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Povzetek: The major obstacle to the application of mesenchymal stromal cells (MSCs) in regenerative medicine is the expansion of the donor-derived cells in vitro to obtain high cell numbers in the shortest possible time. However, MSCs gradually undergo replicative senescence after a variable number of divisions that reduce their therapeutic efficacy, which needs to be determined before administration. The authors developed a fast and simple evaluation assay testing two senescence inducers, mitoxantrone (Mxt) and trichostatin A (TSA), to predict the onset of spontaneous replicative senescence of adipose-derived mesenchymal stromal cells (ASCs) and have confirmed the correlation between induced senescence and spontaneous replicative senescence in the assay using Mxt. This protocol facilitates the standardization of therapeutic ASCs and MSCs from other origins before application.
Ključne besede: adipose mesenchymal stromal (stem) cells (ASCs), cell longevity, induced senescence, mesenchymal stromal (stem) cells (MSCs), mitoxantrone, replicative senescence, trichostatin A
Objavljeno v DiRROS: 16.07.2024; Ogledov: 1; Prenosov: 1
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Povzetek: Purpose: Glioblastoma (GBM) is the most common primary brain tumour and one of the deadliest cancers. In addition to late diagnosis and inadequate treatment, the extremely low survival rate is also due to the lack of appropriate therapeutic biomarkers and corresponding therapeutic agents. One of the potential therapeutic biomarkers is the intermediate filament vimentin, which is associated with epithelial-mesenchymal transition (EMT). The purpose of this study was to analyse the effect of the anti-vimentin nanobody Nb79 on cell invasion in vitro and in vivo. To further our understanding of the mechanism of action, we investigated the association between Nb79 and EMT in GBM and GBM stem cells by analysing the expression levels of key EMT-related proteins. Methods: The expression of vimentin in glioma tissues and cells was determined by RT-qPCR. An invasion assay was performed on differentiated glioblastoma cell line U-87 MG and stem cell line NCH421k in vitro as well as in vivo in zebrafish embryos. The effect of Nb79 on expression of EMT biomarkers beta-catenin, vimentin, ZEB-1 and ZO1 was determined by Western blot and immunocytochemistry. Results: Our study shows that vimentin is upregulated in glioblastoma tissue compared to lower grade glioma and non-tumour brain tissue. We demonstrated that treatment with Nb79 reduced glioblastoma cell invasion by up to 64% in vitro and up to 21% in vivo. In addition, we found that the tight junction protein ZO-1 had higher expression on the cell membrane, when treated with inhibitory anti-vimentin Nb79 compared to control. Conclusion: In conclusion, our results suggest that anti-vimentin nanobody Nb79 is a promising tool to target glioblastoma cell invasion.
Ključne besede: glioblastoma, vimentin, nanobody
Objavljeno v DiRROS: 12.07.2024; Ogledov: 52; Prenosov: 27
Celotno besedilo (2,43 MB)
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Ključne besede: cathepsins, glioblastoma, immunohistochemistry, patient survival, cancer stem cell niches
Objavljeno v DiRROS: 11.06.2024; Ogledov: 84; Prenosov: 26
Celotno besedilo (1,91 MB)

Ključne besede: brain tumors, tumor microenvironment, animal models, xenotransplantation
Objavljeno v DiRROS: 09.05.2024; Ogledov: 185; Prenosov: 448
Celotno besedilo (1,35 MB)

Povzetek: Background. Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stemlike cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF.Meterials and methods. To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification.Results. Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).Conclusions. We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.
Ključne besede: glioblastoma, stem cell cultures, bFGF
Objavljeno v DiRROS: 03.04.2024; Ogledov: 213; Prenosov: 86
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