Astrocytes derived from trisomic human embryonic stem cells express markers of astrocytic cancer cells and premalignant stem-like progenitors
© Gopalakrishna-Pillai and Iverson; licensee BioMed Central Ltd. 2010
Received: 20 March 2009
Accepted: 27 April 2010
Published: 27 April 2010
Trisomic variants of human embryonic stem cells (hESCs) arise spontaneously in culture. Although trisomic hESCs share many properties with diploid hESCs, they also exhibit features of cancer stem cells. Since most hESC-based therapies will utilize differentiated derivatives, it is imperative to investigate the potential of trisomic hESCs to undergo malignant transformation during differentiation prior to their use in the clinical setting.
Diploid and trisomic hESCs were differentiated into astrocytic progenitors cells (APCs), RNA extracted and hybridized to human exon-specific microarrays. Global gene expression profiles of diploid and trisomic APCs were compared to that of an astrocytoma cell line and glioblastoma samples, analyzed by others, using the same microarray platform.
Bioinformatic analysis of microarray data indicates that differentiated trisomic APCs exhibit global expression profiles with similarities to the malignant astrocytoma cell line. An analogous trend is observed in comparison to glioblastoma samples indicating that trisomic APCs express markers of astrocytic cancer cells. The analysis also allowed identification of transcripts predicted to be differentially expressed in brain tumor stem cells. These data indicate that in vitro differentiation of trisomic hESCs along astrocytic pathways give rise to cells exhibiting properties of premalignant astrocytic stem/progenitor cells.
Given their occult nature, opportunities to study premalignant stem/progenitor cells in human have been few. The ability to propagate and direct the differentiation of aneuploid hESCs provides a powerful in vitro system for investigating biological properties of human cells exhibiting features of premalignant stem cells. This in vitro culture system can be used to elucidate changes in gene expression occurring enroute to malignant transformation and to identify molecular markers of cancer stem/progenitor cells. These markers are invaluable for diagnostic purposes and may be novel targets for therapeutic intervention.
Human embryonic stem cells (hESCs) are a source of pluripotent cells that can be differentiated in vitro into cells of numerous lineages . The use of hESCs in regenerative medicine requires caution since aneuploid variants of hESCs spontaneously arise in culture. Trisomy for chromosomes X, 12 and/or 17 is one type of aneuploidy frequently observed in hESC lines . Trisomic hESC variants exhibit many properties indistinguishable from their diploid counterparts; they self-renew, express 'stem' markers characteristic of diploid hESCs, retain pluripotency, differentiate in vitro and produce teratomas in mice [3, 4]. Trisomic variants appear karyotypically stable over time in culture and microarray and RT-PCR analyses indicate that gene expression patterns of trisomic hESCs are similar to the diploid hESC lines from which they were derived [3, 4]. However, trisomic hESC variants also display characteristics similar to cancer stem cells; they exhibit a reduced doubling time and teratomas arising from trisomic hESC injection contain a higher percentage of undifferentiated cells similar to teratocarcinomas formed following embryonal carcinoma cell injection . Many similarities in gene expression profiles have been reported for normal and cancer stem cells, suggesting that changes in expression of relatively few genes may be sufficient to drive transformation of normal stem cells into cancer stem cells . Recent evidence indicates that neural precursors derived from variant hESC lines exhibit early features of neoplastic transformation, including increased proliferative capacity and ~20 fold increase in the frequency of tumor initiating cells when assayed by injection into NOD-SCID mice . Since cultured hESCs are genetically unstable and exhibit a propensity to develop spontaneous trisomy [2, 7], it is imperative to evaluate the potential tumorigenicity of trisomic hESC variants.
In general, hESC-based cell-replacement strategies will rely on hESC-derived differentiated cells rather than the pluripotent stem cells. Thus, the potential of aneuploid hESC variants to undergo malignant transformation in the clinical setting is more instructively evaluated by comparing expression profiles of differentiated derivatives of diploid and trisomic hESCs. The primary objective of this study was to determine if similarities in gene expression patterns of diploid and trisomic pluripotent hESCs are retained following in vitro directed differentiation. To investigate this question, an in vitro culture system was developed in which hESCs were differentiated into homogenous populations of human astrocytic progenitor cells (APCs) suitable for global gene expression profiling using high-density exon-specific microarrays. If expression patterns of trisomic hESCs diverge from diploid hESCs following differentiation, then the next objective was to determine if trisomic derivatives exhibit expression profiles similar to malignant cell lines and/or primary tumor samples of the same lineage. Given the difficulty of isolating sufficient quantities of human premalignant progenitors for sophisticated molecular characterization, the final objective of this study was to determine if expression patterns of differentiated derivatives of aneuploid hESCs express markers of previously identified astrocytic cancer stem/progenitor cells. The results of this analysis indicate that in vitro differentiated astrocytes derived from a trisomic hESC line exhibit global gene expression profiles similar to astrocytomas and astrocytic cancer stem/progenitor cells. The results demonstrate that the combination of in vitro directed differentiation of hESCs, global gene expression profiling and robust bioinformatic analyses provides a powerful model system that can be used to identify differentially expressed biomarkers in stem/progenitor cells in heterogeneous tumors.
