Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells

<p>Abstract</p> <p>Background</p> <p>Although extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation – allowing for an increase in percentage yield of neuronal cells.</p> <p>Results</p> <p>Uncommitted GFAP and SOX2 positive neural progenitors – so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100β-, SOX2-], or mixture of neural and non-neural cells.</p> <p>In spite of successfully increasing the percentage yield of glial and neuronal <it>vs</it>. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells – i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively.</p> <p>Conclusion</p> <p>We suggest that biotechnologists attempting to enrich <it>in vitro </it>neural cell cultures in one type of cells – such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.</p

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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 <it>EGFR </it>amplification analysis, LOH/MSI analysis, and <it>P53 </it>nucleotide sequence analysis were performed.</p> <p>Results</p> <p><it>In vitro </it>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.</p> <p>Conclusion</p> <p>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 <it>in vitro </it>multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.</p

Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies

Glioblastoma cell cultures in vitro are frequently used for investigations on the biology of tumors or new therapeutic approaches. Recent reports have emphasized the importance of cell culture type for maintenance of tumor original features. Nevertheless, the ability of GBM cells to preserve EGFR overdosage in vitro remains controversial. Our experimental approach was based on quantitative analysis of EGFR gene dosage in vitro both at DNA and mRNA level. Real-time PCR data were verified with a FISH method allowing for a distinction between EGFR amplification and polysomy 7. We demonstrated that EGFR amplification accompanied by EGFRwt overexpression was maintained in spheroids, but these phenomena were gradually lost in adherent culture. We noticed a rapid decrease of EGFR overdosage already at the initial stage of cell culture establishment. In contrast to EGFR amplification, the maintenance of polysomy 7 resulted in EGFR locus gain and stabilization even in long-term adherent culture in serum presence. Surprisingly, the EGFRwt expression pattern did not reflect the latter phenomenon and we observed no overexpression of the tested gene. Moreover, quantitative analysis demonstrated that expression of the truncated variant of receptor—EGFRvIII was preserved in GBM-derived spheroids at a level comparable to the initial tumor tissue. Our findings are especially important in the light of research using glioblastoma culture as the experimental model for testing novel EGFR-targeted therapeutics in vitro, with special emphasis on the most common mutated form of receptor—EGFRvIII

Innovative biodegradable dibutyrylchitin dressing for the treatment of ulcers occurring during chronic venous insufficiency in patients with type 2 diabetes

The aim of this study was to assess the course of the healing process following the use of dibutyrylchitin (DBC) dressing, a fully degradable material used in the treatment of ulcers which occur during chronic venous insufficiency common in patients suffering from type 2 diabetes. These diseases have a significant impact on the patients’ standard of living, including the potential employment, and on the declining attendance at the current workplace. The implementation of this innovative therapeutic solution may positively affect the above-mentioned difficulties. An analysis of the healing process, following the application of the DBC dressing, was performed. Once the dressing was positioned on the wound, the analysis indicated that it underwent a process of degradation facilitated by the enzymes occurring naturally in the wound. When fully degraded, a further layer was applied. This process was repeated until the wound was fully healed. The study group consisted of 4 patients previously diagnosed with type 2 diabetes. During the observation period, the ulcers in all 4 cases had healed. The examined wound dressings adhered well to the wound surface and degraded within it. No side effects or adverse effects of the applied innovative therapy were observed. An addition of the biodegradable DBC dressing to the standard therapy procedure of ulcers occurring during chronic venous insufficiency among patients with type 2 diabetes indicate safe and effective treatment, which may have a direct reflection in the patient’s professional capacity enhancement. It resulted in the complete healing of all ulcers in each of the observed cases