Proper fetal development requires tight regulation of serotonin concentrations within the fetoplacental unit. This homeostasis is partly maintained by the placental transporter OCT3/SLC22A3, which takes up serotonin from the fetal circulation. Metformin, an antidiabetic drug commonly used to treat gestational diabetes mellitus, was shown to inhibit OCT3. We, therefore, hypothesized that its use during pregnancy could disrupt placental serotonin homeostasis. This hypothesis was tested using three experimental model systems: primary trophoblast cells isolated from the human term placenta, fresh villous human term placenta fragments, and rat term placenta perfusions. Inhibition of serotonin transport by metformin at three concentrations (1 μM, 10 μM, and 100 μM) was assessed in all three models. The OCT3 inhibitor decynium-22 (100 μM) and paroxetine (100 μM), a dual inhibitor of SERT and OCT3, were used as controls. In primary trophoblasts, paroxetine exhibited the strongest inhibition of serotonin uptake, followed by decynium-22. Metformin showed a concentration-dependent effect, reducing serotonin uptake by up to 57 % at the highest concentration. Its inhibitory effect was less pronounced in fresh villous fragments but remained statistically significant at all concentrations. In the perfused rat placenta, metformin demonstrated a concentration-dependent effect, reducing placental serotonin uptake by 44 % at the highest concentration tested. Our findings across all experimental models show inhibition of placental OCT3 by metformin, resulting in reduced serotonin uptake by the trophoblast. This sheds light on mechanisms that may underpin metformin-mediated effects on fetal development.

• MeSH
• biologický transport účinky léků   MeSH
• hypoglykemika farmakologie   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• metformin * farmakologie   MeSH
• oktamerní transkripční faktor 3 metabolismus   MeSH
• placenta * metabolismus   účinky léků   MeSH
• potkani Wistar MeSH
• proteiny přenášející organické kationty MeSH
• serotonin * metabolismus   MeSH
• těhotenství MeSH
• trofoblasty * metabolismus   účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Embryonic stem cells and induced pluripotent stem cells provided us with fascinating new knowledge in recent years. Mechanistic insight into intricate regulatory circuitry governing pluripotency stemness and disclosing parallels between pluripotency stemness and cancer instigated numerous studies focusing on roles of pluripotency transcription factors, including Oct4, Sox2, Klf4, Nanog, Sall4 and Tfcp2L1, in cancer. Although generally well substantiated as tumour-promoting factors, oncogenic roles of pluripotency transcription factors and their clinical impacts are revealing themselves as increasingly complex. In certain tumours, both Oct4 and Sox2 behave as genuine oncogenes, and reporter genes driven by composite regulatory elements jointly recognized by both the factors can identify stem-like cells in a proportion of tumours. On the other hand, cancer stem cells seem to be biologically very heterogeneous both among different tumour types and among and even within individual tumours. Pluripotency transcription factors are certainly implicated in cancer stemness, but do not seem to encompass its entire spectrum. Certain cancer stem cells maintain their stemness by biological mechanisms completely different from pluripotency stemness, sometimes even by engaging signalling pathways that promote differentiation of pluripotent stem cells. Moreover, while these signalling pathways may well be antithetical to stemness in pluripotent stem cells, they may cooperate with pluripotency factors in cancer stem cells - a paradigmatic example is provided by the MAPK-AP-1 pathway. Unexpectedly, forced expression of pluripotency transcription factors in cancer cells frequently results in loss of their tumour-initiating ability, their phenotypic reversion and partial epigenetic normalization. Besides the very different signalling contexts operating in pluripotent and cancer stem cells, respectively, the pronounced dose dependency of reprogramming pluripotency factors may also contribute to the frequent loss of tumorigenicity observed in induced pluripotent cancer cells. Finally, contradictory cell-autonomous and non-cell-autonomous effects of various signalling molecules operate during pluripotency (cancer) reprogramming. The effects of pluripotency transcription factors in cancer are thus best explained within the concept of cancer stem cell heterogeneity.

