The impact of neurotrophic factors on the expression of neuroectodermal markers in skin mesenchymal stem cells

Kultivēšanas barotnes sastāvs ir faktors, kas stimulē cilmes šūnu diferenciāciju in vitro. Neirotrofie faktori virza diferenciāciju neiroektodermas šūnu virzienā. Bakalaura darba mērķis bija analizēt neirotrofo faktoru ietekmi uz neiroektodermas marķieru ekspresiju ādas mezenhimālajās cilmes šūnās (Ā-MCš). Neiroektodermas gēnu ekspresija Ā-MCš tika noteikta ar RT-PCR metodi pēc diferencēšanas trīs barotnēs. Tika izveidots 15 neiroektodermas gēnu praimeru panelis. Sox10, NES un TUBB3 gēni tika analizēti arī ar kvantitatīvo PCR (qPCR) metodi. RT-PCR rezultāti uzrādīja atšķirības Pax6 un S100β gēnu ekspresijā starp barotņu sastāviem. qPCR analīze parādīja, ka Švāna šūnu barotnē pieauga Sox10 ekspresija, neirodiferenciācijas barotnē pieauga TUBB3 ekspresija un NES ekspresija palielinājās gan neirodiferenciācijas, gan Švāna šūnu barotnēs.The composition of cultivation medium is a factor that stimulates stem cell differentiation in vitro. Neurotrophic factors promote differentiation in the direction of neuroectodermal cells. The aim of bachelor work was to analyze the impact of neurotrophic factors on the expression of neuroectodermal markers in skin mesenchymal stem cells (S-MCs). Neuroectodermal gene expression in S-MCs was determined by RT-PCR method after differentiation in three mediums. The panel of 15 neuroectodermal gene primers was created. Sox10, NES, TUBB3 genes were also analyzed by quantitative PCR (qPCR) method. RT-PCR results showed differences in Pax6 and S100β gene expression between medium compositions. qPCR analysis showed that Sox10 expression was increased in Schwann cell medium, TUBB3 expression was increased in neural differentiation medium and NES expression was increased in neural differentiation and in Schwann cell media

Phenotypic characterization of skin mesenchymal stem cells fallowing the neurotrophic factor induced neurodifferentiation

Ādas mezenhimālās cilmes šūnas (Ā-MCš), pakļautas specifiskiem faktoriem, spēj diferencēties par neironiem un glijas šūnām. Maģistra darba mērķis bija analizēt Ā-MCš fenotipu pēc neirotrofo faktoru inducētas neirodiferenciācijas. Neiroektodermas gēnu ekspresiju Ā-MCš analizēja ar qPCR, cilmes šūnu pluripotences un mezenhimālo marķieru ekspresiju ar plūsmas citometriju, Sigma-1 receptora ekspresiju (SigmaR1) ar qPCR un IF, kā arī BDNF sekrēciju ar ELISA. Ā-MCš fenotipu salīdzināja ar ReNcell CX šūnām. Retīnskābes indukcijas barotnē novēroja nervu kores gēnu ekspresijas pieaugumu. Cilmes šūnu marķieru ekspresija pēc diferenciācijas kritās. Mezenhimālo marķieru ekspresija samazinājās vienā no testētajām donoru šūnu līnijām. SigmaR1 visaugstāk ekspresēja nediferencētas Ā-MCš. Švāna šūnu barotnē šūnas pastiprināti sekretēja BDNF.Skin mesenchymal stem cells (S-MSc) in the presence of specific growth factors may differentiate to neurons and glial cells. The aim of the master thesis was to analyse the phenotype of S-MSc after neurotrophic factor induced neurodifferentiation. S-MSc were analysed for neuroectodermal gene expression with qPCR, for stem cell pluripotency and mesenchymal markers with flow cytometry, for Sigma-1 receptor (SigmaR1) expression and for BDNF secretion with ELISA. The phenotype of S-MSc was compared to ReNcell CX cell line. The increase of neural gene expression in S-MSc was observed in retinoic acid induction medium. Stem cell marker expression was decreased after differentiation. Mesenchymal stem cell marker expression was decreased in one of the three tested donor cell lines. The highest expression of SigmaR1 was observed in undifferentiated S-MSc. Cells cultivated in Schwann cells medium enhanced the secretion of BDNF

Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

Abstract. Background Macrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. Extracellular vesicles (EVs) are membrane-bound vesicles containing different biomolecules that are involved in cell to cell signal transfer. Accumulating evidence suggests that cancer-derived EVs are taken up by macrophages and modulate their phenotype and cytokine profile. However, the interactions of cancer-derived EVs with monocytes and macrophages at various differentiation and polarization states are poorly understood. In the current study, we have analyzed the uptake and functional effects of primary (SW480) and metastatic (SW620) isogenic colorectal cancer (CRC) cell line-derived EVs on monocytes (M), inactive macrophages (M0) and M1 and M2 polarized macrophages. Methods THP-1 monocytes were differentiated into M0 macrophages by addition of phorbol-12-myristate-13-acetate. Then M0 macrophages were further polarized into M1 and M2 macrophages in the presence of LPS, IFN- γ, IL-4, and IL-13 respectively. Internalization of SW480 and SW620-derived EVs was analyzed by flow cytometry and fluorescence microscopy. Changes in monocyte and macrophage immunophenotype and secretory profile upon EV exposure were analyzed by flow cytometry, quantitative PCR and Luminex assays. Results THP-1 monocytes and M0 macrophages efficiently take up SW480 and SW620-derived EVs, and our results indicate that dynamin-dependent endocytic pathways may be implicated. Interestingly, SW480 and SW620-derived EVs increased CD14 expression in M0 macrophages whereas SW480-derived EVs decreased HLA-DR expression in M1 and M2 polarized macrophages. Moreover, SW480-derived EVs significantly increased CXCL10 expression in monocytes and M0 macrophages. In contrast, SW620-derived EVs induced secretion of IL-6, CXCL10, IL-23 and IL-10 in M0 macrophages. However, addition of CRC cell line-derived EVs together with LPS, IFN- γ (M1) and IL-4, IL-13 (M2) stimuli during macrophage polarization had no additional effect on cytokine expression in M1 and M2 macrophages. Conclusion Our results suggest that CRC cell line-derived EVs are internalized and reprogram the immunophenotype and secretory profile in monocytes and inactive macrophages inducing mixed M1 and M2 cytokine response. Although CRC EVs decreased HLA-DR expression in M1, M2 polarized macrophages, their effect on the secretory profile of M1 and M2 polarized macrophages was negligible

Additional file 4: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

TNFα, IL-23, IL-6, IL-1 β, CXCL10, CCL22, IL-10 and MMP9 secretion profile at different monocyte-macrophage differentiation stages. The graphs represent average biomolecule concentrations SEM (n = 3). Statistical analysis carried out with one-way ANOVA test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and **** ≤ 0.0001 vs. untreated cell control of the respective monocyte-macrophage cell subset. (PDF 63 kb

Additional file 2: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

SW480 and SW620-derived EV effect on monocyte (M) and macrophage (M0, M1, M2) viability. a OD values at 450 nm which are in direct proportion of viable cell counts. b SW480 and SW620 EV cytotoxicity on THP-1 monocytes and M0, M1 and M2 macrophages. The graphs represent mean ± SEM (n = 3). Statistical analysis carried out with the t-test. *p ≤ 0.05, **p ≤ 0.01 vs. untreated cell control of the respective monocyte-macrophage cell subset. (PDF 50 kb

Additional file 3: of Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

Effect of temperature on the SW480 EV uptake in THP-1 monocytes. Flow cytometry histograms showing Syto RNA Select fluorescence intensities of untreated (left) and Syto RNA Select-labeled SW480 EV-treated THP-1 monocytes following incubation at 4 °C (middle) and 37 °C (right). Histogram markers show the percentage of Syto RNA Select-positive cells. (PDF 53 kb