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. 2010 Nov 17;31(3):337–343. doi: 10.1007/s10571-010-9627-4

Identification of CD133−/Telomeraselow Progenitor Cells in Glioblastoma-Derived Cancer Stem Cell Lines

Fabian Beier 1, Christoph P Beier 2,✉, Ines Aschenbrenner 3, Gerhard C Hildebrandt 1,4, Tim H Brümmendorf 5, Dagmar Beier 2
PMCID: PMC11498608  PMID: 21082235

Abstract

Glioblastoma multiforme (GBM) is paradigmatic for the investigation of cancer stem cells (CSC) in solid tumors. The CSC hypothesis implies that tumors are maintained by a rare subpopulation of CSC that gives rise to rapidly proliferating progenitor cells. Although the presence of progenitor cells is crucial for the CSC hypothesis, progenitor cells derived from GBM CSC are yet uncharacterized. We analyzed human CD133+ CSC lines that were directly derived from CD133+ primary astrocytic GBM. In these CSC lines, CD133+/telomerasehigh CSC give rise to non-tumorigenic, CD133−/telomeraselow progenitor cells. The proliferation of the progenitor cell population results in significant telomere shortening as compared to the CD133+ compartment comprising CSC. The average difference in telomere length as determined by a modified multi-color flow fluorescent in situ hybridization was 320 bp corresponding to 4–8 cell divisions. Taken together, we demonstrate that CD133+ primary astrocytic GBM comprise proliferating, CD133−/telomeraselow progenitor cell population characterized by low telomerase activity and shortened telomeres as compared to CSC.

Keywords: Glioblastoma multiforme, Cancer stem cells, Telomere, Telomerase, Progenitor cells

Introduction

Glioblastoma multiforme (GBM) is one of the most devastating tumor entities with a median survival of 14.6 months despite multimodal therapy (Stupp et al. 2005). Since the first discovery of CD133+ cancer stem cells (CSC), GBM became paradigmatic for the investigation of CSC in solid malignancies (Beier et al. 2007; Galli et al. 2004; Singh et al. 2004). In these tumors, only CD133+, but not CD133−, cells reconstituted the initial tumor in vivo when injected into nude mice (Bao et al. 2006; Beier et al. 2007; Singh et al. 2004; Son et al. 2009). Even though GBM CSC have been initially identified as CD133+ tumor cells, CD133 expression does not seem to be an ultimate prerequisite, since CD133− GBM CSC have recently been identified as well (Beier et al. 2007; Gunther et al. 2008). GBM-derived CSC lines provide a powerful tool to elucidate the biology of GBM (Lee et al. 2006). Based on the similarity of CSC and neural stem cells (NSC) multiple authors have postulated the existence of rapid proliferating progenitor cells with limited proliferative potential (e.g., Reya et al. 2001). Slowly proliferating NSC give rise to a series of progressively more lineage-restricted rapidly proliferating progenitor cells with limited proliferative potential. The progenitor cells then terminally differentiate into highly specialized cells expressing markers of their respective neural lineages (Ravin et al. 2008; Reynolds and Weiss 1992; Rietze et al. 2001). The presence of rapidly proliferating progenitor cells is a key prediction of the CSC hypothesis (Reya et al. 2001) because their depletion explains the shrinkage of tumors (elimination of progenitor cells) without curing the patients (failure to target CSC).