• MeSH
• buněčná diferenciace genetika   MeSH
• embryonální kmenové buňky MeSH
• indukované pluripotentní kmenové buňky * metabolismus   MeSH
• lidé MeSH
• nádory * genetika   metabolismus   MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• pluripotentní kmenové buňky * MeSH
• přeprogramování buněk genetika   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Protein syntheses at appropriate timings are important for promoting diverse biological processes and are controlled at the levels of transcription and translation. Pou5f1/Oct4 is a transcription factor that is essential for vertebrate embryonic development. However, the precise timings when the mRNA and protein of Pou5f1/Oct4 are expressed during oogenesis and early stages of embryogenesis remain unclear. We analyzed the expression patterns of mRNA and protein of Pou5f1/Oct4 in mouse oocytes and embryos by using a highly sensitive in situ hybridization method and a monoclonal antibody specific to Pou5f1/Oct4, respectively. Pou5f1/Oct4 mRNA was detected in growing oocytes from the primary follicle stage to the fully grown GV stage during oogenesis. In contrast, Pou5f1/Oct4 protein was undetectable during oogenesis, oocyte maturation and the first cleavage stage but subsequently became detectable in the nuclei of early 2-cell-stage embryos. Pou5f1/Oct4 protein at this stage was synthesized from maternal mRNAs stored in oocytes. The amount of Pou5f1/Oct4 mRNA in the polysomal fraction was small in GV-stage oocytes but was significantly increased in fertilized eggs. Taken together, our results indicate that the synthesis of Pou5f1/Oct4 protein during oogenesis and early stages of embryogenesis is controlled at the level of translation and suggest that precise control of the amount of this protein by translational regulation is important for oocyte development and early embryonic development.

• MeSH
• embryonální vývoj genetika   MeSH
• myši inbrední ICR MeSH
• myši MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• oogeneze genetika   MeSH
• těhotenství MeSH
• vývojová regulace genové exprese genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Duchenne muscular dystrophy (DMD) affects 1:3500-5000 newborn boys and manifests with progressive skeletal muscle wasting, respiratory failure and eventual heart failure. Symptoms show different onset from patients' childhood to the second decade of age. We reprogrammed fibroblasts from two independent DMD patients with a complete loss of dystrophin expression, carrying deletions of exons 45-50 and 48-50. The resulting hiPSCs show expression of pluripotency markers (NANOG, OCT4, SSEA4), differentiation capacity into all three germ layers, normal karyotype, genetic identity to the originating parental fibroblasts and the patient-specific dystrophin mutation.

• MeSH
• buněčná diferenciace MeSH
• buněčné linie cytologie   metabolismus   MeSH
• dítě MeSH
• Duchennova muskulární dystrofie genetika   metabolismus   patofyziologie   MeSH
• dystrofin genetika   metabolismus   MeSH
• exony MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• mladiství MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• sekvenční delece MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Loss of totipotentcy in an early embryo is directed by molecular processes responsible for cell fate decisions. Three dimensional genome organisation is an important factor linking chromatin architecture with stage specific gene expression patterns. Little is known about the role of chromosome organisation in gene expression regulation of lineage specific factors in mammalian embryos. Using bovine embryos as a model we have described these interactions at key developmental stages. Three bovine chromosomes (BTA) that differ in size, number of carried genes, and contain loci for key lineage regulators OCT4, NANOG and CDX2, were investigated. The results suggest that large chromosomes regardless of their gene density (BTA12 gene-poor, BTA5 gene-rich) do not significantly change their radial position within the nucleus. Gene loci however, may change its position within the chromosome territory (CT) and relocate its periphery, when stage specific process of gene activation is required. Trophectoderm specific CDX2 and epiblast precursor NANOG loci tend to locate on the surface or outside of the CTs, at stages related with their high expression. We postulate that the observed changes in CT shape reflect global alternations in gene expression related to differentiation.

