Background: Wolfram syndrome (WFS) is a rare autosomal recessive syndrome in which diabetes mellitus and neurodegenerative disorders occur as a result of Wolframin deficiency and increased ER stress. In addition, WFS1 deficiency leads to calcium homeostasis disturbances and can change mitochondrial dynamics. The aim of this study was to evaluate protein levels and changes in gene transcription on human WFS cell model under experimental ER stress. Methods: We performed transcriptomic and proteomic analysis on WFS human cell model—skin fibroblasts reprogrammed into induced pluripotent stem (iPS) cells and then into neural stem cells (NSC) with subsequent ER stress induction using tunicamycin (TM). Results were cross-referenced with publicly available RNA sequencing data in hippocampi and hypothalami of mice with WFS1 deficiency. Results: Proteomic analysis identified specific signal pathways that differ in NSC WFS cells from healthy ones. Next, detailed analysis of the proteins involved in the mitochondrial function showed the down-regulation of subunits of the respiratory chain complexes in NSC WFS cells, as well as the up-regulation of proteins involved in Krebs cycle and glycolysis when compared to the control cells. Based on pathway enrichment analysis we concluded that in samples from mice hippocampi the mitochondrial protein import machinery and OXPHOS were significantly down-regulated. Conclusions: Our results show the functional and morphological secondary mitochondrial damage in patients with WFS. Graphical

Cellular origin of glioblastoma (GB) is constantly discussed and remains a controversial subject. Unfortunately, neurobiologists are not consistent in defining neural stem cells (NSC) complicating this issue even further. Nevertheless, some suggestions referring to GB origin can be proposed based on comparing GB to central nervous system (CNS) cells. Firstly, GB cells showin vitrodifferentiation pattern similar to GFAP positive neural cells, rather than classical (GFAP negative) NSC. GB cells in primary cultures become senescentin vitro, similar to GFAP positive neural progenitors, whereas classical NSC proliferatein vitroinfinitely. Classical NSC apoptosis triggered by introduction of IDH1R132H undermines hypothesis stating that IDH-mutant (secondary) GB origins from these NSC. Analysis of biological role of typical IDH-wildtype (primary) GB oncogene such as EGFRvIII also favors GFAP positive cells rather than classical NSC as source of GB. Single-cell NGS and single-cell transcriptomics also suggest that GFAP positive cells are GB origin. Considering the above-mentioned and other discussed in articles data, we suggest that GFAP positive cells (astrocytes, radial glia, or GFAP positive neural progenitors) are more likely to be source of GB than classical GFAP negative NSC, and furtherin vitroassays should be focused on these cells. It is highly possible that several populations of tumor initiating cells (TIC) exist within GB, adjusting their phenotype and even genotype to various environmental conditions including applied therapy and periodically going through different TIC states as well as non-TIC state. This adjustment is driven by changes in number and types of amplicons. The existence of various populations of TIC would enable creating neoplastic foci in different environments and increase tumor aggressiveness.

Background: Glioblastoma (GB) is considered one of the most lethal tumors. Extensive research at the molecular level may enable to gain more profound insight into its biology and thus, facilitate development and testing of new therapeutic approaches. Unfortunately, stable glioblastoma cell lines do not reflect highly heterogeneous nature of this tumor, while its primary cultures are difficult to maintain in vitro. We previously reported that senescence is one of the major mechanisms responsible for primary GB cells stabilization failure, to a lesser extent accompanied by apoptosis and mitotic catastrophe-related cell death. Methods: We made an attempt to circumvent difficulties with glioblastoma primary cultures by testing 3 different approaches aimed to prolong their in vitro maintenance, on a model of 10 patient-derived tumor specimens. Results: Two out of ten analyzed GB specimens were successfully stabilized, regardless of culture approach applied. Importantly, cells transduced with immortalizing factors or cultured in neural stem cell-like conditions were still undergoing senescence/apoptosis. Sequential in vivo/in vitro cultivation turned out to be the most effective, however, it only enabled to propagate cells with preserved molecular profile up to 3^rd mice transfer. Nevertheless, it was the only method that impeded these phenomena long enough to provide sufficient amount of material for in vitro/in vivo targeted analyses. Interestingly, our data additionally demonstrated that some subpopulations of several stabilized GB cell lines undergo idiopathic senescence, however, it is counterbalanced by simultaneous proliferation of other cell subpopulations. Conclusions: In the majority of primary glioma cultures, there has to be an imbalance towards apoptosis and senescence, following few weeks of rapid proliferation. Our results indicate that it has to be associated with the mechanisms other than maintenance of glioblastoma stem cells or dependence on proteins controlling cell cycle.

