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Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors.

Rieske P, Golanska E, Zakrzewska M, Piaskowski S, Hulas-Bigoszewska K, Wolańczyk M, Szybka M, Witusik-Perkowska M, Jaskolski DJ, Zakrzewski K, Biernat W, Krynska B, Liberski PP - BMC Cancer (2009)

Bottom Line: In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP).Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin.Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Lodz, Poland. piotrrieske@yahoo.com

ABSTRACT

Background: Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed.

Methods: Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed.

Results: In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum.

Conclusion: Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

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Characterization of GFAP+NNP and GBM cells showing discordant phenotype. a, GFAP+NNP cells negative for CD133 and GFAP positive; b, GBM4 cells positive for GFAP, Beta III-tubulin and SOX-2 and cells SOX-2, Beta III-tubulin and GFAP negative; c, GBM3 cells forming aggregate; d, GBM1 cells released from aggregate positive for GFAP and MAP2; e, aggregated and released from aggregate cells positive for CD44 and Nestin (GBM 3); f, GBM2 cells original culture cells positive for CD133, and cells positive for GFAP.
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Figure 1: Characterization of GFAP+NNP and GBM cells showing discordant phenotype. a, GFAP+NNP cells negative for CD133 and GFAP positive; b, GBM4 cells positive for GFAP, Beta III-tubulin and SOX-2 and cells SOX-2, Beta III-tubulin and GFAP negative; c, GBM3 cells forming aggregate; d, GBM1 cells released from aggregate positive for GFAP and MAP2; e, aggregated and released from aggregate cells positive for CD44 and Nestin (GBM 3); f, GBM2 cells original culture cells positive for CD133, and cells positive for GFAP.

Mentions: Cells isolated from eight glioblastomas, as well as GFAP+NNP, were grown in the same culture conditions: first in expansion medium, then in serum-starvation medium, and finally in neural differentiation medium, as described in Materials and methods. All undifferentiated GFAP+NNP presented the multilineage phenotype, defined as co-expression of GFAP, CD44, Beta III-tubulin, MAP2 and Nestin, SOX-2, Vimentin [5-7]. GFAP+NNP were CD133 negative (Fig. 1a). In initial monolayer cultures isolated from eight glioblastomas, 10% (GBM6) to 95% (GBM1) of cells presented an evident multilineage phenotype (Table 2). All glioblastoma cells with multilineage phenotype were also SOX-2 positive (Fig. 1b). In addition to the population of cells with multilineage phenotype, cells with one or more of the following phenotypes were also observed in different glioblastoma cultures: MAP2+high/GFAP+/CD44-; CD44+/GFAP-/MAP2-; GFAP+/CD44+/MAP2-; MAP2+high/GFAP-/CD44-; CD133+/CD44+/MAP2+/GFAP+ and CD133+/MAP2-/GFAP- (Table 2).


Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors.

Rieske P, Golanska E, Zakrzewska M, Piaskowski S, Hulas-Bigoszewska K, Wolańczyk M, Szybka M, Witusik-Perkowska M, Jaskolski DJ, Zakrzewski K, Biernat W, Krynska B, Liberski PP - BMC Cancer (2009)

Characterization of GFAP+NNP and GBM cells showing discordant phenotype. a, GFAP+NNP cells negative for CD133 and GFAP positive; b, GBM4 cells positive for GFAP, Beta III-tubulin and SOX-2 and cells SOX-2, Beta III-tubulin and GFAP negative; c, GBM3 cells forming aggregate; d, GBM1 cells released from aggregate positive for GFAP and MAP2; e, aggregated and released from aggregate cells positive for CD44 and Nestin (GBM 3); f, GBM2 cells original culture cells positive for CD133, and cells positive for GFAP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657909&req=5

Figure 1: Characterization of GFAP+NNP and GBM cells showing discordant phenotype. a, GFAP+NNP cells negative for CD133 and GFAP positive; b, GBM4 cells positive for GFAP, Beta III-tubulin and SOX-2 and cells SOX-2, Beta III-tubulin and GFAP negative; c, GBM3 cells forming aggregate; d, GBM1 cells released from aggregate positive for GFAP and MAP2; e, aggregated and released from aggregate cells positive for CD44 and Nestin (GBM 3); f, GBM2 cells original culture cells positive for CD133, and cells positive for GFAP.
Mentions: Cells isolated from eight glioblastomas, as well as GFAP+NNP, were grown in the same culture conditions: first in expansion medium, then in serum-starvation medium, and finally in neural differentiation medium, as described in Materials and methods. All undifferentiated GFAP+NNP presented the multilineage phenotype, defined as co-expression of GFAP, CD44, Beta III-tubulin, MAP2 and Nestin, SOX-2, Vimentin [5-7]. GFAP+NNP were CD133 negative (Fig. 1a). In initial monolayer cultures isolated from eight glioblastomas, 10% (GBM6) to 95% (GBM1) of cells presented an evident multilineage phenotype (Table 2). All glioblastoma cells with multilineage phenotype were also SOX-2 positive (Fig. 1b). In addition to the population of cells with multilineage phenotype, cells with one or more of the following phenotypes were also observed in different glioblastoma cultures: MAP2+high/GFAP+/CD44-; CD44+/GFAP-/MAP2-; GFAP+/CD44+/MAP2-; MAP2+high/GFAP-/CD44-; CD133+/CD44+/MAP2+/GFAP+ and CD133+/MAP2-/GFAP- (Table 2).

Bottom Line: In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP).Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin.Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Lodz, Poland. piotrrieske@yahoo.com

ABSTRACT

Background: Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed.

Methods: Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed.

Results: In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum.

Conclusion: Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

Show MeSH
Related in: MedlinePlus