HESC and other cell culture
HESC lines H9 (WiCell) and BG01V (American Type Culture Collection) were grown under feeder-independent conditions on matrigel-coated dishes (BD) in medium containing basal DMEM/F-12 with 1 mM glutamine, 20% knockout serum replacement (KSR; Invitrogen), 2 mM non-essential amino acids and 8 ng/ml FGF (Invitrogen). To obtain non-adherent embryoid bodies, small pieces of undifferentiated hESC colonies (~100-200 cells) were mechanically dissected and cultured on low attachment plates in the same media used for maintaining pluripotent hESCs, except KSR was removed and replaced with 10% Fetal Bovine Serum (FBS; Hyclone). Neurospheres were derived from 4-5 day old embryoid bodies and grown in suspension for two weeks in medium containing DMEM/F-12 with 2 mM L-glutamine, 10 μl/ml BIT9500 (StemCell Technologies) supplemented with 10 ng/ml FGF, 10 ng/ml EGF (Millipore). To obtain astrocytic progenitor cells, neurospheres were allowed to adhere on matrigel-coated plates and differentiated in the presence of CCF-STTG1 conditioned media supplemented with 10 ng/ml EGF. CCF-STTG1 cells, a grade IV human astrocytoma cell line, were obtained from American Type Culture Collection and cultured in growth medium containing DMEM/F-12 with 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate supplemented with 10% FBS, under 5% CO2 at 37°C.
Human ESCs grown on matrigel-coated LabTek chamber slides were rinsed with 1× PBS and fixed in 4% paraformaldehyde for 30 minutes at room temperature. Immunofluorescence staining was performed according to instructions provided in the Stem Cell Characterization Sample Kit (Millipore). The following primary antibodies were used at 1:200 dilutions: anti-TRA-1-60 (Millipore) and OCT-4 (Cell Signaling). Secondary antibodies, rhodamine-conjugated anti-mouse IgG or IgM or Cy2-conjugated anti-mouse IgM or IgG (Jackson Immunoresearch Laboratories Inc.) were used to detect primary antibody at 1:500 dilutions. H9 and BG01V APCs were cultured in matrigel-coated 24-well plates, the cells rinsed with 1× PBS and fixed in 4% paraformaldehyde for 30 minutes at room temperature. An astrocyte-specific primary antibody directed against glial fibrillary acidic protein (α-GFAP, Chemicon) was used at 1:200 dilution. Secondary antibody, rhodamine-conjugated anti-mouse IgG (Jackson Immunoresearch laboratories Inc.) was used to detect GFAP positive cells. Images were visualized with an Inverted IX81 (Olympus) fluorescence microscope and captured on a Retiga 2000R cooled CCD color camera (QImaging).
Exon microarrays and data analysis
Affymetrix Human Exon 1.0ST microarrays were selected as the platform for global gene expression profiling because they contain 1.4 million probesets, including four oligonucleotides for each known or predicted exon in the human genome, and are expected to be more comprehensive than Affymetrix U133 Plus 2 microarrays, where most probesets are clustered in and around 3' regions of genes. Additional information can be obtained from the Affymetrix website http://www.affymetrix.com. RNA was isolated from three independent cultures of each cell type using the Trizol method (Invitrogen) and RNA quality determined by Nanodrop technique (NanoDrop Technologies). One μg of total RNA was used for preparation of individual samples. Microarray hybridization, scanning and data acquisition were performed at the City of Hope Microarray Core Facility. All microarray data were analyzed using Partek Genomic Suite software. Signal estimates obtained from CEL files were quantile-sketch normalized using the RMA algorithm for core probeset intensities. They were adjusted for "Detection Above Background", using surrogate GC mismatch intensities. Imported log2-transformed exon intensities were subjected to one-way ANOVA (analysis of variance) for comparison between the three biological replicates of each cell population, except glioblastoma samples where the 23 individual patient samples were treated as one cell population. Unsupervised hierarchical cluster analysis, heat maps and individual gene dot plots were generated using Partek Genomic Suite software. High-end (over) or low-end (under) expression is indicated by red and blue, respectively, in the heat maps. Expression level of each gene in each cell population was measured relative to the median expression level across the three replicates and variations between cell populations graphically depicted using individual gene dot plots, where the horizontal line within the colored bar represents the median expression level within each population and the vertical length of the bar represents SEM. Supervised hierarchical analyses, filtered at p value < 0.02 as described in detail in Results, was used to generate gene lists. Please see the Partek website http://www.partek.com/ for a more detailed description of statistical analysis of microarray data using the Partek Genomic Suite applications. Exon array data of 23 glioblastoma samples were extracted from GEO database: GSE9385 . Exon microarray data of H9 APC, BG01V APC and CCF-STTG1 cells have been deposited at ArrayExpress http://www.ebi.ac.uk/arrayexpress: accession number is E-MEXP-2633.
