26 research outputs found

    Comparative Analysis of Nuclear Transfer Embryo Derived Mouse Embryonic Stem Cells. Part I: Cellular characterization

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    Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. Karyotype analyses and cell growth studies did not reveal any significant variations. Despite some differences observed, the present study revealed that ntESC lines had similar differentiation competences compared to other ESCs. The results indicate that the observed differences may be related to the genotype rather than to the nuclear transfer technology

    Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy

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    The cellular and molecular bases of neurological diseases have been studied for decades; however, the underlying mechanisms are not yet fully elucidated. Compared with other disorders, diseases of the nervous system have been very difficult to study mainly due to the inaccessibility of the human brain and live neurons in vivo or in vitro and difficulties in examination of human postmortem brain tissue. Despite the availability of various genetically engineered animal models, these systems are still not adequate enough due to species variation and differences in genetic background. Human induced pluripotent stem cells (hiPSCs) reprogrammed from patient somatic cells possess the potential to differentiate into any cell type, including neural progenitor cells and postmitotic neurons; thus, they open a new area to in vitro modeling of neurological diseases and their potential treatment. Currently, many protocols for generation of various neuronal subtypes are being developed; however, most of them still require further optimization. Here, we highlight accomplishments made in the generation of dopaminergic and cholinergic neurons, the two subtypes most affected in Alzheimer's and Parkinson's diseases and indirectly affected in Huntington's disease. Furthermore, we discuss the potential role of hiPSC-derived neurons in the modeling and treatment of neurological diseases related to dopaminergic and cholinergic system dysfunction

    Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos

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    Background: The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. [br/] Results: The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types. [br/] Conclusion: In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This information may be well applicable to investigate further the possible phylogenetic role and the regulation of POU5F1 gene

    TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

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    Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors

    Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy

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    The cellular and molecular bases of neurological diseases have been studied for decades; however, the underlying mechanisms are not yet fully elucidated. Compared with other disorders, diseases of the nervous system have been very difficult to study mainly due to the inaccessibility of the human brain and live neurons in vivo or in vitro and difficulties in examination of human postmortem brain tissue. Despite the availability of various genetically engineered animal models, these systems are still not adequate enough due to species variation and differences in genetic background. Human induced pluripotent stem cells (hiPSCs) reprogrammed from patient somatic cells possess the potential to differentiate into any cell type, including neural progenitor cells and postmitotic neurons; thus, they open a new area to in vitro modeling of neurological diseases and their potential treatment. Currently, many protocols for generation of various neuronal subtypes are being developed; however, most of them still require further optimization. Here, we highlight accomplishments made in the generation of dopaminergic and cholinergic neurons, the two subtypes most affected in Alzheimer's and Parkinson's diseases and indirectly affected in Huntington's disease. Furthermore, we discuss the potential role of hiPSC-derived neurons in the modeling and treatment of neurological diseases related to dopaminergic and cholinergic system dysfunction

    Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy

    No full text
    The cellular and molecular bases of neurological diseases have been studied for decades; however, the underlying mechanisms are not yet fully elucidated. Compared with other disorders, diseases of the nervous system have been very difficult to study mainly due to the inaccessibility of the human brain and live neurons in vivo or in vitro and difficulties in examination of human postmortem brain tissue. Despite the availability of various genetically engineered animal models, these systems are still not adequate enough due to species variation and differences in genetic background. Human induced pluripotent stem cells (hiPSCs) reprogrammed from patient somatic cells possess the potential to differentiate into any cell type, including neural progenitor cells and postmitotic neurons; thus, they open a new area to in vitro modeling of neurological diseases and their potential treatment. Currently, many protocols for generation of various neuronal subtypes are being developed; however, most of them still require further optimization. Here, we highlight accomplishments made in the generation of dopaminergic and cholinergic neurons, the two subtypes most affected in Alzheimer’s and Parkinson’s diseases and indirectly affected in Huntington’s disease. Furthermore, we discuss the potential role of hiPSC-derived neurons in the modeling and treatment of neurological diseases related to dopaminergic and cholinergic system dysfunction

    Gene targeting and Calcium handling efficiencies in mouse embryonic stem cell lines

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    AIM: To compare gene targeting efficiencies, expression profiles, and Ca2+ handling potentials in two widely used mouse embryonic stem cell lines

    Brain-derived neurotrophic factor increases cell number of neural progenitor cells derived from human induced pluripotent stem cells

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    Background Several pieces of evidence from in vitro studies showed that brain-derived neurotrophic factor (BDNF) promotes proliferation and differentiation of neural stem/progenitor cells (NSCs) into neurons. Moreover, the JAK2 pathway was proposed to be associated with mouse NSC proliferation. BDNF could activate the STAT-3 pathway and induce proliferation in mouse NSCs. However, its effects on proliferation are not fully understood and JAK/STAT pathway was proposed to play a role in this activity. Methods In the present study, the effects of BDNF on cell proliferation and neurite outgrowth of Alzheimer’s disease (AD) induced pluripotent stem cells (iPSCs)-derived human neural progenitor cells (hNPCs) were examined. Moreover, a specific signal transduction pathway important in cell proliferation was investigated using a JAK2 inhibitor (AG490) to clarify the role of that pathway. Results The proliferative effect of BDNF was remarkably observed as an increase in Ki-67 positive cells. The cell number of hNPCs was significantly increased after BDNF treatment represented by cellular metabolic activity of the cells measured by MTT assay. This noticeable effect was statistically shown at 20 ng/ml of BDNF treatment. BDNF, however, did not promote neurite outgrowth but increased neuronal cell number. It was found that AG490 suppressed hNPCs proliferation. However, this inhibitor partially decreased BDNF-induced hNPCs proliferation. These results demonstrated the potential role of BDNF for the amelioration of AD through the increase of AD-derived hNPCs number

    Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres Are Suitable for Neurotoxicity Screening

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    We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications
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