We aimed to identify progenitor cell populations downstream of CD133+ CSC by investigating telomerase activity and telomere length in the CD133+ subgroup of primary astrocytic GBM. Telomeres act as a protective cap to avoid chromosomal instability and shorten with each cell division unless the cells express telomerase, an enzyme reinstating lost DNA telomere sequences (Levy et al. 1992). In telomerase negative cells, telomere length reflects the replicative history (Allsopp et al. 1992) and the determination of telomere length is a useful tool to decipher the pathogenesis of various diseases (Beier et al. 2005; Calado and Young 2009; Drummond et al. 2007). In the haematopoietic system and during neurogenesis, there is a marked difference in telomerase activity and telomere length of haematopoietic/NSC, stem cell-derived progenitor cells, and fully differentiated cells (Drummond et al. 2007; Engelhardt et al. 1997; Kruk et al. 1996; Van Ziffle et al. 2003). We used an adopted multi-color flow fluorescent in situ hybridization (FISH) protocol (Beier et al. 2005) to investigate telomere length in CSC and putative progenitor cells and provide experimental evidence for the presence of CD133−/telomeraselow progenitor cells in CD133+ CSC lines derived from primary astrocytic GBM.

Materials and Methods

Culture of Primary GBM Cells and Spheres

The generation and propagation of all CSC lines used have been previously described (Beier et al. 2007). In brief, CSC lines were derived from freshly resected astrocytic GBM. After dissociation, tumor cells were grown in stem cell permissive DMEM-F12 medium (Invitrogen, USA) supplemented with 20 ng/ml of each human recombinant epidermal growth factor, human recombinant basic fibroblast growth factor (both R&D Systems, Germany), leukemia inhibitory factor (Chemicon, USA), and 2% B27 Supplement (Life Technologies, USA).

Fluorescence Activated Cell Sorting (FACS)

Cells were stained using CD133/2-PE (1:100, Miltenyi Biotech, Germany) or isotype control antibody (1:100 mIgGk-PE, Caltag, Germany). FACS was performed on BD FACSAria (Becton–Dickinson, USA) and analyzed as described in the figure legends.

Detection of Telomerase Activity

Telomerase activity from 2 × 105 thawed tumor cells was determined using the TeloTAGGG PCR ELISA plus kit according to the manufacturer’s instruction (Roche, Germany).

Detection of Telomere Length

Telomere length was determined as described previously (Beier et al. 2005) with minor modifications: CD133 was stained using the CD133/2-biotin labeled antibody (Miltenyi Biotech, Germany) and detected using streptavidin-Cy5 and a mouse-specific secondary Cy5-labeled antibody (Becton Dickenson, USA). Telomere length of cow thymocytes was determined using southern blot (Beier et al. 2005).

Results

GBM CSC Lines Show Hierarchical Organization

We used four previously published CSC lines (R11, R18, R28, and R52) derived from CD133+ primary astrocytic GBM for investigation of the hierarchical organization of tumor cells (Beier et al. 2007, 2008; Lottaz et al. 2010). In these CSC lines, the clonogenic index of CD133+ and CD133− subpopulations of tumor cells differed significantly, and only a very small subset of cells mainly within the CD133+ fraction of tumor cells featured stem cell properties (Fig. 1a and data published in Beier et al. 2007). Conversely and in line with previous publications (Singh et al. 2004), the CD133+ subpopulation of tumor cells which has been shown to comprise the small subpopulation of CSC was highly tumorigenic when injected into T-lymphocyte deficient mice. The CD133− population showed reduced or no tumorigenicity (published in Beier et al. 2008).

Fig. 1.

Fig. 1

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Hierarchical organization of the GBM CSC lines. a 1000 freshly sorted CD133+ and CD133− cells were plated out and clonogenicity was determined as doublet after 14 days in three different cell lines (R11, R18, and R28). Mean and SE are given. b CD133+ (left panel) and CD133− cells (right panels) were purified by FACS and analyzed for CD133 expression after 1, 2, 4, and 7 days (representative data of the CSC line R11 are given). c Cell cycle analysis of tumor cells (gate R1) is given. Cells in G1 and G2 phases are further distinguished based on CD133 expression (horizontal line gives level of background fluorescence, data of CSC line R11 is shown)