• MeSH
• buněčné jádro genetika   MeSH
• buněčný rodokmen MeSH
• embryonální vývoj MeSH
• hybridizace in situ fluorescenční MeSH
• nanog genetika   metabolismus   MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• savčí chromozomy genetika   MeSH
• skot MeSH
• transkripční faktor CDX2 genetika   metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ectopic expression of defined sets of genetic factors can reprogramme somatic cells to induced pluripotent stem cells (iPSCs) that closely resemble embryonic stem cells. However, the low reprogramming efficiency is a significant handicap for mechanistic studies and potential clinical application. In this study, we used human bone marrow-derived mesenchymal stem cells (hBMMSCs) as target cells for reprogramming and investigated efficient iPSC generation from hBMMSCs using the compounds of p53 siRNA, valproic acid (VPA) and vitamin C (Vc) with four transcription factors OCT4, SOX2, KLF4, and c-MYC (compound induction system). The synergetic mechanism of the compounds was studied. Our results showed that the compound induction system could efficiently reprogramme hBMMSCs to iPSCs. hBMMSC-derived iPSC populations expressed pluripotent markers and had multi-potential to differentiate into three germ layer-derived cells. p53 siRNA, VPA and Vc had a synergetic effect on cell reprogramming and the combinatorial use of these substances greatly improved the efficiency of iPSC generation by suppressing the expression of p53, decreasing cell apoptosis, up-regulating the expression of the pluripotent gene OCT4 and modifying the cell cycle. Therefore, our study highlights a straightforward method for improving the speed and efficiency of iPSC generation and provides versatile tools for investigating early developmental processes such as haemopoiesis and relevant diseases. In addition, this study provides a paradigm for the combinatorial use of genetic factors and molecules to improve the efficiency of iPSC generation.

• MeSH
• biologické markery metabolismus   MeSH
• buněčné kultury metody   MeSH
• buněčný cyklus účinky léků   genetika   MeSH
• buňky kostní dřeně cytologie   účinky léků   metabolismus   MeSH
• down regulace genetika   účinky záření   MeSH
• indukované pluripotentní kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• kyselina askorbová farmakologie   MeSH
• kyselina valproová farmakologie   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• mezenchymální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• multipotentní kmenové buňky cytologie   metabolismus   MeSH
• myši inbrední ICR MeSH
• myši MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• přeprogramování buněk účinky léků   genetika   MeSH
• upregulace účinky léků   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency.

