38 research outputs found
Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk Activity in Mouse Embryonic Stem Cells
Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways
Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells
Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1) is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES) cell maintenance. We employed wild-type and HIF-1α-deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP) 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A) regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS) level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1
Modulation of differentiation of embryonic stem cells by polypyrrole: The impact on neurogenesis
The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecularweight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Czech Science FoundationGrant Agency of the Czech Republic [19-16861S, 17-05466S]; internal grant agency of Tomas Bata University in Zlin [IGA/CPS/2021/001]; Slovenian Research AgencySlovenian Research Agency - Slovenia [P2-0082]; Ministry of Education, Youth and Sports of the Czech Republic-DKRVO [RP/CPS/2020/001]RP/CPS/2020/001; Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT; Grantová Agentura České Republiky, GA ČR: 17-05466S, 19-16861S; Javna Agencija za Raziskovalno Dejavnost RS, ARRS: P2-0082; Univerzita Tomáše Bati ve Zlíně: IGA/CPS/2021/00
Alkaline Phosphatase in Stem Cells
Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells
Neural Differentiation Is Inhibited through HIF1α/β-Catenin Signaling in Embryoid Bodies
Extensive research in the field of stem cells and developmental biology has revealed evidence of the role of hypoxia as an important factor regulating self-renewal and differentiation. However, comprehensive information about the exact hypoxia-mediated regulatory mechanism of stem cell fate during early embryonic development is still missing. Using a model of embryoid bodies (EBs) derived from murine embryonic stem cells (ESC), we here tried to encrypt the role of hypoxia-inducible factor 1α (HIF1α) in neural fate during spontaneous differentiation. EBs derived from ESC with the ablated gene for HIF1α had abnormally increased neuronal characteristics during differentiation. An increased neural phenotype in Hif1α−/− EBs was accompanied by the disruption of β-catenin signaling together with the increased cytoplasmic degradation of β-catenin. The knock-in of Hif1α, as well as β-catenin ectopic overexpression in Hif1α−/− EBs, induced a reduction in neural markers to the levels observed in wild-type EBs. Interestingly, direct interaction between HIF1α and β-catenin was demonstrated by immunoprecipitation analysis of the nuclear fraction of wild-type EBs. Together, these results emphasize the regulatory role of HIF1α in β-catenin stabilization during spontaneous differentiation, which seems to be a crucial mechanism for the natural inhibition of premature neural differentiation
Neural differentiation of mouse embryonic stem cells grown in monolayer
To drive neural differentiation of mouse embryonic stem (ES) cells, various culture
protocols have been previously developed that all require the formation of embryoid
bodies, usually combined with a treatment by all-trans retinoic acid (aRA). Here,
we investigated whether or not neural differentiation can also occur in a simplified
monolayer culture. Mouse ES cells were plated in serum-containing DMEM media with
and without aRA and grown under these conditions for 2 days. Then, the cells were
transferred to fresh serum-containing DMEM media and/or to serum-free DMEM/F12 media
supplemented with a mixture of insulin, transferrin, selenium, and fibronectin (ITSF)
for further culture. The changes in cell morphology and in the expression of selected
molecular markers were monitored. Generally, in contrast to all the others, the
protocol consisting of a 2-day culture in serum-containing DMEM followed by continuous
exposure to the ITSF supplement in DMEM/F12 drove a vast majority of ES cells to generate
phenotypic signs of neural lineage. Altogether, neural differentiation can be achieved
in vitro without the step involving the formation of embryoid bodies as well as the
treatment by aRA
The biocompatibility of polyaniline and polypyrrole: A comparative study of their cytotoxicity, embryotoxicity and impurity profile
Conducting polymers (CP), namely polyaniline (PANI) and polypyrrole (PPy), are promising materials applicable for the use as biointerfaces as they intrinsically combine electronic and ionic conductivity. Although a number of works have employed PANI or PPy in the preparation of copolymers, composites, and blends with other polymers, there is no systematic study dealing with the comparison of their fundamental biological properties. The present study, therefore, compares the biocompatibility of PANI and PPy in terms of cytotoxicity (using NIH/3T3 fibroblasts and embryonic stem cells) and embryotoxicity (their impact on erythropoiesis and cardiomyogenesis within embryonic bodies). The novelty of the study lies not only in the fact that embryotoxicity is presented for the first time for both studied polymers, but also in the elimination of inter-laboratory variations within the testing, such variation making the comparison of previously published works difficult. The results clearly show that there is a bigger difference between the biocompatibility of the respective polymers in their salt and base forms than between PANI and PPy as such. PANI and PPy can, therefore, be similarly applied in biomedicine when solely their biological properties are considered. Impurity content detected by mass spectroscopy is presented. These results can change the generally accepted opinion of the scientific community on better biocompatibility of PPy in comparison with PANI. © 2018 Elsevier B.V.LO1504, NPU, Northwestern Polytechnical University; 17-05095S, GACR, Grantová Agentura České Republiky; MŠMT, Ministerstvo Školství, Mládeže a TělovýchovyCzech Science Foundation [17-05095S]; Ministry of Education, Youth and Sports of the Czech Republic - Program NPU I [LO1504
Conductive Polymer PEDOT:PSS-Based Platform for Embryonic Stem-Cell Differentiation
Organic semiconductors are constantly gaining interest in regenerative medicine. Their tunable physico-chemical properties, including electrical conductivity, are very promising for the control of stem-cell differentiation. However, their use for combined material-based and electrical stimulation remains largely underexplored. Therefore, we carried out a study on whether a platform based on the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) can be beneficial to the differentiation of mouse embryonic stem cells (mESCs). The platform was prepared using the layout of a standard 24-well cell-culture plate. Polyethylene naphthalate foil served as the substrate for the preparation of interdigitated gold electrodes by physical vapor deposition. The PEDOT:PSS pattern was fabricated by precise screen printing over the gold electrodes. The PEDOT:PSS platform was able to produce higher electrical current with the pulsed-direct-current (DC) electrostimulation mode (1 Hz, 200 mV/mm, 100 ms pulse duration) compared to plain gold electrodes. There was a dominant capacitive component. In proof-of-concept experiments, mESCs were able to respond to such electrostimulation by membrane depolarization and elevation of cytosolic calcium. Further, the PEDOT:PSS platform was able to upregulate cardiomyogenesis and potentially inhibit early neurogenesis per se with minor contribution of electrostimulation. Hence, the present work highlights the large potential of PEDOT:PSS in regenerative medicine