Semi-quantitative RT-PCR, DNA sequencing and quantitative RT-PCR analyses
H9 and BG01V hESCs cultured on matrigel were harvested after five to six days in culture. APCs were harvested on the fifth passage after neurospheres were differentiated into astrocytic progenitors on matrigel-coated plates. Total RNA was extracted using TRIzol according to the manufacturer's instructions (Invitrogen). Gene-specific PCR primers located within exons or spanning exon junctions were designed based on human gene sequences obtained from Ensembl http://www.ensembl.org/. Sequences of PCR primers are shown in Additional file 1, Table S1. Synthesis of cDNA was performed using 2 μg of total RNA, SuperScript II reverse transcriptase (Invitrogen) and random primers. Semi-quantitative RT-PCR reactions used 1 μl of cDNA template and exon-specific primers. PCR products were resolved by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining. Data were recorded using QuantityOne software (BioRad). PCR product of interest was excised, purified and cloned into StrataClone vector using the PCR Cloning Kit (Stratagene) and the DNA insert sequenced using T3 or T7 primers. Quantitative PCR reactions (25 μl) were performed in an iCycler (BioRad) using 1 μl cDNA template, exon primers for the gene in question and SYBR green PCR mix (Applied Biosystems). Relative quantification was determined using the ΔΔCT method  according to the manufacturer's protocol (BioRad). Quantitative RT-PCR analysis using Human Cancer Pathway Finder PCR Arrays (SuperArray, Biosciences Corporation) containing proprietary RT-PCR primers for a number of cancer-associated genes was carried out according to the manufacturer's instructions using 1 μl cDNA template.
Derivation of astrocytic progenitor cells from hESCs
Gene expression profile of trisomic APCs is similar to CCF-STTG1 astrocytoma cells
Numerous transcripts exhibit differences in relative expression levels (either over or under expression) in each of the three individual pair wise comparisons (GvC: 2929 differentially expressed transcripts, DvC: 4019 and GvD: 5193). Because the BG01V trisomic hESC line was not derived from the diploid H9 hESC line, and neither hESC line is related to the CCF-STTG1 astrocytoma cell line, many of these differences in expression levels undoubtedly arise from inherent genetic differences between the three distinct cell lines. It is for this reason that one cannot rely solely on the individual pair wise comparisons to identify the most statistically significant differences in gene expression. Rather, we focused on those gene transcripts exhibiting consistent sign changes in expression levels in both the BG01V APCs (G) and CCF-STTG1 astrocytoma cell line (D) relative to the H9 APCs (C) (i.e. both over expressed in BG01V APCs and CCF-STTG1 cells relative to H9 APCs or both under expressed in BG01V APCs and CCF-STTG1 cells relative to H9 APCs). Those transcripts exhibiting consistent sign changes in relative expression levels are identified by performing an analysis of variance (ANOVA) of the supervised hierarchical clustering; i.e. an ANOVA of the intersection of the two individual data sets (or the group comparison).
The complete list of 1416 transcripts that meet this higher threshold of significance is shown in Additional file 2, Table S2 (the GDvC comparison), where gene transcripts are listed in descending order on the basis of the combined average fold-change in expression levels in BG01V APC (samples G) and CCF-STTG1 astrocytoma cells (samples D) relative to H9 APCs (samples C). Although differences in mean expression levels of several transcripts are observed between BG01V APCs and CCF-STTG1 astrocytoma cells, visual inspection of the Additional file 2, Table S2 data set indicates that the ANOVA of the group comparison successfully eliminated most false positives. More than 96% of the 1,416 identified transcripts exhibit consistent sign changes; ~33% are over expressed and ~63% are under expressed in both BG01V APCs and CCF-STTG1 astrocytoma cells relative to diploid H9 APCs. Fewer than 4% of the transcripts show inconsistent sign changes (i.e. under expressed in BG01V APCs and over expressed in CCF-STTG1 cells relative to H9 APCs or over expressed in BG01V APCs and under expressed in CCF-STTG1 cells relative to H9 APCs). Most of the inconsistent transcripts exhibit only minor changes in relative expression levels (~10% to 20% increase or decrease) and relatively insignificant p values. In contrast, approximately 130 of the consistently over expressed transcripts show at least a two-fold increase in mean expression level in each individual pair wise comparison, BG01V APCs (samples G) vs. H9 APCs (samples C) and CCF-STTG1 (samples D) vs. H9 APCs (samples C), and far greater significance with p values less than ~2 × 10-9.
Many of the consistently and abundantly over expressed transcripts in trisomic BG01V APCs and CCF-STTG1 astrocytoma cells encode proteins previously implicated in cancer in general or associated with astrocytomas specifically, including HSPA1A, HOXD10, GPNMB, GUCY1B3, GUCY1A3, HDAC9, APOE, CTSH, THRB, RAB38 and PIK3R1 [10–21]. Transcripts exhibiting significant under expression in trisomic BG01V APCs and CCF-STTG1 astrocytoma cells relative to diploid H9 APCs include several markers of normal differentiated astrocytes, including TRPA1, GABRA2, BDNF, BDKRB1 and BDKRB2 [22–24]. This result suggests that directed differentiation of trisomic hESCs along an astrocytic lineage produces astrocytic progenitor cells with an intermediate phenotype; although BG01V APCs continue to express many biomarkers of normal, differentiated astrocytes (similar to diploid H9 APCs), they also express numerous markers that are characteristic of the malignant astrocytoma cell line (CCF-STTG1).
