In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.
- MeSH
- blastocysta účinky léků metabolismus MeSH
- fertilizace in vitro MeSH
- fibroblastový růstový faktor 2 * farmakologie MeSH
- insulinu podobný růstový faktor I * farmakologie MeSH
- kultivační média * chemie farmakologie MeSH
- leukemický inhibiční faktor * farmakologie MeSH
- oocyty MeSH
- prasata embryologie genetika MeSH
- proteomika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Early mouse neural stem cells (NSCs) first appear in embryonic day E5.5 and express pluripotency markers Oct4, Sox2, Nanog and early neural marker Sox1. Early NSCs are a good model for understanding the role of various pathways that control initial neural commitment. However, a protocol for differentiation of mouse embryonic stem cells (ESCs) into early NSCs by adherent monolayer culture has not yet been established. Hence, in this study, we identified the combination of growth factors and small molecules that differentiated mouse ESCs into early NSCs and supported their proliferation. Leukaemia inhibitory factor (LIF) was the first factor to be tested and it was found that ESCs can differentiate into early neurogenic lineage in the presence of LIF. However, we found that the induction is weaker in the presence of LIF as compared to cells differentiated in its absence. GSK-3 inhibitor, along with BMP and TGF-β pathway inhibitor (dual SMAD inhibition), are commonly used to sequentially direct ESCs towards NSCs. However, when we used this combination, mouse ESCs failed to differentiate into early NSCs. We observed that by adding Wnt inhibitor to the combination of GSK-3 inhibitor, BMP inhibitor, TGF-β inhibitor and LIF, it was possible to differentiate ESCs into early NSCs. qRT-PCR analysis of early NSCs illustrated that they expressed key pluripotency genes Oct4 and Nanog, albeit at levels lower than non-differentiated ESCs, along with early neural markers Sox1 and Pax6.
- MeSH
- buněčná diferenciace MeSH
- embryonální kmenové buňky MeSH
- kinasa 3 glykogensynthasy * MeSH
- kostní morfogenetické proteiny MeSH
- leukemický inhibiční faktor farmakologie MeSH
- myši MeSH
- nervové kmenové buňky * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Differentiation of pancreatic β-cells is regulated by a wide range of signalling pathways. The aim of our current work was to evaluate the effect of the Jak/Stat signalling pathway on the differentiation of human non-endocrine pancreatic cells into insulin-producing cells. Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor (LIF) stimulated differentiation of C-peptide-negative human non-endocrine pancreatic cells into insulin-producing cells in 6.3 ± 2.0 % cells (N = 5) and induced expression of pro-endocrine transcription factor neurogenin 3, Notch signalling pathway suppressor HES6 and stimulator of β-cell neogenesis REG3A. The expression of the REG3A gene and increased rate of differentiation into insulin-producing cells (10.2 ± 2.1 %) were further stimulated by a combination of LIF with nicotinamide and dexamethasone. Glucose-stimulated (5 vs. 20 mM) C-peptide secretion confirmed proper insulin secretory function of trans-differentiated insulin-producing cells (0.51 vs. 2.03 pmol C-peptide/μg DNA, P < 0.05). Our results indicate that Jak/Stat signalling critically contributes to trans-differentiation of non-endocrine pancreatic cells into functional insulin-producing cells. The positive effect of the Jak/Stat signalling pathway on trans-differentiation is mediated by the key genes that activate differentiation of pancreatic β-cells.
- MeSH
- beta-buňky cytologie MeSH
- buněčná diferenciace účinky léků MeSH
- C-peptid MeSH
- imunohistochemie MeSH
- Janus kinasy genetika metabolismus MeSH
- kultivované buňky MeSH
- leukemický inhibiční faktor farmakologie MeSH
- lidé MeSH
- pankreas cytologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- signální transdukce účinky léků MeSH
- transkripční faktory STAT genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