We then questioned, if CD133 was only assigned to CSC and some generations of cells directly derived from CSC, or if CD133− tumor cells could give rise to CD133+ tumor cells as well. Tumor cells were sorted for their CD133 expression and CD133+ and CD133− fractions of tumor cells were cultured separately. FACS analysis of each fraction was repeated four times a week. As suggested by the CSC hypothesis, CD133+ cells gave rise to CD133− cells (Fig. 1b). Within 24 h the ratio of CD133+ and CD133− cells normalized to the original values before the FACS. In contrast, CD133− cells did not re-acquire CD133 expression within 7 days (Fig. 1b). According to the cancer stem cell hypothesis and in line with our results so far, the proliferative capacity of tumor cells is inversely correlated with the degree of differentiation. However, committed progenitor cells have been shown to cycle more actively as opposed to both immature stem cells as well as to terminally differentiated cells. Knowing the impaired proliferative capacity of CD133− cells (Fig. 1a), we determined the actual rate of proliferation of CD133+ and CD133− cells and performed cell cycle analysis of both fractions within the CSC lines. A relevant proportion (median 42.9% range 19.9–77.7%) of proliferating cells did not express CD133 indicating CD133−, cycling, non-tumorigenic cells with limited proliferative capacity (Fig. 1c).

Telomere Shortening in CD133− Cells Indicates Limited Proliferative Capacity and Lack of Effective Telomere Maintenance

NSC but not NSC-derived progenitor cells effectively maintain telomere length by the expression of telomerase (Kruk et al. 1996). We could recently show that different types of GBM CSC lines feature high similarities either with adult or fetal NSC on a transcriptional level (Lottaz et al. 2010). Therefore, we questioned if the different fractions of tumor cells within the CD133+ GBM CSC line maintain telomere length by expression of telomerase and if telomerase decreases during differentiation of tumor cells. In sorted CD133+ and CD133− fractions of tumor cells, CD133+ cells showed significantly higher telomerase activity (CD133+/telomerasehigh) as compared to the CD133− subpopulation (CD133−/telomeraselow, Fig. 2a).

Fig. 2.

Fig. 2

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Telomerase activity and telomere length in CD133+ and CD133− cells. a Relative telomerase activity of 105 sorted CD133+ and CD133− GBM cells is shown. The mean and SD of three independent experiments are given (* P < 0.05 two-sided Student’s t test, data of CSC line R11 is given). b The telomere length in CD133+ and CD133− cells of the CSC lines R11 were analyzed by multi-color flow FISH. Diploid cells (gate R1) are gated together with the cow thymocytes (used as internal control) based on size and PI staining and further differentiated based on size and granularity (gate R2). In gate R3, the telomere length analysis of cow thymocytes used as internal control is shown: the right peak represents the telomere fluorescence while the left peak gives the background autofluorescence. GBM cells (gate R2) were further separated based on their Cy5-labeled CD133 of or isotype antibody staining. Telomere length analysis of CD133+ (gate R4) and CD133− cells (gate R5). CD133 negative cells from R5 are represented marked as dark gray peaks while CD133 positive cells from R4 are shown as white grey peaks (n = 4 independent experiments with the CSC line R11). c The difference of the telomere length between CD133+and CD133− cells was reproduced in four CD133+ GBM tumor cell lines (R18, R28, R52, and R11). The mean difference was 0.32 kbp ± 0.09 SE (P = 0.043, paired Student’s t test)

Because CD133− cells showed significantly lower telomerase expression and a restricted proliferative capacity, telomere length was suspected to have undergone accelerated shortening in CD133− as opposed to CD133+ tumor cells upon differentiation. We therefore analyzed telomere length of CD133+ and CD133− cells using an adapted multi-color flow-FISH protocol (Beier et al. 2005). Indeed, telomere length in CD133− cells was found to be significantly shorter as compared to their CD133+ counterparts (Fig. 2b). In the investigated CSC lines, the average difference of telomere length was 320 bp (Fig. 2b). Assuming a telomere loss per cell division of 50–100 bp (Calado and Young 2009; Drummond et al. 2007) telomere difference suggests 4–8 additional cell divisions of CD133−/telomeraselow progenitor cells (Fig. 2c). Together, our data suggests the presence of proliferating CD133−/telomeraselow progenitor cells derived from CD133+/telomerasehigh CSC (Fig. 3) and hereby provides experimental proof-of-concept of postulated CSC-derived progenitor cells.