RT-PCR validation of differentially expressed transcripts in trisomic and diploid hESC-derived APCs
Trisomic APCs express markers of astrocytic cancer cells
Markers over expressed in astrocytic cancer cells (GND > C).
Trisomic APCs express markers of astrocytic cancer stem/progenitor cells
Markers over expressed in premalignant astrocytic stem-like/progenitor cells (GN > CD).
In this study, an in vitro culture system was developed to differentiate diploid and trisomic hESCs into astrocytic progenitor cells (APCs), which were used to determine if gene expression profiles of trisomic APCs remain similar to, or deviate from, diploid APCs following astrocytic differentiation. The data indicate that expression profiles of trisomic BG01V APCs diverge considerably from diploid H9 APCs. Analysis of high-density microarray data revealed numerous, highly significant differences in transcript expression levels in trisomic BG01V APCs relative to diploid H9 APCs. Similar differences were observed when the human astrocytoma cell line, CCF-STTG1, was compared to diploid H9 APCs. Several expression level changes, initially detected by microarray analysis, were subsequently confirmed by qRT-PCR validation. A remarkably similar trend was observed when trisomic BG01V APCs were compared to human glioblastoma patient samples. Taken together, the data suggest that following differentiation along an astrocytic pathway trisomic BG01V APCs exhibit a global gene expression profile that is more similar to astrocytic cancer cells that to normal diploid hESC-derived APCs.
Although trisomic BG01V APCs continue to express markers of differentiated astrocytes, they are clearly distinct from diploid H9 APCs. Despite the high PROM1 expression in BG01V APCs when cultured under adherent conditions, there is insufficient evidence to classify trisomic BG01V APCs as brain tumor-initiating cells [25, 26]. Although trisomic BG01V APCs were derived from multipotent neurospheres that give rise to oligodendrocytes or neurons under different culture conditions (Gopalakrishna-Pillai and Iverson, unpublished), it is also incorrect to consider BG01V APCs equivalent to transformed neural stem cells. Unlike the CCF-STTG1 astrocytoma cells, BG01V APCs do not readily dedifferentiate into non-adherent neurospheres (not shown). Thus, it may be more appropriate to consider trisomic BG01V APCs as one type of premalignant astrocytic stem/progenitor cell. BG01V APCs exhibit an increased rate of proliferation relative to diploid H9 APCs and display gene expression patterns more similar to the astrocytoma cell line and glioblastoma patient samples. Trisomic BG01V APCs may be predisposed to becoming astrocytic cancer cells, but have not yet acquired the full spectrum of mutations required for malignant transformation.
Because the BG01V trisomic hESC line studied here was not derived from the diploid H9 hESC line used for comparison, the results of this analysis cannot be used to identify those changes in gene expression that are predicted to be initiators of premalignant transformation or tumorigenesis (i.e. changes in gene expression that cause transformation). However, group analyses of the data sets can be used to identify differentially expressed transcripts in normal differentiated astrocytes/astrocytic progenitors relative to astrocytic cancer cells. For example, highly significant decreases in expression levels of several transcripts encoding known markers of normal astrocytes including TRPA1, BDNF and MGMT were detected by the analyses. Down regulation of MGMT transcript levels, via hypermethylation of the MGMT promoter, is associated with malignant progression of astrocytomas and is being used in the clinic to identify patients that may benefit the most from treatment with temozolomide [29, 30]. Thus, transcripts exhibiting highly significant over expression in all classes of abnormal astrocytes, including trisomic BG01V APCs, CCF-STTG1 astrocytoma cells and glioblastoma patient samples with respect to diploid H9 APCs (Table 1, GND > C) may be diagnostic markers of transformation and/or potential therapeutic targets (i.e. changes in gene expression that are consequences of transformation). This list includes cell surface expressed markers such as GPRC5, signaling molecules such PIK3R1 and the histone deacetylase, HDAC9, for which small molecule inhibitors are currently under investigation in glioblastoma clinical trials [13, 31].
Neural stem cells with astrocytic character arising in the sub ventricular zone are thought to be one source of origin of gliomas [32, 33]. Donor-derived neural stem cells were recently demonstrated to give rise to tumors of glioneuronal origin in an Ataxia Telangiectasia patient . Slowly cycling cancer stem cells, which are refractory to conventional radiation and chemotherapy, are thought to be a source of both the original tumor as well as recurrent tumors. These astrocytic cancer stem cells create an obstacle for effective treatment of malignant gliomas, yet identification of molecular markers characteristic of brain tumor stem cells is a major challenge complicated by their low numbers, elusive nature, heterogeneity of brain tumors and the difficulty of obtaining abundant quantities of normal human astrocytes that can serve as controls for global expression analyses . As a result, gene expression profiles of rare subpopulations of brain tumor stem cells cannot be readily identified by microarray analyses of brain tumor samples. The system used in this study describes methods for both obtaining sufficient quantities of suitable control cells via directed differentiation of hESCs and using gene expression profiles of these cells to refine the list of putative biomarkers of the astrocytic cancer stem cells through rigorous bioinformatic analyses of the microarray data.
Given their astrocytic, premalignant and stem-like properties, the class of transcripts predicted to be biomarkers of premalignant astrocytic stem/progenitor cells are the GN > CD transcripts (Table 2, Additional file 6, Table S5, Figure 8), and include transcripts encoding numerous potential therapeutic targets and/or cell-surface expressed proteins. In addition to PROM1, CHI3L1 was also identified by the analysis. CHI13L1 (YKL-40) is expressed in a small percentage of glioblastoma cells upon initial diagnosis, but exhibits profound up regulation upon tumor recurrence . The regulator of G protein signaling 5, RGS5, is over expressed in highly angiogenic astrocytomas and RGS5 expression is specifically up regulated in the vasculature of premalignant lesions . FKBP5 is over expressed in gliomas and down regulation of FKBP5 expression using siRNAs suppresses glioma cell growth . IGFBP2 over expression has been demonstrated to promote glioma growth as well as progression from low to high grade in mouse models . Over expression of transcripts encoding transmembrane proteins KCNMB4, a neural-specific β subunit of a large-conductance, calcium-sensitive potassium channel associated with glioma cell growth , and LPHN2, a putative G-protein coupled receptor, were both identified by the comparative microarray analysis. As were ASTN1, an adhesion molecule associated with neuronal migration along astroglial fibers , and GPC4, a cell-surface expressed proteoglycan that may play a role in controlling cell division . Additional over expressed transcripts identified by comparative global microarray analysis using in vitro differentiated trisomic BG01V APCs include those encoding signaling molecules such as the protein phosphatase, PPP2R2B, the purinergic receptor, P2RY5, the ras homolog, RHOU, and others not previously associated with astrocytomas or astrocytic cancer stem cells.
Inherent genetic instability of cultured hESCs renders them susceptible to gain or loss of entire chromosomes and/or discrete chromosomal regions. Gain of chromosomes 12 or 17 has been reported in several other hESC lines [2, 3, 7]. Amplification or deletion of discrete regions of chromosome 20 and 5 as well as mosaic gain of chromosome 12 has also been reported to arise spontaneously in cultured hESC lines . Significantly, neural stem cells derived from some of these hESC variants have been clearly demonstrated to exhibit several features of neoplastic transformation in vivo. Thus, it is highly unlikely that trisomic BG01V hESCs line are unique in their ability to differentiate into premalignant astrocytic stem/progenitor cells upon in vitro directed differentiation. The in vivo evidence of neoplastic transformation of differentiated hESC variants suggests that the propensity toward transformation may be a somewhat common occurrence and underscores the absolute necessity of subjecting all hESC-derived cells to functional characterization prior to their use in therapeutic regimens . Although BG01V hESCs may not be unique in exhibiting features of premalignant transformation following differentiation, the conspicuous differences in expression profiles of BG01V APCs and H9 APCs, combined with the striking similarities in expression profiles of BG01V APCs and glioblastoma samples suggest that gain of chromosomes X, 12 and/or 17 may be one of several routes by which transformation can be initiated in astrocytic progenitor cells. However, we cannot rule out the possibility that genetic events other than trisomy played a role in the initiation of premalignant transformation observed here since the trisomic hESC line, BG01V, is not a derivative of the diploid hESC line, H9.
That a constellation of gain-of-function and/or loss-of-function mutations in multiple genes is required for malignant transformation has been known for decades [41, 42]. Aneuploidy, however, has been associated with cancer for more than a century . Given the high-degree of aneuploidy observed in glioblastoma patient samples, it is difficult to distinguish those genes or chromosomal regions associated with tumor initiation or propagation from those representing random events arising from the inevitable genetic instability common to these high-grade tumors. Comprehensive analysis of chromosomal aberrations in 141 glioma samples identified approximately 35 broad and focal regions of gene amplifications and deletions demonstrating statistically significant associations in human gliomas , and revealed that amplification of a number of chromosomal regions, including chromosomes 12 and 17, met the threshold for significance in these glioma samples, including secondary glioblastomas arising from low-grade gliomas. High-resolution copy number analysis of glioma samples also revealed recurrent gain of multiple sub-regions of chromosome 12 in secondary glioblastomas arising from lower grade astrocytomas . Since recurrent gain of chromosomes 12 or 17 has been observed in a number of karyotypically abnormal hESC lines [2–4, 7], this also suggests that other aneuploid hESC variants might exhibit properties similar to trisomic BG01V cells upon differentiation into astrocytes.
