Damaged neural tissue is regenerated by neural stem cells (NSCs), which represent a rare and difficult-to-culture cell population. Therefore, alternative sources of stem cells are being tested to replace a shortage of NSCs. Here we show that mouse adipose tissue-derived mesenchymal stem cells (MSCs) can be effectively differentiated into cells expressing neuronal cell markers. The differentiation protocol, simulating the inflammatory site of neural injury, involved brain tissue extract, fibroblast growth factor, epidermal growth factor, supernatant from activated splenocytes and electrical stimulation under physiological conditions. MSCs differentiated using this protocol displayed neuronal cell morphology and expressed genes for neuronal cell markers, such as neurofilament light (Nf-L), medium (Nf-M) and heavy (Nf-H) polypeptides, synaptophysin (SYP), neural cell adhesion molecule (NCAM), glutamic acid decarboxylase (GAD), neuron-specific nuclear protein (NeuN), βIII-tubulin (Tubb3) and microtubule-associated protein 2 (Mtap2), which are absent (Nf-L, Nf-H, SYP, GAD, NeuN and Mtap2) or only slightly expressed (NCAM, Tubb3 and Nf-M) in undifferentiated cells. The differentiation was further enhanced when the cells were cultured on nanofibre scaffolds. The neural differentiation of MSCs, which was detected at the level of gene expression, was confirmed by positive immunostaining for Nf-L protein. The results thus show that the simulation of conditions in an injured neural tissue and inflammatory environment, supplemented with electrical stimulation under physiological conditions and cultivation of cells on a three-dimensional (3D) nanofibre scaffold, form an effective protocol for the differentiation of MSCs into cells with neuronal markers. Copyright © 2015 John Wiley & Sons, Ltd.

• MeSH
• buněčná diferenciace * MeSH
• diferenciační antigeny biosyntéza   MeSH
• mezenchymální kmenové buňky metabolismus   patologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nervová tkáň metabolismus   patologie   MeSH
• nervové kmenové buňky metabolismus   patologie   MeSH
• zánět metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Neurodegenerative diseases are devastating disorders and the demands on their treatment are set to rise in connection with higher disease incidence. Knowledge of the spatiotemporal profile of cellular protein expression during neural differentiation and definition of a set of markers highly specific for targeted neural populations is a key challenge. Intracellular proteins may be utilized as a readout for follow-up transplantation and cell surface proteins may facilitate isolation of the cell subpopulations, while secreted proteins could help unravel intercellular communication and immunomodulation. This review summarizes the potential of proteomics in revealing molecular mechanisms underlying neural differentiation of stem cells and presents novel candidate proteins of neural subpopulations, where understanding of their functionality may accelerate transition to cell replacement therapies.

• MeSH
• lidé MeSH
• nervové kmenové buňky cytologie   metabolismus   transplantace   MeSH
• neurodegenerativní nemoci metabolismus   terapie   MeSH
• neurogeneze * MeSH
• proteom metabolismus   MeSH
• transplantace kmenových buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Embryonic neural stem cells (NSCs), comprising neuroepithelial and radial glial cells, are indispensable precursors of neurons and glia in the mammalian developing brain. Since the process of neurogenesis occurs in a hypoxic environment, the question arises of how NSCs deal with low oxygen tension and whether it affects their stemness. Genes from the hypoxia-inducible factors (HIF) family are well known factors governing cellular response to hypoxic conditions. In this study, we have discovered that the endogenous stabilization of hypoxia-inducible factor 1α (Hif1α) during neural induction is critical for the normal development of the NSCs pool by preventing its premature depletion and differentiation. The knock-out of the Hif1α gene in mESC-derived neurospheres led to a decrease in self-renewal of NSCs, paralleled by an increase in neuronal differentiation. Similarly, neuroepithelial cells differentiated in hypoxia exhibited accelerated neurogenesis soon after Hif1α knock-down. In both models, the loss of Hif1α was accompanied by an immediate drop in neural repressor Hes1 levels while changes in Notch signaling were not observed. We found that active Hif1α/Arnt1 transcription complex bound to the evolutionarily conserved site in Hes1 gene promoter in both neuroepithelial cells and neural tissue of E8.5 - 9.5 embryos. Taken together, these results emphasize the novel role of Hif1α in the regulation of early NSCs population through the activation of neural repressor Hes1, independently of Notch signaling.

• MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• hypoxie MeSH
• nervové kmenové buňky * MeSH
• neurogeneze MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. MATERIALS AND METHODS: We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. RESULTS: Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. CONCLUSION: Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders.

• MeSH
• buněčná diferenciace * MeSH
• fibroblasty cytologie   MeSH
• imunoenzymatické techniky MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• kontrastní látky chemie   MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• lysin chemie   MeSH
• magnetická rezonanční tomografie metody   MeSH
• magnetické nanočástice chemie   MeSH
• neurony cytologie   MeSH
• plíce cytologie   MeSH
• plod cytologie   MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The mechanisms that regulate the maintenance of stem cell self-renewal versus differentiation are complex and remain mostly unknown. Understanding neurogenesis and neural cell differentiation presents a unique challenge for the treatment of nervous system disorders. To gain more insight into molecular mechanisms of the differentiation of neural cells, we combined the advantage of porcine fetal neural stem cells (NSCs) in vitro differentiation model and proteomic analysis. Using 2-DE followed by MS, we profiled constituent proteins of NSCs and their differentiated progenies at first and then indicated protein species that were significantly up- or down-regulated during the differentiation. The largest identified group of constituent proteins was related to RNA and protein metabolism and processing, including chaperones, and the second largest consisted of proteins involved in cell organization (cytoskeleton and annexins). Differentiation of neural cells was found to be accompanied by changes in the expression of proteins involved in DNA and RNA binding, mRNA processing and transport, stress responses, iron storage, and redox regulation. Additional immunoblot analysis verified the induction of alpha-B crystallin and heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and A2/B1. Furthermore, immunocytochemistry demonstrated specific localization of alpha-B crystallin in the cytoplasm or nucleus of glial cells and confirmed cellular expression patterns of hnRNPs A1 and A2/B1. These findings represent a significant step towards understanding neural cell differentiation and identification of the regulatory proteins associated with this process.

• MeSH
• 2D gelová elektroforéza MeSH
• alfa-krystaliny - řetězec B metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• financování organizované MeSH
• heterogenní jaderné ribonukleoproteiny skupiny A-B metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• kmenové buňky cytologie   fyziologie   MeSH
• kultivované buňky MeSH
• mozek cytologie   MeSH
• neurony cytologie   fyziologie   MeSH
• prasata MeSH
• proteomika MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

• MeSH
• buněčná diferenciace MeSH
• buňky kostní dřeně cytologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• hematopoetické kmenové buňky cytologie   MeSH
• hematopoéza MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• neurony cytologie   MeSH
• slezina cytologie   chirurgie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

Neural stem cells (NSCs) are defined by their dual ability to self-renew through mitotic cell division or differentiate into the varied neural cell types of the CNS. DISP3/PTCHD2 is a sterol-sensing domain-containing protein, highly expressed in neural tissues, whose expression is regulated by thyroid hormone. In the present study, we used a mouse NSC line to investigate what effect DISP3 may have on the self-renewal and/or differentiation potential of the cells. We demonstrated that NSC differentiation triggered significant reduction in DISP3 expression in the resulting astrocytes, neurons and oligodendrocytes. Moreover, when DISP3 expression was disrupted, the NSC "stemness" was suppressed, leading to a larger population of cells undergoing spontaneous neuronal differentiation. Conversely, overexpression of DISP3 resulted in increased NSC proliferation. When NSCs were cultured under differentiation conditions, we observed that the lack of DISP3 augmented the number of NSCs differentiating into each of the neural cell lineages and that neuronal morphology was altered. In contrast, DISP3 overexpression resulted in impaired cell differentiation. Taken together, our findings imply that DISP3 may help dictate the NSC cell fate to either undergo self-renewal or switch to the terminal differentiation cell program.

