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-molecular-weight 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.

• Keywords
• biocompatibility, conducting polymer, neurogenesis, polypyrrole, stem cells,
• MeSH
• Cell Differentiation drug effects   genetics   MeSH
• Cell Line MeSH
• Embryoid Bodies cytology   drug effects   MeSH
• Gene Expression drug effects   MeSH
• Molecular Structure MeSH
• Mouse Embryonic Stem Cells cytology   drug effects   metabolism   MeSH
• Mice MeSH
• Neural Stem Cells cytology   drug effects   metabolism   MeSH
• Neurogenesis drug effects   genetics   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Polymers chemistry   pharmacology   MeSH
• Pyrroles chemistry   pharmacology   MeSH
• PAX6 Transcription Factor genetics   MeSH
• Basic Helix-Loop-Helix Transcription Factors genetics   MeSH
• SOXB1 Transcription Factors genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ascl1 protein, mouse MeSH Browser
• Polymers MeSH
• polypyrrole MeSH Browser
• Pyrroles MeSH
• PAX6 Transcription Factor MeSH
• Basic Helix-Loop-Helix Transcription Factors MeSH
• SOXB1 Transcription Factors MeSH

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.

• MeSH
• Down-Regulation MeSH
• Hypoxia-Inducible Factor 1, alpha Subunit metabolism   MeSH
• Mitogen-Activated Protein Kinase Kinases metabolism   MeSH
• Mouse Embryonic Stem Cells metabolism   MeSH
• Mice MeSH
• Reactive Oxygen Species metabolism   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Hypoxia-Inducible Factor 1, alpha Subunit MeSH
• Mitogen-Activated Protein Kinase Kinases MeSH
• Reactive Oxygen Species MeSH

The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.

• MeSH
• Acetylcysteine administration & dosage   MeSH
• Actinin genetics   MeSH
• Embryonic Stem Cells cytology   MeSH
• Homeobox Protein Nkx-2.5 genetics   MeSH
• Myocytes, Cardiac cytology   MeSH
• Culture Media, Serum-Free * MeSH
• Cells, Cultured MeSH
• Myosins genetics   MeSH
• Mice MeSH
• In Vitro Techniques MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylcysteine MeSH
• Actinin MeSH
• Homeobox Protein Nkx-2.5 MeSH
• Culture Media, Serum-Free * MeSH
• Myosins MeSH
• Nkx2-5 protein, mouse MeSH Browser

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.

• MeSH
• Acetophenones pharmacology   MeSH
• Extracellular Signal-Regulated MAP Kinases metabolism   MeSH
• Phosphatidylinositol 3-Kinases metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Mouse Embryonic Stem Cells drug effects   metabolism   MeSH
• Mice MeSH
• NADPH Oxidases genetics   metabolism   MeSH
• Onium Compounds pharmacology   MeSH
• Oxidative Stress drug effects   MeSH
• Cell Proliferation drug effects   MeSH
• Wnt Proteins metabolism   MeSH
• Proto-Oncogene Proteins c-akt metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Drug Synergism MeSH
• STAT3 Transcription Factor metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetophenones MeSH
• acetovanillone MeSH Browser
• diphenyleneiodonium MeSH Browser
• Extracellular Signal-Regulated MAP Kinases MeSH
• Phosphatidylinositol 3-Kinases MeSH
• NADPH Oxidases MeSH
• Onium Compounds MeSH
• Wnt Proteins MeSH
• Proto-Oncogene Proteins c-akt MeSH
• Reactive Oxygen Species MeSH
• STAT3 Transcription Factor MeSH

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general.

• MeSH
• Principal Component Analysis MeSH
• Cell Line MeSH
• Mass Spectrometry methods   MeSH
• Calibration MeSH
• Coculture Techniques MeSH
• Humans MeSH
• Human Embryonic Stem Cells physiology   MeSH
• Multivariate Analysis MeSH
• Mice MeSH
• Neural Networks, Computer * MeSH
• Specimen Handling MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• Publication type
• Journal Article MeSH
• Review MeSH

The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for self-renewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson's disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells.

• Keywords
• Chromatin, Differentiation, Embryonic stem cells, Epigenetics, Nucleus, Pluripotency,
• Publication type
• Journal Article MeSH