Dot plots for several additional markers associated with gliomas are shown in Additional file 7, Figure S2. Amplification of EGFR (or gain of chromosome 7) is often seen in high-grade gliomas . Not surprisingly, over expression of EGFR in some glioblastoma samples is also observed here (Additional file 7, Figure S2, panel A). Loss of chromosome 10 (or regions of chromosome 10 encoding the PTEN locus) is associated with some de novo glioblastomas, and reduced PTEN expression in some glioblastoma samples is seen here (panel B). IL13RA2 is reportedly over expressed in ~90% of glioblastoma samples. Exon microarray analysis indicates bifurcation of patient samples into two distinct groups; one group exhibits significant over expression of IL13RA2 transcripts, whereas IL13RA2 is expressed at levels similar to controls in the other group of patient samples (D). Relative expression levels of PDGFB (F), PDGFRA (G) PDGFRB (H) are also shown in Additional file 7, Figure S2. Although median expression level of PDGFRB is decreased relative to controls, there is a wide range of expression levels, which may also reflect distinct glioma subtypes. Mutations in a number of genes have also been associated with gliomagenesis, although many are somatic mutations that do not necessarily result in expression level changes . Several are shown in Additional file 7, Figure S2, including AKT1 (C), NRG1 (E), TP53 (I), MDM2 (J), NF1 (K) and RB1 (L). There is no obvious correlation in relative expression levels of these markers in glioblastoma samples and/or premalignant astrocytic progenitors, with perhaps the exception of NRG1 (HER2 ligand) where expression levels are lower in all samples relative to the diploid H9 APCs. Although trisomy for chromosomes 12 and 17 may be one potential precipitating event resulting in transformation of BG01V hESCs into premalignant APCs during differentiation, not all differences in relative gene expression levels can be explained by the trisomy. Dot plots for a number of genes that are known to be expressed in astrocytes, known to be associated with cancer and known to map to chromosomes 12 or 17 are also shown in Additional file 7, Figure S2, including STAT3 (panel M, chromosome 17) where expression levels in glioblastoma, BG01V APCs and CCF-STTG1 samples are higher than H9 APCs, ERBB2 (HER2 - panel N - chromosome 17) where expression levels are higher in BG01V APCs and glioblastoma samples but also high in diploid H9 APCs, and both RARA (panel O, chromosome 17) and RARG (panel P, chromosome 12), where expression levels are higher in BG01V APCs relative to H9 APCs but also lower in glioblastoma samples. Thus, not all transcripts encoded by genes that map to chromosomes X, 12 and/or 17 are over expressed in BG01V APCs and not all over expressed gene transcripts in BG01V APCs map to chromosomes X, 12 and/or 17. We think that most gene transcripts that are over expressed in BG01V APCs relative to H9 APCs are consequences of premalignant transformation rather than causes of premalignant transformation.
Nonetheless, gain of chromosomes X, 12 and/or 17 has also been observed in carcinoma in situ of the testis, which is thought to be the common pre-invasive progenitor cell (or precancerous stem cell) that gives rise to both seminoma and non-seminomatous testicular germ cell tumors . Recurrent amplification of 17q23.2 in some breast tumors has recently been reduced to a 249 kb minimal region including potential tumor driver genes, RPS6KB1 and mir-21 . Finding gains of similar chromosomal regions in tumors whose origins are unrelated to the nervous system suggests that trisomy for these chromosomal regions is a type of moderate aneuploidy common to other precancerous progenitor or stem cells and hypothesized to be an initiator of tumorigenesis [48–50]. This suggests that directed differentiation of trisomic hESC variants along other lineages might be used to simulate early molecular events occurring enroute to malignant transformation of other cancers and to identify diagnostic markers and/or molecular targets amenable to therapeutic intervention.
An in vitro culture system was developed in which diploid and trisomic hESCs were differentiated into homogenous populations of human astrocytic progenitor cells (APCs) suitable for global gene expression profiling using high-density microarrays. Expression profiles of the hESC-derived APCs were compared to a malignant astrocytoma cell line and glioblastoma tumor samples, and used to demonstrate that trisomic APCs share many properties with the astrocytoma cell line and glioblastoma samples. The bioinformatic analysis employed here facilitated identification of numerous differentially expressed transcripts that are characteristic of astrocytic cancer cells. This analysis was also used to identify biomarkers of the subpopulation of astrocytic cancer stem cells that comprise only a small fraction of diverse cell types found in heterogeneous brain tumors. Directed differentiation of trisomic hESCs is a powerful in vitro model system for investigating changes in gene expression occurring enroute to malignant transformation and for identifying molecular markers characteristic of premalignant stem/progenitor cells that may be amenable for therapeutic intervention for patients with astrocytomas. The results of this analysis further underscore the need for exercising extreme caution when utilizing stem cells in regenerative medicine.
- Non standard abbreviations used in the manuscript include hESC:
(human embryonic stem cell)
(astrocytic progenitor cell).
The authors are grateful to Giau M. Hua, M.S. for outstanding technical services in culturing hESCs, City of Hope Microarray, Light Microscopy, Cytogenetics and DNA Sequencing Comprehensive Cancer Center Core Facilities, Dr. Richard Jove for providing funds for microarray chips used in this study and Drs. Adam Bailis and Timothy O'Connor for comments on the manuscript. This research was supported by a National Cancer Institute Cancer Center Support Grant, 5P30CA033572, awarded to the City of Hope.