• MeSH
• astrocyty cytologie   metabolismus   MeSH
• buněčná diferenciace genetika   MeSH
• buněčné linie MeSH
• buněčný cyklus genetika   MeSH
• fenotyp MeSH
• lidé MeSH
• membránové proteiny genetika   MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neurony cytologie   metabolismus   MeSH
• oligodendroglie cytologie   metabolismus   MeSH
• proliferace buněk MeSH
• vývojová regulace genové exprese * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Neural stem cells (NSC) capable of differentiating into neurons, astrocytes and oligodendrocytes are a promising source of cells for the treatment of central nervous system diseases. Access to signaling proteins present in such cells in low copies and with specific regulatory functions has been very restrictive until now as judged by classical proteomic approaches and limitations due to scarcity of stem cell populations. Hence, we utilized the Kinex Antibody Microarray analysis where profiles of the proliferating porcine NSC and differentiated counterparts were compared and selected changes were verified by immunoblotting. Differentiated neural cells exhibited an increased level of RafB proto-oncogene-encoded protein-serine kinase, MAP kinase protein-serine kinase 3, heme oxygenase 2 (HO2) and protein phosphatase 4 catalytical subunit. On the other hand, relatively high level of G protein-coupled receptor-serine kinase 2 and enhanced phosphorylations of alphaB-crystallin (S45), protein-serine kinase C gamma (T655), protein-serine kinase D (PKCmu; S738+S742) together with eukaryotic translation initiation factor 2 alpha (eIF2alpha) (S51) raised intriguing questions as regards their potential functionality within stem cells. In-depth study of HO2 and phospho-S45 alphaB-crystallin confirmed expression profiles and intense cytoplasmic localization of HO2 in neurons but a weaker signal in glial cells. Phospho-S45 alphaB-crystallin was localized in nuclei of differentiated neural cells. Computer simulation of possible interaction network connecting regulated proteins, exposed additional relationships including direct interactions of HO2 with amyloid precursor protein or huntingtin-associated protein 1.

• MeSH
• alfa-krystaliny - řetězec B metabolismus   MeSH
• biologické markery metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné jádro metabolismus   MeSH
• financování organizované MeSH
• fosforylace MeSH
• hemová oxygenasa (decyklizující) metabolismus   MeSH
• imunoblotting MeSH
• kmenové buňky fyziologie   MeSH
• kultivované buňky MeSH
• mapování interakce mezi proteiny MeSH
• neurony fyziologie   MeSH
• počítačová simulace MeSH
• prasata MeSH
• proteiny fyziologie   metabolismus   MeSH
• protilátky MeSH
• signální transdukce MeSH
• tretinoin farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cell (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular 'raw material' needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.

• MeSH
• biokompatibilní potahované materiály farmakologie   MeSH
• buněčná adheze účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• extracelulární matrix - proteiny metabolismus   MeSH
• lidé MeSH
• molekuly buněčné adheze metabolismus   MeSH
• myši MeSH
• nervové kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• neurony cytologie   účinky léků   metabolismus   MeSH
• peptidy farmakologie   MeSH
• pluripotentní kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• vitronektin farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The isolation of neural stem cells from fetal and adult mammalian CNS and the demonstration of functional neurogenesis in adult CNS have offered perspectives for treatment of many devastating hereditary and acquired neurological diseases. Due to this enormous potential, neural stem cells are a subject of extensive molecular profiling studies with a search for new markers and regulatory pathways governing their self-renewal as opposed to differentiation. Several in-depth proteomic studies have been conducted on primary or immortalized cultures of neural stem cells and neural progenitor cells, and yet more remains to be done. Additionally, neurons and glial cells have been obtained from embryonic stem cells and mesenchymal stem cells, and proteins associated with the differentiation process have been characterized to a certain degree with a view to further investigations. This review summarizes recent findings relevant to the proteomics of neural stem cells and discusses major proteins significantly regulated during neural stem cell differentiation with a view to their future use in cell-based regenerative and reparative therapy.

• MeSH
• buněčná diferenciace MeSH
• financování organizované MeSH
• kmenové buňky cytologie   chemie   MeSH
• lidé MeSH
• neurony cytologie   chemie   MeSH
• proteiny fyziologie   genetika   MeSH
• proteomika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH