neural differentiation
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DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. In the present study we used a multipotent cerebellar progenitor cell line, C17.2, to investigate the impact of DISP3 on the proliferation and differentiation of neural precursors. We found that ectopically expressed DISP3 promotes cell proliferation and alters expression of genes that are involved in tumorigenesis. Finally, the differentiation profile of DISP3-expressing cells was altered, as evidenced by delayed expression of neural specific markers and a reduced capacity to undergo neural differentiation.
- MeSH
- buněčná diferenciace * MeSH
- buněčné linie MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- metabolismus lipidů MeSH
- mozek cytologie MeSH
- nervové kmenové buňky cytologie metabolismus MeSH
- proliferace buněk MeSH
- regulace genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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
Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.
- MeSH
- biologické markery MeSH
- buněčná diferenciace * MeSH
- buněčné linie MeSH
- buněčný rodokmen genetika MeSH
- hmotnostní spektrometrie MeSH
- imunohistochemie MeSH
- lidé MeSH
- nervové kmenové buňky cytologie metabolismus MeSH
- neuroglie MeSH
- neurony MeSH
- stanovení celkové genové exprese MeSH
- vývojová regulace genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
Cílem studie bylo pomocí nepřímé imunohistochemie zjistit expresi galektinu-3 (Gal3), cytokeratinu 19 (CK19), neural cell adhesion molecule (NCAM) a E-cadherinu (Ecad) ve variantách papilárního karcinomu štítné žlázy (PTC) s ohledem na možné využití těchto markerů v bioptické diagnostice. Soubor tvořilo 84 případů – 36 klasických variant PTC (cPTC), 26 folikulárních variant PTC (fPTC) a 22 papilárních mikrokarcinomů (mPTC). Gal3 byl exprimován ve 36/36 (100 %) cPTC, ve 24/26 (92 %) fPTC a v 19/22 (86 %) mPTC. Exprese CK19 byla zastižena ve 34/36 (94 %) cPTC, v 17/26 (65 %) fPTC a ve 13/22 (59 %) mPTC. Exprese NCAM byla prokázána v 5/36 (14 %) cPTC, v 7/26 (27 %) fPTC a v 9/22 (41 %) mPTC. Ecad byl exprimován ve 23/36 (64 %) cPTC, v 17/26 (65 %) fPTC a v 18/22 (82 %) mPTC. V expresi CK19 byl zjištěn signifikantní rozdíl mezi cPTC versus fPTC a mPTC (p < 0,001). Dále byla prokázána signifikantní korelace mezi expresí CK19 a ztrátou exprese Ecad ve vztahu k šíření nádoru mimo štítnou žlázu (p = 0,001, p = 0,04). Detekci exprese Gal3 a CK19 lze tedy doporučit jako pomocné kritérium v diagnostice PTC, nicméně je třeba upozornit na nižší expresi CK19 ve fPTC a v mPTC. K detailnímu posouzení úlohy CK19 a Ecad při šíření PTC mimo štítnou žlázu je třeba dalších studií.
The immunohistochemical expression of galectin-3 (Gal3), cytokeratin 19 (CK19), neural cell adhesion molecule (NCAM), and E-cadherin (Ecad) was evaluated to assess their use in diagnostics of papillary thyroid carcinoma (PTC). A total of 84 PTCs - 36 classical variants (cPTCs), 26 follicular variants (fPTCs), and 22 papillary microcarcinomas (mPTCs) were studied. Expression of Gal3 was found in 36/36 (100%) cPTCs, 24/26 (92%) fPTCs, and 19/22 (86%) mPTCs. CK19 expression was detected in 34/36 (94%) cPTCs, 17/26 (65%) fPTCs, and 13/22 (59%) mPTCs. Expression of NCAM was seen in 5/36 (14%) cPTCs, 7/26 (27%) fPTCs, and 9/22 (41%) mPTCs. Ecad expression was found in 23/36 (64%) cPTCs, 17/26 (65%) fPTCs, and 18/22 (82%) mPTCs. A significant difference in CK19 expression was observed between cPTC and both fPTC and mPTC (p < 0.001). Furthermore, extrathyroid tumor spread significantly correlated with both level of CK19 expression and loss of Ecad expression (p = 0.001, p = 0.04). Our findings suggest that Gal3 and CK19 are useful markers for PTC, although decreased CK19 expression in mPTC and fPTC must be considered. Furthermore, CK19 and Ecad may play a role in extrathyroid tumor spread.
- MeSH
- diferenciální diagnóza MeSH
- finanční podpora výzkumu jako téma MeSH
- galektin 3 diagnostické užití MeSH
- kadheriny diagnostické užití MeSH
- keratin-19 diagnostické užití MeSH
- molekuly buněčné adheze nervové diagnostické užití MeSH
- nádory štítné žlázy klasifikace patologie MeSH
- papilární karcinom klasifikace patologie MeSH
- Publikační typ
- hodnotící studie MeSH
- MeSH
- audiometrie metody přístrojové vybavení MeSH
- lidé MeSH
- poruchy sluchu diagnóza MeSH
- Check Tag
- lidé 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
- bolest diagnóza MeSH
- diferenciální diagnóza metody MeSH
- lidé MeSH
- lokální anestezie využití MeSH
- management bolesti MeSH
- Check Tag
- lidé MeSH
