Šárka Kubinová

Background: Cytokine licensing with pro-inflammatory molecules, such as tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), has emerged as a promising strategy to enhance the therapeutic potential of multipotent mesenchymal stromal cells (MSCs). While licensing has demonstrated benefits for immunomodulation, its effects on other key MSC functions, including differentiation and paracrine activity, remain incompletely explored. In this study, we evaluated the transcriptomic, metabolomic, and functional changes induced by short-term TNF-α/IFN-γ priming of Wharton’s jelly-derived MSCs (WJ-MSCs). Methods: WJ-MSCs were expanded and exposed to TNF-α and IFN-γ (10 ng/ml each) for 24 h. Transcriptomic analysis was performed using RNA sequencing to identify differentially expressed genes related to immune modulation and lineage commitment. Metabolomic profiling was conducted using high-resolution mass spectrometry to assess changes in metabolic pathways. Functional assays evaluated the effects of cytokine priming on induced differentiation and growth factor secretion. Results: Cytokine licensing induced notable alterations in gene expression, upregulating pathways linked to immune response, inflammation, and cytokine signalling. However, short-term cytokine treatment significantly attenuated the osteogenic and adipogenic differentiation of MSCs, as evidenced by the reduced expression of RUNX2, ALP, CEBPA, and PPARG. The priming had a negligible effect on EGF, FGF-2, HGF, LIF, and SCF secretion. The production of VEGF-A and VEGF-C was elevated, although the levels remained low. Metabolomic analysis revealed enhanced kynurenine pathway activity, indicative of increased tryptophan catabolism, accompanied by elevated levels of fatty acids and polyamines. Conclusions: Our findings demonstrate that TNF-α/IFN-γ priming reprograms WJ-MSCs by enhancing their immunomodulatory capacity at the expense of differentiation potential. These results highlight the need for tailored strategies to optimize MSC functionality for specific clinical applications.

Recent studies undoubtedly show that the mammalian target of rapamycin (mTOR) and the Hippo–Yes-associated protein 1 (YAP) pathways are important mediators of mechanical cues. The crosstalk between these pathways as well as de-regulation of their signaling has been implicated in multiple tumor types, including liver tumors. Additionally, physical cues from 3D microenvironments have been identified to alter gene expression and differentiation of different cell lineages. However, it remains incompletely understood how physical constraints originated in 3D cultures affect cell plasticity and what the key mediators are of such process. In this work, we use collagen scaffolds as a model of a soft 3D microenvironment to alter cellular size and study the mechanotransduction that regulates that process. We show that the YAP-mTOR axis is a downstream effector of 3D cellular culture-driven mechanotransduction. Indeed, we found that cell mechanics, dictated by the physical constraints of 3D collagen scaffolds, profoundly affect cellular proliferation in a YAP–mTOR-mediated manner. Functionally, the YAP–mTOR connection is key to mediate cell plasticity in hepatic tumor cell lines. These findings expand the role of YAP–mTOR-driven mechanotransduction to the control hepatic tumor cellular responses under physical constraints in 3D cultures. We suggest a tentative mechanism, which coordinates signaling rewiring with cytoplasmic restructuring during cell growth in 3D microenvironments.

Chondroitin sulphates belong to a group of naturally occurring glycosaminoglycans and play a role in many physiological processes including ageing and the effects of various diseases. Research into chondroitin sulphates has found that the most important analytes are 4- and 6-sulphated disaccharides. We developed an HPLC method for the separation and quantification of underivatized chondroitin/dermatan sulphates—unsaturated disaccharides (4- and 6-sulphated disaccharides). This method is based on the separation of disaccharides by amido as well as amino columns under acidic conditions. These columns enabled the successful separation of 4- and 6-sulphated disaccharides using 50 (amido column) and 25 mmol/L (amino column) phosphate buffer, pH 4.25 (detection at 230 nm), at retention times of less than 10 min. The limit of quantification was 0.5 μg/mL. The applicability of this method was demonstrated through analysis of unsaturated disaccharides produced from the enzymatic digestion of chondroitin/dermatan sulphates of the solubilized extracellular matrix produced from porcine urinary bladder and human umbilical cord.

The emerged field of non-thermal plasma (NTP) shows great potential in the alteration of cell redox status, which can be utilized as a promising therapeutic implication. In recent years, the NTP field considerably progresses in the modulation of immune cell function leading to promising in vivo results. In fact, understanding the underlying cellular mechanisms triggered by NTP remains incomplete. In order to boost the field closer to real-life clinical applications, there is a need for a critical overview of the current state-of-the-art. In this review, we conduct a critical analysis of the NTP-triggered modulation of immune cells. Importantly, we analyze pitfalls in the field and identify persisting challenges. We show that the identification of misconceptions opens a door to the development of a research strategy to overcome these limitations. Finally, we propose the idea that solving problems highlighted in this review will accelerate the clinical translation of NTP-based treatments.

Iron oxide-based nanoparticles have been repeatedly shown to affect lysosomal-mediated signaling. Recently, nanoparticles have demonstrated an ability to modulate autophagic flux via lysosome-dependent signaling. However, the precise underlying mechanisms of such modulation as well as the impact of cellular genetic background remain enigmatic. In this study, we investigated how lysosomal-mediated signaling is affected by iron oxide nanoparticle uptake in three distinct hepatic cell lines. We found that nanoparticle-induced lysosomal dysfunction alters sub-cellular localization of pmTOR and p53 proteins. Our data indicate that alterations in the sub-cellular localization of p53 protein induced by nanoparticle greatly affect the autophagic flux. We found that cells with high levels of Bcl-2 are insensitive to autophagy initiated by nanoparticles. Altogether, our data identify lysosomes as a central hub that control nanoparticle-mediated responses in hepatic cells. Our results provide an important fundamental background for the future development of targeted nanoparticle-based therapies.