• MeSH
• antigen Ki-67 metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné jádro genetika   metabolismus   MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• epigeneze genetická MeSH
• FRAP MeSH
• G2 fáze MeSH
• histony metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• lysin metabolismus   MeSH
• metylace MeSH
• myši MeSH
• nika kmenových buněk MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• western blotting MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Oct4 is a specific marker of embryonic stem cell (ESC) pluripotency. However, little is known regarding how Oct4 responds to DNA damage. Here, we investigated whether Oct4 recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4. These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: We used time-lapse confocal microscopy, microirradiation by UV laser (355 nm), induction of DNA lesions by specific agents, and GFP technology to study the Oct4 response to DNA damage. We found that Oct4 accumulates in UV-damaged regions immediately after irradiation in an adenosine triphosphate-dependent manner. Intriguingly, this event was not accompanied by pronounced Nanog and c-MYC recruitment to the UV-damaged sites. The accumulation of Oct4 to UV-damaged chromatin occurred simultaneously with H3K9 deacetylation and H2AX phosphorylation (γH2AX). Moreover, we observed an ESC-specific nuclear distribution of γH2AX after interference to cellular processes, including histone acetylation, transcription, and cell metabolism. Inhibition of histone deacetylases mostly prevented pronounced Oct4 accumulation at UV-irradiated chromatin. CONCLUSIONS/SIGNIFICANCE: Our studies demonstrate pluripotency-specific events that accompany DNA damage responses. Here, we discuss how ESCs might respond to DNA damage caused by genotoxic injury that might lead to unwanted genomic instability.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• chromatin metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• embryonální kmenové buňky cytologie   MeSH
• fibroblasty metabolismus   MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• histony chemie   MeSH
• kinetika MeSH
• myši MeSH
• oktamerní transkripční faktor 3 metabolismus   MeSH
• poškození DNA MeSH
• regenerativní lékařství metody   MeSH
• regulace genové exprese MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The present study was designed to extensively characterize cell lines derived from porcine blastocysts by several methodical approaches, including morphological observation, cytogenetic analysis, estimation of alkaline phosphatase activity and detection of specific marker expression at the mRNA/protein level. A comparison was made between the properties of cell lines isolated from in vivo- and in vitro-obtained blastocysts. Our results showed that 57.1% of the in vivo-obtained blastocysts attached to the feeder layer and that 33.3% of them started to grow in a monolayer. The percentage of attached in vitro-produced blastocysts was lower (24.6%), and only 6.9% of them started to grow. Outgrowths from the in vitro-produced blastocysts formed mainly trophectoderm or epithelial-like monolayer, whereas the in vivo-obtained blastocysts formed heterogeneous outgrowths that also contained cells with embryonic stem (ES)-like morphology. Detailed analyses showed that the primary outgrowths with ES-like morphology expressed the pluripotency markers OCT-4 and NANOG and revealed intensive alkaline phosphatase staining, while they did not express markers of differentiation. The majority of passaged cells, including those with ES-like morphology, lacked OCT-4 protein and revealed expression of specific differentiation markers (cytokeratin 18, lamins A/C, transferrin, α-fetoprotein and GATA-4), although they still expressed NANOG and exhibited weak alkaline phosphatase activity. Moreover, these cells spontaneously differentiated into neural, fibroblast or epithelial-like cells, even in the presence of leukaemia inhibitory factor. Our results show that complex analysis of markers of pluripotency as well as differentiation markers is necessary for proper interpretation of data in porcine embryonic stem cell studies.

• MeSH
• biologické markery analýza   metabolismus   MeSH
• blastocysta cytologie   metabolismus   MeSH
• buněčné linie MeSH
• embryo savčí MeSH
• fertilizace in vitro MeSH
• fertilizace genetika   fyziologie   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• hypoxanthinfosforibosyltransferasa genetika   metabolismus   MeSH
• kultivace embrya MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• prasata embryologie   genetika   metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• těhotenství MeSH
• validační studie jako téma MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

The aim of our study was to characterize mouse embryonal carcinoma (EC) cells P19 in different stages of retinoic acid induced neurodifferentiation by two methods, immunocytochemistry and RT qPCR. The characterization of the cells is crucial before any transplantation into any model, e.g. in our case into the mouse brain with the aim to treat a neurodegenerative disease. Specific protein markers (MAP-2, OCT-4, FORSE-1) were detected by immunocytochemistry in the cell cultures. The mRNA expression levels of PAX-6, MASH-1, Brachyury, GATA-4 and AFP were determined by RT qPCR method. HPRT was used as a housekeeping gene. The degree of differentiation can be characterized by expression of analyzed genes. The presence of OCT-4 and FORSE-1 proteins in undifferentiated pluripotent cells and the presence of dendrite specific MAP-2 in neuroprogenitors was detected. The expression levels of PAX-6 and MASH-1 increased and expression of Brachyury decreased during the neurodifferentiation process. The expression levels of GATA-4 and AFP were the highest after induction of differentiation with retinoic acid. Detailed characterization of cells before transplantation experiments can contribute to better understanding of their effect.

• MeSH
• antigeny povrchové genetika   metabolismus   MeSH
• buněčná diferenciace genetika   účinky léků   MeSH
• exprese genu MeSH
• financování organizované MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• imunohistochemie MeSH
• kmenové buňky embryonálního karcinomu fyziologie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• oční proteiny genetika   metabolismus   MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny asociované s mikrotubuly genetika   metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• transkripční faktory bHLH metabolismus   MeSH
• transkripční faktory paired box genetika   metabolismus   MeSH
• tretinoin farmakologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH