Limits...
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.

Show MeSH

Related in: MedlinePlus

GFAP+NNP vs. GBM neural differentiation. a, GFAP+NNP cells at passage 1, three days of neural differentiation. Cells positive for CD44 and GFAP, and cells positive for MAP2 and GFAP but negative for CD44. MAP2 dominates but GFAP is visible. Cells CD44+, GFAP+ are also presented. b, GFAP+NNP cells at passage 1, four days of neural differentiation. Two types of cells are visible: positive for CD44 and GFAP, and cells MAP2+; GFAP+, CD44-. c, GFAP+NNP cells at passage 1, 5 days of neural differentiation: GFAP+ MAP2- and MAP2+ GFAP- cells. d, GBM2: CD44, GFAP, MAP2 positive cells- cells sustaining discordant phenotype. e, GBM 1 cells released from aggregate, 15 days of neural differentiation: cells GFAP- MAP2- and MAP2+ GFAP+ are visible. f, GBM3: CD44 negative, GFAP and MAP2 positive cells in the centre. g, GBM8: strongly MAP2 positive cells and GFAP, CD44 positive cells released from the aggregate. h, GBM4: GFAP and MAP2 positive cells sustaining Nestin expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2657909&req=5

Figure 5: GFAP+NNP vs. GBM neural differentiation. a, GFAP+NNP cells at passage 1, three days of neural differentiation. Cells positive for CD44 and GFAP, and cells positive for MAP2 and GFAP but negative for CD44. MAP2 dominates but GFAP is visible. Cells CD44+, GFAP+ are also presented. b, GFAP+NNP cells at passage 1, four days of neural differentiation. Two types of cells are visible: positive for CD44 and GFAP, and cells MAP2+; GFAP+, CD44-. c, GFAP+NNP cells at passage 1, 5 days of neural differentiation: GFAP+ MAP2- and MAP2+ GFAP- cells. d, GBM2: CD44, GFAP, MAP2 positive cells- cells sustaining discordant phenotype. e, GBM 1 cells released from aggregate, 15 days of neural differentiation: cells GFAP- MAP2- and MAP2+ GFAP+ are visible. f, GBM3: CD44 negative, GFAP and MAP2 positive cells in the centre. g, GBM8: strongly MAP2 positive cells and GFAP, CD44 positive cells released from the aggregate. h, GBM4: GFAP and MAP2 positive cells sustaining Nestin expression.

Mentions: Next, we replaced the serum-starvation medium with neural differentiation medium in GFAP+NNP cultures as well as glioblastoma cell aggregates. Under these conditions, GFAP+NNP rarely formed aggregates. If aggregates appeared, they were unstable and completely dispersed after one or two transfers due to massive release of differentiating cells. The dispersed cells showed features of differentiation and grew in monolayer (Fig. 3a,b). Using triple immunocytochemical staining, we showed that at early stages of differentiation (after 2–3 days of incubation in the neural differentiation medium), in 25–35% of the GFAP+NNP derivatives, MAP2+high/GFAP+low/CD44- phenotype could be induced. Cells with upregulated MAP2 and Beta III-tubulin expression, and downregulated GFAP expression, express TH (tyrosine hydroxylase), an enzyme required for catecholamines synthesis, which further confirms their neuronal characteristics (Fig. 4a–h). The morphology of these cells, lack of CD44 and high expression of MAP2 and Beta III-tubulin, suggested that they were potential neuronal intermediates (Fig. 5a). Moreover, their percentage was almost as high as the percentage of neuronal cells observed two days later. Among cells showing GFAP and MAP2 co-expression, most (about 70%) presented only the remnants of GFAP, which constitutes additional evidence that the neuronal cells originated from the cells initially showing a multilineage phenotype. The existence of MAP2+, GFAP+, CD44- cells has already been presented by our group [7]. We showed that the percentage of those cells after 5–7 days of neural differentiation did not exceed 5% [7].


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)

GFAP+NNP vs. GBM neural differentiation. a, GFAP+NNP cells at passage 1, three days of neural differentiation. Cells positive for CD44 and GFAP, and cells positive for MAP2 and GFAP but negative for CD44. MAP2 dominates but GFAP is visible. Cells CD44+, GFAP+ are also presented. b, GFAP+NNP cells at passage 1, four days of neural differentiation. Two types of cells are visible: positive for CD44 and GFAP, and cells MAP2+; GFAP+, CD44-. c, GFAP+NNP cells at passage 1, 5 days of neural differentiation: GFAP+ MAP2- and MAP2+ GFAP- cells. d, GBM2: CD44, GFAP, MAP2 positive cells- cells sustaining discordant phenotype. e, GBM 1 cells released from aggregate, 15 days of neural differentiation: cells GFAP- MAP2- and MAP2+ GFAP+ are visible. f, GBM3: CD44 negative, GFAP and MAP2 positive cells in the centre. g, GBM8: strongly MAP2 positive cells and GFAP, CD44 positive cells released from the aggregate. h, GBM4: GFAP and MAP2 positive cells sustaining Nestin expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: GFAP+NNP vs. GBM neural differentiation. a, GFAP+NNP cells at passage 1, three days of neural differentiation. Cells positive for CD44 and GFAP, and cells positive for MAP2 and GFAP but negative for CD44. MAP2 dominates but GFAP is visible. Cells CD44+, GFAP+ are also presented. b, GFAP+NNP cells at passage 1, four days of neural differentiation. Two types of cells are visible: positive for CD44 and GFAP, and cells MAP2+; GFAP+, CD44-. c, GFAP+NNP cells at passage 1, 5 days of neural differentiation: GFAP+ MAP2- and MAP2+ GFAP- cells. d, GBM2: CD44, GFAP, MAP2 positive cells- cells sustaining discordant phenotype. e, GBM 1 cells released from aggregate, 15 days of neural differentiation: cells GFAP- MAP2- and MAP2+ GFAP+ are visible. f, GBM3: CD44 negative, GFAP and MAP2 positive cells in the centre. g, GBM8: strongly MAP2 positive cells and GFAP, CD44 positive cells released from the aggregate. h, GBM4: GFAP and MAP2 positive cells sustaining Nestin expression.
Mentions: Next, we replaced the serum-starvation medium with neural differentiation medium in GFAP+NNP cultures as well as glioblastoma cell aggregates. Under these conditions, GFAP+NNP rarely formed aggregates. If aggregates appeared, they were unstable and completely dispersed after one or two transfers due to massive release of differentiating cells. The dispersed cells showed features of differentiation and grew in monolayer (Fig. 3a,b). Using triple immunocytochemical staining, we showed that at early stages of differentiation (after 2–3 days of incubation in the neural differentiation medium), in 25–35% of the GFAP+NNP derivatives, MAP2+high/GFAP+low/CD44- phenotype could be induced. Cells with upregulated MAP2 and Beta III-tubulin expression, and downregulated GFAP expression, express TH (tyrosine hydroxylase), an enzyme required for catecholamines synthesis, which further confirms their neuronal characteristics (Fig. 4a–h). The morphology of these cells, lack of CD44 and high expression of MAP2 and Beta III-tubulin, suggested that they were potential neuronal intermediates (Fig. 5a). Moreover, their percentage was almost as high as the percentage of neuronal cells observed two days later. Among cells showing GFAP and MAP2 co-expression, most (about 70%) presented only the remnants of GFAP, which constitutes additional evidence that the neuronal cells originated from the cells initially showing a multilineage phenotype. The existence of MAP2+, GFAP+, CD44- cells has already been presented by our group [7]. We showed that the percentage of those cells after 5–7 days of neural differentiation did not exceed 5% [7].

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