IntroductionThe prospect of therapeutic applications of the induced pluripotent stem cells (iPSCs) is based on their ability to generate virtually any cell type present in human body. Generation of iPSCs from somatic cells has opened up new possibilities to investigate stem cell biology, to better understand pathophysiology of human diseases, and to design new therapy approaches in the field of regenerative medicine. In this study, we focus on the ability of the episomal system, a non-viral and integration-free reprogramming method to derive iPSCs from somatic cells of various origin.MethodsCells originating from neonatal and adult tissue, renal epithelium, and amniotic fluid were reprogrammed by using origin of replication/Epstein-Barr virus nuclear antigen-1 (oriP/EBNA-1)-based episomal vectors carrying defined factors. The iPSC colony formation was evaluated by using immunocytochemistry and alkaline phosphatase assay and by investigating gene expression profiles. The trilineage formation potential of generated pluripotent cells was assessed by embryoid body-mediated differentiation. The impact of additionally introduced factors on episome-based reprogramming was also investigated.ResultsReprogramming efficiencies were significantly higher for the epithelial cells compared with fibroblasts. The presence of additional factor miR 302/367 in episomal system enhanced reprogramming efficiencies in fibroblasts and epithelial cells, whereas the downregulation of Mbd3 expression increased iPSC colony-forming efficiency in fibroblasts solely.ConclusionsIn this study, we performed a side-by-side comparison of iPSC colony-forming efficiencies in fibroblasts and epithelial cells transiently transfected with episomal plasmids and demonstrated that iPSC generation efficiency was highest when donor samples were derived from epithelial cells. We determined that reprogramming efficiency of episomal system could be further improved. Considering results obtained in the course of this study, we believe that episomal reprogramming provides a simple, reproducible, and efficient tool for generating clinically relevant pluripotent cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0112-3) contains supplementary material, which is available to authorized users.

Background:It has recently been reported by several sources that original (i.e., present in vivo) glioma cell phenotypes or genotypes cannot be maintained in vitro. For example, glioblastoma cell lines presenting EGFR amplification cannot be established.Methods and results:IDH1 sequencing and loss of heterozygosity analysis was performed for 15 surgery samples of astrocytoma and early and late passages of cells derived from those and for 11 archival samples. We were not able to culture tumour cells presenting IDH1 mutations originating from currently proceeded 10 tumours; the same results were observed in 7 samples of archival material.Conclusion:The IDH1 mutation is expected to be almost mutually exclusive with EGFR amplification, so glioma cells with IDH1 mutations seem to represent a new group of tumour cells, which cannot be readily analysed in vitro because of their elimination. The reasons for this intriguing phenomenon should be investigated since its understanding can help to define a new therapeutic approach based on simulating in vivo conditions, responsible for tumour cells elimination in vitro. Moreover, a new model for culturing glioma cells in vitro should be designed since the current one does not provide conditions corresponding to in vivo growth.

Glial fibrillary acidic protein (GFAP)-positive cells derived from the neurogenic areas of the brain can be stem/progenitor cells and give rise to new neurons in vitro and in vivo. We report here that a population of GFAP-positive cells derived from fetal human brain parenchyma coexpress markers of early neural and neuronal cells, and have neural progenitor cell characteristics. We used a monolayer culture system to expend and differentiate these cells. During the initial proliferative phase, all cells expressed GFAP, nestin and low levels of betaIII-tubulin. When these cells were cultured in serum and then basic fibroblast growth factor, they generated two distinct progenies: (i) betaIII-tubulin- and nestin-positive cells and (ii) GFAP- and nestin-positive cells. These cells, when subsequently cultured in serum-free media without growth factors, ceased to proliferate and differentiated into two major neural cell classes, neurons and glia. In the cells of neuronal lineage, nestin expression was down-regulated and betaIII-tubulin expression became robust. Cells of glial lineage differentiated by down-regulating nestin expression and up-regulating GFAP expression. These data suggest that populations of parenchymal brain cells, initially expressing both glial and neuronal markers, are capable of differentiating into single neuronal and glial lineages through asymmetric regulation of gene expression in these cells, rather than acquiring markers through differentiation.

Epidermal growth factor receptor variant III (EGFRvIII) seems to constitute the perfect therapeutic target for glioblastoma (GB), as it is specifically present on up to 28–30% of GB cells. In case of other tumor types, expression and possible role of this oncogene still remain controversial. In spite of EGFRvIII mechanism of action being crucial for the design of small active anticancer molecules and immunotherapies, i.e., CAR-T technology, it is yet to be precisely defined. EGFRvIII is known to be resistant to degradation, but it is still unclear whether it heterodimerizes with EGF-activated wild-type EGFR (EGFRWT) or homodimerizes (including covalent homodimerization). Constitutive kinase activity of this mutated receptor is relatively low, and some researchers even claim that a nuclear, but not a membrane function, is crucial for its activity. Based on the analyses of recurrent tumors that are often lacking EGFRvIII expression despite its initial presence in corresponding primary foci, this oncogene is suggested to play a marginal role during later stages of carcinogenesis, while even in primary tumors EGFRvIII expression is detected only in a small percentage of tumor cells, undermining the rationality of EGFRvIII-targeting therapies. On the other hand, EGFRvIII-positive cells are resistant to apoptosis, more invasive, and characterized with enhanced proliferation rate. Moreover, expression of this oncogenic receptor was also postulated to be a marker of cancer stem cells. Opinions regarding the role that EGFRvIII plays in tumorigenesis and for tumor aggressiveness are clearly contradictory and, therefore, it is crucial not only to determine its mechanism of action, but also to unambiguously define its role at early and advanced cancer stages.

Cell line analysis is an important element of cancer research. Despite the progress in glioblastoma cell culturing, the cells isolated from the majority of specimens cannot be propagated infinitely in vitro. The aim of this study was to identify the processes responsible for the stabilization failure. Therefore, we analyzed 56 primary GB cultures, 7 of which were stabilized. Our results indicate that senescence is primarily responsible for the glioblastoma cell line stabilization failure, while mitotic catastrophe and apoptosis play a minor role. Moreover, a new technical approach allowed for a more profound analysis of the senescent cells in primary cultures, including the distinction between tumor and normal cells. In addition, we observed that glioblastoma cells in primary cultures have a varied potential to undergo spontaneous in vitro senescence, which is often higher than that of the normal cells infiltrating the tumor. Thus, this is the first report of GB cells in primary cell cultures (including both monolayer and spheroid conditions) rapidly and spontaneously becoming senescent. Intriguingly, our data also suggest that nearly half of GB cell lines have a combination of TP53 mutation and CDKN2A homozygous deletion, which are considered as mutually exclusive in glioblastoma. Moreover, recognition of the mechanisms of senescence and mitotic catastrophe in glioblastoma cells may be a step towards a potential new therapeutic approach.