- Lerou PH, Daley GQ: Therapeutic potential of embryonic stem cells. Blood Rev. 2005, 19 (6): 321-31. 10.1016/j.blre.2005.01.005.View ArticlePubMed
- Draper JS, et al: Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol. 2004, 22 (1): 53-4. 10.1038/nbt922.View ArticlePubMed
- Herszfeld D, et al: CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells. Nat Biotechnol. 2006, 24 (3): 351-7. 10.1038/nbt1197.View ArticlePubMed
- Plaia TW, et al: Characterization of a new NIH-registered variant human embryonic stem cell line, BG01V: a tool for human embryonic stem cell research. Stem Cells. 2006, 24 (3): 531-46. 10.1634/stemcells.2005-0315.View ArticlePubMed
- Reya T, et al: Stem cells, cancer, and cancer stem cells. Nature. 2001, 414 (6859): 105-11. 10.1038/35102167.View ArticlePubMed
- Werbowetski-Ogilvie TE, et al: Characterization of human embryonic stem cells with features of neoplastic progression. Nat Biotechnol. 2009, 27 (1): 91-7. 10.1038/nbt.1516.View ArticlePubMed
- Mitalipova MM, et al: Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol. 2005, 23 (1): 19-20. 10.1038/nbt0105-19.View ArticlePubMed
- French PJ, et al: Identification of differentially regulated splice variants and novel exons in glial brain tumors using exon expression arrays. Cancer Res. 2007, 67 (12): 5635-42. 10.1158/0008-5472.CAN-06-2869.View ArticlePubMed
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25 (4): 402-8. 10.1006/meth.2001.1262.View ArticlePubMed
- Kuan CT, et al: Glycoprotein nonmetastatic melanoma protein B, a potential molecular therapeutic target in patients with glioblastoma multiforme. Clin Cancer Res. 2006, 12 (7 Pt 1): 1970-82. 10.1158/1078-0432.CCR-05-2797.View ArticlePubMed
- The Cancer Genome Atlas (TCGA), Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008, 455 (7216): 1061-8. 10.1038/nature07385.
- Abdel-Fattah R, et al: Differential expression of HOX genes in neoplastic and non-neoplastic human astrocytes. J Pathol. 2006, 209 (1): 15-24. 10.1002/path.1939.View ArticlePubMed
- Entin-Meer M, et al: Butyric acid prodrugs are histone deacetylase inhibitors that show antineoplastic activity and radiosensitizing capacity in the treatment of malignant gliomas. Mol Cancer Ther. 2005, 4 (12): 1952-61. 10.1158/1535-7163.MCT-05-0087.View ArticlePubMed
- Hwang SL, et al: The expression of thyroid hormone receptor isoforms in human astrocytomas. Surg Neurol. 2008, 70 (S1): 4-8. 10.1016/j.surneu.2008.03.033.View Article
- Murakami M, et al: Immunohistochemical localization of apolipoprotein E in human glial neoplasms. J Clin Invest. 1988, 82 (1): 177-88. 10.1172/JCI113568.PubMed CentralView ArticlePubMed
- Nylandsted J, Brand K, Jaattela M: Heat shock protein 70 is required for the survival of cancer cells. Ann N Y Acad Sci. 2000, 926: 122-5. 10.1111/j.1749-6632.2000.tb05605.x.View ArticlePubMed
- Rohde M, et al: Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms. Genes Dev. 2005, 19 (5): 570-82. 10.1101/gad.305405.PubMed CentralView ArticlePubMed
- Rousseau A, et al: Expression of oligodendroglial and astrocytic lineage markers in diffuse gliomas: use of YKL-40, ApoE, ASCL1, and NKX2-2. J Neuropathol Exp Neurol. 2006, 65 (12): 1149-56. 10.1097/01.jnen.0000248543.90304.2b.View ArticlePubMed
- Saino M, et al: Inhibition of angiogenesis in human glioma cell lines by antisense RNA from the soluble guanylate cyclase genes, GUCY1A3 and GUCY1B3. Oncol Rep. 2004, 12 (1): 47-52.PubMed
- Sivaparvathi M, et al: Expression and the role of cathepsin H in human glioma progression and invasion. Cancer Lett. 1996, 104 (1): 121-6. 10.1016/0304-3835(96)04242-5.View ArticlePubMed
- Walton SM, et al: Spontaneous CD8 T cell responses against the melanocyte differentiation antigen RAB38/NY-MEL-1 in melanoma patients. J Immunol. 2006, 177 (11): 8212-8.View ArticlePubMed
- Grimaldi M, Maratos M, Verma A: Transient receptor potential channel activation causes a novel form of [Ca 2+]I oscillations and is not involved in capacitative Ca 2+ entry in glial cells. J Neurosci. 2003, 23 (11): 4737-45.PubMed
- Cheng A, et al: Truncated tyrosine kinase B brain-derived neurotrophic factor receptor directs cortical neural stem cells to a glial cell fate by a novel signaling mechanism. J Neurochem. 2007, 100 (6): 1515-30.PubMed
- Lee CJ, et al: Astrocytic control of synaptic NMDA receptors. J Physiol. 2007, 581 (Pt 3): 1057-81. 10.1113/jphysiol.2007.130377.PubMed CentralView ArticlePubMed
- Galli R, et al: Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res. 2004, 64 (19): 7011-21. 10.1158/0008-5472.CAN-04-1364.View ArticlePubMed
- Singh SK, et al: Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003, 63 (18): 5821-8.PubMed
