Neural differentiation
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The involvement of microRNAs (miRNAs) in orchestrating self-renewal and differentiation of stem cells has been revealed in a number of recent studies. And while in human pluripotent stem cells, miRNAs have been directly linked to the core pluripotency network, including the cell cycle regulation and the maintenance of the self-renewing capacity, their role in the onset of differentiation in other contexts, such as determination of neural cell fate, remains poorly described. To bridge this gap, we used three model cell types to study miRNA expression patterns: human embryonic stem cells (hESCs), hESCs-derived self-renewing neural stem cells (NSCs), and differentiating NSCs. The comprehensive miRNA profiling presented here reveals novel sets of miRNAs differentially expressed during human neural cell fate determination in vitro. Furthermore, we report a miRNA expression profile of self-renewing human NSCs, which has been lacking to this date. Our data also indicates that miRNA clusters enriched in NSCs share the target-determining seed sequence with cell cycle regulatory miRNAs expressed in pluripotent hESCs. Lastly, our mechanistic experiments confirmed that cluster miR-17-92, one of the NSCs-enriched clusters, is directly transcriptionally regulated by transcription factor c-MYC.
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
Adenosine deaminase acting on RNA 1 (ADAR1) is the principal enzyme for the adenosine-to-inosine RNA editing that prevents the aberrant activation of cytosolic nucleic acid sensors by endogenous double stranded RNAs and the activation of interferon-stimulated genes. In mice, the conditional neural crest deletion of Adar1 reduces the survival of melanocytes and alters the differentiation of Schwann cells that fail to myelinate nerve fibers in the peripheral nervous system. These myelination defects are partially rescued upon the concomitant removal of the Mda5 antiviral dsRNA sensor in vitro, suggesting implication of the Mda5/Mavs pathway and downstream effectors in the genesis of Adar1 mutant phenotypes. By analyzing RNA-Seq data from the sciatic nerves of mouse pups after conditional neural crest deletion of Adar1 (Adar1cKO), we here identified the transcription factors deregulated in Adar1cKO mutants compared to the controls. Through Adar1;Mavs and Adar1cKO;Egr1 double-mutant mouse rescue analyses, we then highlighted that the aberrant activation of the Mavs adapter protein and overexpression of the early growth response 1 (EGR1) transcription factor contribute to the Adar1 deletion associated defects in Schwann cell development in vivo. In silico and in vitro gene regulation studies additionally suggested that EGR1 might mediate this inhibitory effect through the aberrant regulation of EGR2-regulated myelin genes. We thus demonstrate the role of the Mda5/Mavs pathway, but also that of the Schwann cell transcription factors in Adar1-associated peripheral myelination defects.
- MeSH
- adenosindeaminasa * genetika metabolismus MeSH
- buněčná diferenciace * genetika MeSH
- crista neuralis * metabolismus MeSH
- IFIH1 genetika metabolismus MeSH
- myelinová pochva metabolismus MeSH
- myši knockoutované * MeSH
- myši MeSH
- Schwannovy buňky * metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The unique properties of stem cells to self-renew and differentiate hold great promise in disease modelling and regenerative medicine. However, more information about basic stem cell biology and thorough characterization of available stem cell lines is needed. This is especially essential to ensure safety before any possible clinical use of stem cells or partially committed cell lines. As proteins are the key effector molecules in the cell, the proteomic characterization of cell lines, cell compartments or cell secretome and microenvironment is highly beneficial to answer above mentioned questions. Nowadays, method of choice for large-scale discovery-based proteomic analysis is mass spectrometry (MS) with data-independent acquisition (DIA). DIA is a robust, highly reproducible, high-throughput quantitative MS approach that enables relative quantification of thousands of proteins in one sample. In the current protocol, we describe a specific variant of DIA known as SWATH-MS for characterization of neural stem cell differentiation. The protocol covers the whole process from cell culture, sample preparation for MS analysis, the SWATH-MS data acquisition on TTOF 5600, the complete SWATH-MS data processing and quality control using Skyline software and the basic statistical analysis in R and MSstats package. The protocol for SWATH-MS data acquisition and analysis can be easily adapted to other samples amenable to MS-based proteomics.
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
LIF is a cytokine playing a key role in the regulation of self-renewal and maintenance of undifferentiated state in mouse ES cells. The response of pluripotent cells to LIF is mediated mainly by the STAT3 and ERK signalling pathways. Recently, we have shown that LIF potentiated retinoic acid-induced neural differentiation of pluripotent mouse embryonal carcinoma P19 cells. Here we demonstrate that pro-neural effects of LIF and partially also of retinoic acid are abolished by inhibition of the JAK2->STAT3 signalling pathway. In contrast, inhibition of the MEK1->ERK signalling pathway does not exhibit any effect. These results suggest that in neurogenic regions, cooperative action of LIF and other neuro-differentiation-inducing factors, such as retinoic acid, may be mediated by the STAT3 signalling pathway.
- MeSH
- buněčná diferenciace MeSH
- embryonální karcinom genetika MeSH
- embryonální kmenové buňky MeSH
- finanční podpora výzkumu jako téma MeSH
- interleukin-6 farmakokinetika farmakologie MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- pluripotentní kmenové buňky MeSH
- techniky in vitro MeSH
- transkripční faktor STAT3 farmakokinetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata 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
The neural crest (NC) is a transient dynamic structure of ectodermal origin, found in early vertebrate embryos. The multipotential NC cells migrate along well defined routes, differentiate to various cell types including melanocytes and participate in the formation of various permanent tissues. As there is only limited information about the molecular mechanisms controlling early events in melanocyte specification and development, we exploited the AMV v-Myb transcriptional regulator, which directs differentiation of in vitro chicken NC cells to the melanocyte lineage. This activity is strictly dependent on v-Myb specifically binding to the Myb recognition DNA element (MRE). The two tamoxifen-inducible v-Myb alleles were constructed one which recognizes the MRE and one which does not. These were activated in ex ovo NC cells, and the expression profiles of resulting cells were analyzed using Affymetrix microarrays and RT-PCR. These approaches revealed up-regulation of the BMP antagonist Gremlin 2 mRNA, and down-regulation of mRNAs encoding several epithelial genes including KRT19 as very early events following the activation of melanocyte differentiation by v-Myb. The enforced v-Myb expression in neural tubes of chicken embryos resulted in detectable presence of Gremlin 2 mRNA. However, expression of Gremlin 2 in NC cells did not promote formation of melanocytes suggesting that Gremlin 2 is not the master regulator of melanocytic differentiation.
- MeSH
- aktivace transkripce * MeSH
- alely MeSH
- buněčná diferenciace * MeSH
- crista neuralis cytologie MeSH
- keratin-19 genetika metabolismus MeSH
- kostní morfogenetický protein 5 genetika metabolismus MeSH
- kultivované buňky MeSH
- kuřecí embryo MeSH
- melanocyty fyziologie MeSH
- mezibuněčné signální peptidy a proteiny genetika metabolismus MeSH
- onkogenní proteiny v-myb fyziologie MeSH
- ptačí proteiny genetika metabolismus MeSH
- regulace genové exprese MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