Background: The biological role of EGFRvIII (epidermal growth factor receptor variant three) remains unclear. Methods: Three glioblastoma DK-MG sublines were tested with EGF (epidermal growth factor) and TGFβ (transforming growth factor β). Sublines were characterized by an increased percentage of EGFRvIII-positive cells and doubling time (DK-MG^low to DK-MG^extra-high), number of amplicons, and EGFRvIII mRNA expression. The influence of the growth factors on primary EGFRvIII positive glioblastomas was assessed. Results: The overexpression of exoEGFRvIII in DK-MG^high did not convert them into DK-MG^extra-high, and this overexpression did not change DK-MG^low to DK-MG^high; however, the overexpression of RAS^G12V increased the proliferation of DK-MG^low. Moreover, the highest EGFRvIII phosphorylation in DK-MG^extra-high did not cause relevant AKT (known as protein kinase B) and ERK (extracellular signal-regulated kinase) activation. Further analyses indicate that TGFβ is able to induce apoptosis of DK-MG^high cells. This subline was able to convert to DK-MG^extra-high, which appeared resistant to this proapoptotic effect. EGF acted as a pro-survival factor and stimulated proliferation; however, simultaneous senescence induction in DK-MG^extra-high cells was ambiguous. Primary EGFRvIII positive (and SOX2 (SRY-Box Transcription Factor 2) positive or SOX2 negative) glioblastoma cells differentially responded to EGF and TGFβ. Conclusions: The roles of TGFβ and EGF in the EGFRvIII context remain unclear. EGFRvIII appears as a weak oncogene and not a marker of GSC (glioma stem cells). Hence, it may not be a proper target for CAR-T (chimeric antigen receptor T cells).

Mutation in isocitrate dehydrogenase 1 (IDH1^R132H) occurs in various types of cancer, including low and high grade gliomas. Despite high incidence indicating its central role in tumor initiation and progression there are no targeted therapies directed against this oncogene available in the clinic. This is due to the limited understanding of the role of IDH1^R132H in carcinogenesis, which is further propagated by the lack of appropriate experimental models. Moreover, proper in vitro models for analysis of gliomagenesis are required. In this study, we employed a Tet On system to generate human induced neural stem cells with doxycycline-inducible IDH1^R132H. Equivalent expression of both forms of IDH1 in the presented model remains similar to that described in tumor cells. Additional biochemical analyses further confirmed tightly controlled gene regulation at protein level. Formation of a functional mutant IDH1 enzyme was supported by the production of D-2-hydroxyglutarate (D2HG). All samples tested for MGMT promoter methylation status, including parental cells, proved to be partially methylated. Analysis of biological effect of IDH1^R132H revealed that cells positive for oncogene showed reduced differentation efficiency and viability. Inhibition of mutant IDH1 with selective inhibitor efficiently suppressed D2HG production as well as reversed the effect of mutant IDH1 protein on cell viability. In summary, our model constitutes a valuable platform for studies on the molecular basis and the cell of origin of IDH-mutant glioma (e.g. by editing P53 in these cells and their derivatives), as well as a reliable experimental model for drug testing.

Recent years have seen rapid progress in biological treatments for genetic diseases, as well as conditions like type 1 diabetes that lack an obvious genetic component. The authors sought to explain why this progress has emerged at this particular moment. The best way to illustrate this is by showcasing a wide range of therapies targeting diverse diseases. This progress has been driven by technological advances in genetically modified CAR-T and CAR-NK cells (e.g., using CRISPR or transgenes), which have led to significant improvements in cancer therapy. A key trend now is the emergence of “off-the-shelf” approaches aimed at generating cellular therapies compatible with a range of recipients by mitigating alloreactivity and immune rejection. Different diseases impose distinct biological and logistical limitations; thus, treatment of each patient requires an appropriate strategy. Emerging advances include the modification of therapeutic cells, either ex vivo or in vivo. Current options for transgene delivery mainly comprise lipid nanoparticles (LNPs), adeno-associated virus (AAV) vectors, and lentiviral vectors. Researchers also focus on selecting suitable promoters for specific expression in selected cell types. Altogether, these advances have led to remarkable progress in treating various diseases in recent years. This publication discusses the development of biological therapies, with particular emphasis on cell and gene therapies, illustrated by viable examples across various disorders. It covers implemented solutions for several types of cancer, as well as selected hereditary diseases and syndromes, including Huntington’s disease, carbamoyl phosphate synthetase 1 (CPS1) deficiency, hemiplegia, epidermolysis bullosa, chronic granulomatous disease, and congenital deafness. Emerging applications in heart diseases and diabetes are also summarized, along with therapeutic strategies involving tRNA gene editing. Although numerous strategies exist, only the most representative, practical, and up-to-date examples are emphasized.