- Phillips HS, et al: Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell. 2006, 9 (3): 157-73. 10.1016/j.ccr.2006.02.019.View ArticlePubMed
- Gunther HS, et al: Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria. Oncogene. 2008, 27 (20): 2897-909. 10.1038/sj.onc.1210949.View ArticlePubMed
- Paz MF, et al: CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas. Clin Cancer Res. 2004, 10 (15): 4933-8. 10.1158/1078-0432.CCR-04-0392.View ArticlePubMed
- Murat A, et al: Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma. J Clin Oncol. 2008, 26 (18): 3015-24. 10.1200/JCO.2007.15.7164.View ArticlePubMed
- Yu C, et al: Mitochondrial Bax translocation partially mediates synergistic cytotoxicity between histone deacetylase inhibitors and proteasome inhibitors in glioma cells. Neuro Oncol. 2008, 10 (3): 309-19. 10.1215/15228517-2007-063.PubMed CentralView ArticlePubMed
- Sanai N, Alvarez-Buylla A, Berger MS: Neural stem cells and the origin of gliomas. N Engl J Med. 2005, 353 (8): 811-22. 10.1056/NEJMra043666.View ArticlePubMed
- Roelofs RF, et al: Adult human subventricular, subgranular, and subpial zones contain astrocytes with a specialized intermediate filament cytoskeleton. Glia. 2005, 52 (4): 289-300. 10.1002/glia.20243.View ArticlePubMed
- Amariglio N, et al: Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient. PLoS Med. 2009, 6 (2): e29-10.1371/journal.pmed.1000029.View Article
- Berger M, et al: Regulator of G-protein signaling-5 induction in pericytes coincides with active vessel remodeling during neovascularization. Blood. 2005, 105 (3): 1094-101. 10.1182/blood-2004-06-2315.View ArticlePubMed
- Jiang W, et al: FK506 binding protein mediates glioma cell growth and sensitivity to rapamycin treatment by regulating NF-kappaB signaling pathway. Neoplasia. 2008, 10 (3): 235-43.PubMed CentralView ArticlePubMed
- Dunlap SM, et al: Insulin-like growth factor binding protein 2 promotes glioma development and progression. Proc Natl Acad Sci USA. 2007, 104 (28): 11736-41. 10.1073/pnas.0703145104.PubMed CentralView ArticlePubMed
- Weaver AK, et al: BK channels are linked to inositol 1,4,5-triphosphate receptors via lipid rafts: a novel mechanism for coupling [Ca(2+)](i) to ion channel activation. J Biol Chem. 2007, 282 (43): 31558-68. 10.1074/jbc.M702866200.PubMed CentralView ArticlePubMed
- Fishell G, Hatten ME: Astrotactin provides a receptor system for CNS neuronal migration. Development. 1991, 113 (3): 755-65.PubMed
- Karumanchi SA, et al: Cell surface glypicans are low-affinity endostatin receptors. Mol Cell. 2001, 7 (4): 811-22. 10.1016/S1097-2765(01)00225-8.View ArticlePubMed
- Hanahan D, Weinberg RA: The hallmarks of cancer. Cell. 2000, 100 (1): 57-70. 10.1016/S0092-8674(00)81683-9.View ArticlePubMed
- Armitage P, Doll R: A two-stage theory of carcinogenesis in relation to the age distribution of human cancer. Br J Cancer. 1957, 11 (2): 161-9.PubMed CentralView ArticlePubMed
- Bignold LP, Coghlan BL, Jersmann HP: Hansemann, Boveri, chromosomes and the gametogenesis-related theories of tumours. Cell Biol Int. 2006, 30 (7): 640-4. 10.1016/j.cellbi.2006.04.002.View ArticlePubMed
- Beroukhim R, et al: Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma. Proc Natl Acad Sci USA. 2007, 104 (50): 20007-12. 10.1073/pnas.0710052104.PubMed CentralView ArticlePubMed
- Maher EA, et al: Marked genomic differences characterize primary and secondary glioblastoma subtypes and identify two distinct molecular and clinical secondary glioblastoma entities. Cancer Res. 2006, 66 (23): 11502-13. 10.1158/0008-5472.CAN-06-2072.View ArticlePubMed
- Kristensen DM, et al: Origin of pluripotent germ cell tumours: the role of microenvironment during embryonic development. Mol Cell Endocrinol. 2008, 288 (1-2): 111-8. 10.1016/j.mce.2008.02.018.View ArticlePubMed
- Haverty PM, et al: High-resolution genomic and expression analyses of copy number alterations in breast tumors. Genes Chromosomes Cancer. 2008, 47 (6): 530-42. 10.1002/gcc.20558.View ArticlePubMed
- Li L, et al: Cancer-causing karyotypes: chromosomal equilibria between destabilizing aneuploidy and stabilizing selection for oncogenic function. Cancer Genet Cytogenet. 2009, 188 (1): 1-25. 10.1016/j.cancergencyto.2008.08.016.View ArticlePubMed
- Weaver BA, Cleveland DW: Aneuploidy: instigator and inhibitor of tumorigenesis. Cancer Res. 2007, 67 (21): 10103-5. 10.1158/0008-5472.CAN-07-2266.PubMed CentralView ArticlePubMed
- Torres EM, Williams BR, Amon A: Aneuploidy: cells losing their balance. Genetics. 2008, 179 (2): 737-46. 10.1534/genetics.108.090878.PubMed CentralView ArticlePubMed
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/3/12/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.