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1

Inducing efÞ cient catecholaminergic differentiation of GFAP, SOX2-positive neural progenitors (NHA) by means of kinetic factors

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Rieske P., Witusik M., Wolanczyk M., Krynska B.
Acta Neurobiologiae Experimentalis
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2009
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tom 69
|
nr 1
Extensive research has been performed to control differentiation of neural stem cells. For last three years we have strived to obtain stabilized protocol of catecholaminergic differentiation of GFAP, SOX2- positive neural progenitors (NHA) In 2007 we published fi rst results presenting differentiation of GFAP positive neural progenitors (NHA) in accordance to model of discordant phenotypes suppression (Rieske 2007, Eur J Neurosci). Since the beginning of 2008 we are able to differentiate uncommitted GFAP and SOX2 positive neural progenitors (NHA) in different environmental conditions to: only neural cells consisted of neuronal and glial cells, or fi broblast-like cells, or mixture of neural and fi broblast-like cells (Witusik 2007, Brain Res; Witusik 2008 BMC Biotechnol). In spite of successful blockade of fi broblast-like differentiation by means of environmental changes, we were able to barely increase the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. It was so far also impossible to alter radically ratio catecholaminergic /GABA-ergic cells by means of changing environmental factors (SHH, GDNF, bFGF, EGF, BMPs). It strongly suggested infl uence of stochastic events or so called continuum processes during neuronal versus glial, and catecholaminergic versus GABA-ergic differentiation of described neural progenitors. Nevertheless including kinetic factors to our differentiation protocols allowed to increase percentage of catecholaminergic cells from 10 to 45% of neuronal cells.
2

In vitro cultured 3D aggregates of glioblastoma cells preserve in vivo observed phenotype and genotype of this tumour

51%
Witusik-Perkowska M., Bienkowski M., Jurga M., Stoczynska-Fidelus E., Hulas-Bigoszewska K., Roci I., McGuckin C. P., Banaszyk M., Wolanczyk M., Sikorska B., Liberski P. P., Rieske P.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
|
nr 1
Surprisingly in vitro conditions allowing to culture glioblastoma cells presenting EGFR amplification were not known until now. Our evaluation of EGFR amplification status in glioblastoma (GBM) culture demonstrated that this anomaly was preserved for months in spheroids (aggregated glioblastoma cells) at a level comparable to the earliest passage of cell culture. In contrast, and in accordance with already published data we detected it as completely lost in the adherent culture. Apparently glioblastoma cells presenting EGFR amplification become apoptotic in the regular cell culture conditions. In addition discrepant expression of SOX2 and multilineage phenotype recognized as a markers of neural progenitors was observed in monolayer and 3D culture. Moreover, our analyses showed a decreased invasion potential of adherent GBM loosing EGFR amplification, and spheroids maintaining EGFR amplification. In conclusion, our findings confirm that GBM-derived spheroids seem to be a promising tool to preserve original molecular features of the tumor in vitro, with a special emphasis on EGFR gene aberrations, including EGFRvIII, regarded as novel therapeutic target. Our last unpublished and preliminary data suggest that mechanism responsible for in vitro death of adherent glioblastoma cells showing EGFR amplification seems to be linked to the artificial in vitro cell-cell interaction rather than to the lack of proper autocrine effects. Those suggestion came from analysis of glioblastoma cells in artificial brain tissues system and analysis of aggregated glioblastoma cells only. The present study was supported by the Ministry of Science and Higher Education, Poland, grant No. N N401047337 and Foundation for Polish Science(PARENT-BRIDGE Programme - support for women, No.POMOST_C/15).
3

EGFR involvement in neural and mesenchymal differentiation of glioblastoma progenitor cells

51%
Rieske P., Witusik P., Golanska E., Piaskowski S., Zakrzewski M., Gresner S., Jaskolski D. J., Zakrzewski K., Wolanczyk M., Krynska B., Hulas-Bigoszewska K., Szybka M., Kulczycka-Wojdala D., Liberski P. P.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 1
We have compared differentiation ability of glioblastoma cells with differentiation ability of neural progenitors. An effi cient differentiation arrest was observed in all cell lines isolated from glioblastomas in contrast to normal neural progenitors. However, cells isolated from six glioblastomas showed features of early stages of neural differentiation. Moreover, the cells derived from a majority of glioblastomas (8 out of 10) as well as neural progenitors showed features of non-neural (mesenchymal-like) differentiation. Moreover aggregated cells sustained EGFR amplifi cation, whereas cells grown as a monolayer did not. Cells showing EGFR amplifi cation became apoptotic grown as monolayer. Majority of mesenchymally differentiated glioblastoma cells showed features of senescence. Novel hypotheses which we would like to test are as follows: Neural progenitors could be a potential source of glioblastomas. Glioblastoma presents not only tumor stem cells but also tumor progenitor cells. Stable coexpression of glial and neuronal markers presented by glioblastoma cells results from differentiation arrest. Aggregating glioblastoma cells allows to sustain, EGFR amplifi cation in vitro. Moreover aggregated cells show proliferation ability, and differentiation arrest, whereas monolayer cells can be effi ciently differentiated and fi nally senescent. It suggests that simultaneous analysis of differentiation processes altogether with considering status of genes such as EGFR may help in designing new molecular targets for chemotherapy.
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