEDGMENTS

We thank S. Gutmann and B. Rings for excellent technical assistance, T. Acker and C.D. Lie for helpful discussions, and U. Appelt for assistance with FACS sorting experiments. This work is funded by the Bavarian Research Council (ForNeuroCell). F.A.S. is a fellow of the German National Academic Foundation (Studienstiftung des deutschen Volkes e.V.).

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Associated Data

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Supplementary Materials

Figure S1. Proliferation and viability of cancer stem‐like cells. A. Cell viability assay [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay]. Cell viability during expansion and differentiation was analyzed for non‐transformed precursor cells (NPC) as well as cell lines R2303 and R2902 (n = 6). Shown is the relative viability of R2303 and R2902 compared to NPCs (set = 100%). Both cell lines show increased viability under expansion as well as differentiation conditions, which may be explained by their higher proliferative activity. B. Fluorescence immunostaining for the proliferative marker Ki‐67 in NPCs and R2303 cells shows increased proliferation of R2303 cells under differentiation conditions. Nuclei are counterstained with Hoechst. Magnification 200×. C. Cell numbers of NPCs, R2303 and R2902 cultures under expansion and differentiation conditions. In each condition, 150 000 cells were plated in 5 mL culture medium, cultured for 7 days and counted in a hemocytometer. Cell counts after 7 days in culture (DIC) are normalized to initial cell numbers (0 DIC). Photomicrographs of representative visual fields of the cultures at 7 DIC exemplify differences in cell numbers as well. Magnification 100×.

Figure S2. Expression of Nestin and Musashi‐1 in non‐transformed precursor cells (NPCs) and transformed cell lines. Both NPCs and transformed cell lines R2303 and R2902 continue to express Nestin (red) after 7 days of differentiation. NPCs also show frequent expression of Musashi‐1 (green), while a subpopulation of Musashi‐1 positive cells exists in R2303 and R2902 cell lines. The Musashi‐1 expressing cells differ in morphology from the Musashi‐negative cells. Nuclei are counterstained with Hoechst (blue). Scale bar 50 µm.

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Supporting info item

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