Fig. 3.

Fig. 3

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Overview of the proposed model of CSC-derived progenitor cells

Discussion

The CSC hypothesis is accepted in the haematopoietic system (Bonnet and Dick 1997; Elrick et al. 2005) and may also explain the intratumoral heterogeneity of solid tumors. CSC-derived rapidly dividing progenitor cells are suspected to constitute the major component of actively proliferating malignant tumors (Reya et al. 2001). In contrast to CSC, progenitor cells are supposed to be more sensitive to therapy. However, elimination of the progenitor cell pool remains insufficient to cure GBM unless CSC representing a continued source of relapse even in a minimal residual disease scenario are efficiently targeted (Reya et al. 2001). Only very recently, Chen et al. (2010) proved for the first time the existence of CSC-derived progenitor cells in PTEN deficient GBM cell lines using a clonogenic approach for hierarchy characterization. Surprisingly, but in line with our results, these progenitor did not express the marker CD133. Due to the reasons mentioned above, a better understanding of CSC-derived progenitor cells seems of particular importance, since the mechanisms underlying these transitions could be of immense therapeutic value for targeted treatment approaches.

Using molecular and functional markers, we could experimentally show that amplifying progenitor cells can be defined prospectively in GBM. CD133+/telomerasehigh GBM CSC give rise to proliferating CD133−/telomeraselow cells that were neither tumorigenic nor clonogenic. These results suggest that at least a subgroup of progenitor cells is CD133− (Fig. 3) that may correspond to the type III cells described by Chen et al. (2010). However, our results do not exclude the coexistence of CD133+ progenitor cells or yet uncharacterized cell types.

Further, our data suggest that telomere length and the expression of telomerase might be a crucial difference between CSC and CSC-derived progenitor cells. Similar to NSC and NSC-derived progenitor cells (Richardson et al. 2007; Kruk et al. 1996), the CD133+ compartment comprising CSC expressed significantly higher levels of telomerase activity and showed longer telomeres as compared to more differentiated CD133− progenitor and terminally differentiated cells. Therefore, we propose shorter telomere length and telomerase expression as additional marker for CSC and progenitor cells directly derived from CSC of GBM and as a putative target for new approaches aimed at the targeting of immature tumor cells (Bernhardt et al. 2006; Marian et al. 2010). Conversely, the lower expression of telomerase and shorter telomeres in CD133− proliferating cells seems to characterize progenitor cells. The differing telomerase expression and telomere length in stem cells and progenitor cells is consistent with reports on the haematopoietic system and neurogenesis (Drummond et al. 2007; Kruk et al. 1996). In line with our result, the more lineage-specific downstream precursor cells show no telomerase activity (Drummond et al. 2007; Engelhardt et al. 1997). Our study also allowed clues on the putative number of cell divisions within the progenitor cell compartment. Although the exact number is hard to determine, our data is in line with previous findings in normal NSC and allows an approximation that may be used to improve recently proposed mathematical models (Ganguly and Puri 2006; Kruk et al. 1996).

Together, we here demonstrate the actual existence of CD133−/telomeraselow CSC-derived progenitor cell compartment in GBM and propose telomere length/telomerase activity as a new marker for this important cellular compartment. Further studies will help to elucidate the role of the progenitor cell population in the complex organization of brain tumors and their response to therapies.

Acknowledgments

We would like to thank Gunnar Müller and Elena Spacenko for their excellent technical assistance. This work was supported by the Reform A grant of the University of Regensburg, the NGFNplus Brain Tumor Network (Subproject 7 no. 01GS0887), and the Max Eder grant of the Deutsche Krebshilfe e.V.

Conflict of interest

None.

Footnotes

Fabian Beier and Christoph P. Beier equally contributed to this study.

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