Multipotent mesenchymal stromal cells (MSCs) can be considered an accessible therapeutic tool for regenerative medicine. Here, we compared the growth kinetics, immunophenotypic and immunomodulatory properties, gene expression and secretome profile of MSCs derived from human adult bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and Wharton’s jelly (WJ-MSCs) cultured in clinically-relevant conditions, with the focus on the neuroregenerative potential. All the cell types were positive for CD10/CD29/CD44/CD73/CD90/CD105/HLA-ABC and negative for CD14/CD45/CD235a/CD271/HLA-DR/VEGFR2 markers, but they differed in the expression of CD34/CD133/CD146/SSEA-4/MSCA-1/CD271/HLA-DR markers. BM-MSCs displayed the highest immunomodulatory activity compared to AT- and WJ-MSCs. On the other hand, BM-MSCs secreted the lower content and had the lower gene expression of neurotrophic growth factors compared to other cell lines, which may be caused by the higher sensitivity of BM-MSCs to nutrient limitations. Despite the differences in growth factor secretion, the MSC secretome derived from all cell sources had a pronounced neurotrophic potential to stimulate the neurite outgrowth of DRG-neurons and reduce the cell death of neural stem/progenitor cells after H₂O₂ treatment. Overall, our study provides important information for the transfer of basic MSC research towards clinical-grade manufacturing and therapeutic applications.

Lysosome-activated apoptosis represents an alternative method of overcoming tumor resistance compared to traditional forms of treatment. Pulsed magnetic fields open a new avenue for controlled and targeted initiation of lysosomal permeabilization in cancer cells via mechanical actuation of magnetic nanomaterials. In this study we used a noninvasive tool; namely, a benchtop pulsed magnetic system, which enabled remote activation of apoptosis in liver cancer cells. The magnetic system we designed represents a platform that can be used in a wide range of biomedical applications. We show that liver cancer cells can be loaded with superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs retained in lysosomal compartments can be effectively actuated with a high intensity (up to 8 T), short pulse width (~15 µs), pulsed magnetic field (PMF), resulting in lysosomal membrane permeabilization (LMP) in cancer cells. We revealed that SPION-loaded lysosomes undergo LMP by assessing an increase in the cytosolic activity of the lysosomal cathepsin B. The extent of cell death induced by LMP correlated with the accumulation of reactive oxygen species in cells. LMP was achieved for estimated forces of 700 pN and higher. Furthermore, we validated our approach on a three-dimensional cellular culture model to be able to mimic in vivo conditions. Overall, our results show that PMF treatment of SPION-loaded lysosomes can be utilized as a noninvasive tool to remotely induce apoptosis.

Biological effects of high fluence low-power (HFLP) lasers have been reported for some time, yet the molecular mechanisms procuring cellular responses remain obscure. A better understanding of the effects of HFLP lasers on living cells will be instrumental for the development of new experimental and therapeutic strategies. Therefore, we investigated sub-cellular mechanisms involved in the laser interaction with human hepatic cell lines. We show that mitochondria serve as sub-cellular “sensor” and “effector” of laser light non-specific interactions with cells. We demonstrated that despite blue and red laser irradiation results in similar apoptotic death, cellular signaling and kinetic of biochemical responses are distinct. Based on our data, we concluded that blue laser irradiation inhibited cytochrome c oxidase activity in electron transport chain of mitochondria. Contrary, red laser triggered cytochrome c oxidase excessive activation. Moreover, we showed that Bcl-2 protein inhibited laser-induced toxicity by stabilizing mitochondria membrane potential. Thus, cells that either overexpress or have elevated levels of Bcl-2 are protected from laser-induced cytotoxicity. Our findings reveal the mechanism how HFLP laser irradiation interfere with cell homeostasis and underscore that such laser irradiation permits remote control of mitochondrial function in the absence of chemical or biological agents.

Our previous research revealed that trehalose, a nonreducing disaccharide of glucose and an important stress responsive factor, proved to have anti-inflammatory, antiapoptotic, and particularly antioxidant properties in UVB-irradiated corneas. Trehalose reduced oxidative stress in corneas induced by UVB irradiation, by means of a decrease in the antioxidant/prooxidant imbalance in the corneal epithelium. In this study, we demonstrate that trehalose of 3% or 6% concentration in eye drops directly decreases oxidative stress in UVB-irradiated corneas, by removing the excessive amount of reactive oxygen species (ROS). Trehalose drops applied on corneas during UVB irradiation once daily for four days resulted in a reduction or even absence of the oxidative stress, DNA damage, and peroxynitrite formation (detected by nitrotyrosine residues), seen in buffer-treated corneas. Furthermore, trehalose treatment applied curatively after repeated irradiation for the subsequent fourteen days led to the renewal of corneal transparency and significant suppression or even absence of neovascularization. This was in contrast to buffer-treated irradiated corneas, where the intracorneal inflammation was developed and the untransparent corneas were vascularized. In conclusion, the treatment of UVB-irradiated corneas with trehalose eye drops removed the excessive amount of ROS in the corneal epithelium, leading to the suppression of oxidative stress and favorable corneal healing. The 6% trehalose showed a higher intensive antioxidant effect.