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.

• MeSH
• buněčná diferenciace * MeSH
• buňky kostní dřeně cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• proliferace buněk MeSH
• regenerace nervu * MeSH
• tuková tkáň cytologie   metabolismus   MeSH
• Whartonův rosol cytologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Cell therapies represent a promising approach to slow down the progression of currently untreatable neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease or amyotrophic lateral sclerosis), as well as to support the reconstruction of functional neural circuits after spinal cord injuries. In such therapies, the grafted cells could either functionally integrate into the damaged tissue, partially replacing dead or damaged cells, modulate inflammatory reaction, reduce tissue damage, or support neuronal survival by secretion of cytokines, growth, and trophic factors. Comprehensive characterization of cells and their proliferative potential, differentiation status, and population purity before transplantation is crucial to preventing safety risks, e.g., a tumorous growth due to the proliferation of undifferentiated stem cells. We characterized changes in the proteome and secretome of human neural stem cells (NSCs) during their spontaneous (EGF/FGF2 withdrawal) differentiation and differentiation with trophic support by BDNF/GDNF supplementation. We used LC-MS/MS in SWATH-MS mode for global cellular proteome profiling and quantified almost three thousand cellular proteins. Our analysis identified substantial protein differences in the early stages of NSC differentiation with more than a third of all the proteins regulated (including known neuronal and NSC multipotency markers) and revealed that the BDNF/GDNF support affected more the later stages of the NSC differentiation. Among the pathways identified as activated during both spontaneous and BDNF/GDNF differentiation were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. Our follow-up secretome analysis using Luminex multiplex immunoassay revealed significant changes in the secretion of VEGF and IL-6 during NSC differentiation. Our results further demonstrated an increased expression of neuropilin-1 as well as catenin β-1, both known to participate in the regulation of VEGF signaling, and showed that VEGF-A isoform 121 (VEGF121), in particular, induces proliferation and supports survival of differentiating cells.

• Klíčová slova
• SWATH-MS, VEGF, neural differentiation, neural stem cell, proliferation, proteome, secretome,
• Publikační typ
• časopisecké články MeSH

Melanoma is a skin cancer with permanently increasing incidence and resistance to therapies in advanced stages. Reports of spontaneous regression and tumour infiltration with T-lymphocytes makes melanoma candidate for immunotherapies. Cytokines are key factors regulating immune response and intercellular communication in tumour microenvironment. Cytokines may be used in therapy of melanoma to modulate immune response. Cytokines also possess diagnostic and prognostic potential and cytokine production may reflect effects of immunotherapies. The purpose of this review is to give an overview of recent advances in proteomic techniques for the detection and quantification of cytokines in melanoma research. Approaches covered span from mass spectrometry to immunoassays for single molecule detection (ELISA, western blot), multiplex assays (chemiluminescent, bead-based (Luminex) and planar antibody arrays), ultrasensitive techniques (Singulex, Simoa, immuno-PCR, proximity ligation/extension assay, immunomagnetic reduction assay), to analyses of single cells producing cytokines (ELISpot, flow cytometry, mass cytometry and emerging techniques for single cell secretomics). Although this review is focused mainly on cancer and particularly melanoma, the discussed techniques are in general applicable to broad research field of biology and medicine, including stem cells, development, aging, immunology and intercellular communication.

• Klíčová slova
• T-cell, biomarker, cancer, cytokine, immunoassay, mass spectrometry, melanoma, proteomics, secretome, ultrasensitive,
• MeSH
• čipová analýza proteinů MeSH
• cytokiny analýza   MeSH
• hmotnostní spektrometrie MeSH
• imunoanalýza MeSH
• imunoterapie MeSH
• lidé MeSH
• melanom diagnóza   metabolismus   terapie   MeSH
• nádorové mikroprostředí MeSH
• nádory kůže diagnóza   metabolismus   terapie   MeSH
• proteomika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• cytokiny MeSH

BACKGROUND: Large animal models of Huntington's disease (HD) may increase the reliability of translating preclinical findings to humans. Long live expectancy offers opportunities particularly for disease modifying approaches, but also challenges. The transgenic (tg) HD minipig model assessed in this study exhibits a high genetic homology with humans, similar body weight, and comparable brain structures. To test long-term safety, tolerability, and efficacy of novel therapeutic approaches in this model reliable assessments applicable longitudinally for several years are warranted for all phenotypical domains relevant in HD. OBJECTIVE: To investigate whether the tests proposed assessing motor, cognitive and behavioral domains can be applied repetitively over a 3-year period in minipigs with acceptable variability or learning effects and whether tgHD minipigs reveal changes in these domains compared to wildtype (wt) minipigs suggesting the development of an HD phenotype. METHODS: A cohort of 14 tgHD and 18 wt minipigs was followed for three years. Tests applied every six months included a tongue coordination and hurdle test for the motor domain, a color discrimination test for cognition, and a dominance test for assessing behavior. Statistical analyses were performed using repeated ANOVA for longitudinal group comparisons and Wilcoxon-tests for intra-visit differences between tgHD and wt minipigs. RESULTS: All tests applied demonstrated feasibility, acceptable variance and good consistency during the three-year period. No significant differences between tgHD and wt minipigs were detected suggesting lack of a phenotype before the age of four years. CONCLUSIONS: The assessment battery presented offers measures in all domains relevant for HD and can be applied in long-term phenotyping studies with tgHD minipigs. The observation of this cohort should be continued to explore the timeline of phenotype development and provide information for future interventional studies.

• MeSH
• chování zvířat fyziologie   MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc patofyziologie   MeSH
• jazyk fyziologie   MeSH
• lidé MeSH
• miniaturní prasata fyziologie   MeSH
• prasata fyziologie   MeSH
• protein huntingtin genetika   fyziologie   MeSH
• učení fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH
• Názvy látek
• protein huntingtin MeSH