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.

• Klíčová slova
• Cancer, Differentiation, Lipids, Neural cells, Proliferation,
• 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
• Názvy látek
• DISP3 protein, human MeSH Prohlížeč
• membránové proteiny 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.

• Klíčová slova
• ADAR1, EGR1, MAVS, Schwann cells, differentiation, neural crest,
• 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
• Názvy látek
• ADAR1 protein, mouse MeSH Prohlížeč
• adenosindeaminasa * MeSH
• Ifih1 protein, mouse MeSH Prohlížeč
• IFIH1 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.

• Klíčová slova
• adipose-derived mesenchymal stem cells, electrical stimulation, mouse, nanofibre scaffold, neural differentiation, neural injury,
• 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
• Názvy látek
• diferenciační antigeny 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.

• Klíčová slova
• Cell line characterization, Mass spectrometry, Neural differentiation, Neural stem cell, Protein marker, Selected reaction monitoring,
• 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
• Názvy látek
• biologické markery 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.

• Klíčová slova
• Data independent acquisition, Mass spectrometry, Neural differentiation, Neural stem cell, Proteomics, SWATH-MS, Skyline, Spectral library,
• MeSH
• buněčná diferenciace MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• nervové kmenové buňky * chemie   metabolismus   MeSH
• proteom analýza   MeSH
• proteomika * metody   MeSH
• řízení kvality MeSH
• software * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom 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.

• Klíčová slova
• Hes1, Hif1α, Hypoxia, Neural stem cell, Neuroepithelium, Notch,
• 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

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.

• Klíčová slova
• Cell cycle, Human pluripotent stem cells, Neural stem cells, miRNA sequencing, microRNA,
• MeSH
• buněčná diferenciace genetika   MeSH
• embryonální kmenové buňky MeSH
• lidé MeSH
• mikro RNA * genetika   metabolismus   MeSH
• nervové kmenové buňky * metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA * MeSH

Topographical cues on materials can manipulate cellular fate, particularly for neural cells that respond well to such cues. Utilizing biomaterial surfaces with topographical features can effectively influence neuronal differentiation and promote neurite outgrowth. This is crucial for improving the regeneration of damaged neural tissue after injury. Here, we utilized groove patterns to create neural conduits that promote neural differentiation and axonal growth. We investigated the differentiation of human neural stem cells (NSCs) on silicon dioxide groove patterns with varying height-to-width/spacing ratios. We hypothesize that NSCs can sense the microgrooves with nanoscale depth on different aspect ratio substrates and exhibit different morphologies and differentiation fate. A comprehensive approach was employed, analyzing cell morphology, neurite length, and cell-specific markers. These aspects provided insights into the behavior of the investigated NSCs and their response to the topographical cues. Three groove-pattern models were designed with varying height-to-width/spacing ratios of 80, 42, and 30 for groove pattern widths of 1 μm, 5 μm, and 10 μm and nanoheights of 80 nm, 210 nm, and 280 nm. Smaller groove patterns led to longer neurites and more effective differentiation towards neurons, whereas larger patterns promoted multidimensional differentiation towards both neurons and glia. We transferred these cues onto patterned polycaprolactone (PCL) and PCL-graphene oxide (PCL-GO) composite 'stamps' using simple soft lithography and reproducible extrusion 3D printing methods. The patterned scaffolds elicited a response from NSCs comparable to that of silicon dioxide groove patterns. The smallest pattern stimulated the highest neurite outgrowth, while the middle-sized grooves of PCL-GO induced effective synaptogenesis. We demonstrated the potential for such structures to be wrapped into tubes and used as grafts for peripheral nerve regeneration. Grooved PCL and PCL-GO conduits could be a promising alternative to nerve grafting.

• Klíčová slova
• 3D printing, Differentiation, Groove patterns, Neural stem cells, Neurite guidance cues, Neurite outgrowth, Soft lithography,
• MeSH
• buněčná diferenciace MeSH
• lidé MeSH
• nervové kmenové buňky * MeSH
• neurony fyziologie   MeSH
• oxid křemičitý farmakologie   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• oxid křemičitý MeSH

Cell therapies represent a promising approach to slow down the progression of currently untreatable neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease or amyotrophic lateral sclerosis), as well as to support the reconstruction of functional neural circuits after spinal cord injuries. In such therapies, the grafted cells could either functionally integrate into the damaged tissue, partially replacing dead or damaged cells, modulate inflammatory reaction, reduce tissue damage, or support neuronal survival by secretion of cytokines, growth, and trophic factors. Comprehensive characterization of cells and their proliferative potential, differentiation status, and population purity before transplantation is crucial to preventing safety risks, e.g., a tumorous growth due to the proliferation of undifferentiated stem cells. We characterized changes in the proteome and secretome of human neural stem cells (NSCs) during their spontaneous (EGF/FGF2 withdrawal) differentiation and differentiation with trophic support by BDNF/GDNF supplementation. We used LC-MS/MS in SWATH-MS mode for global cellular proteome profiling and quantified almost three thousand cellular proteins. Our analysis identified substantial protein differences in the early stages of NSC differentiation with more than a third of all the proteins regulated (including known neuronal and NSC multipotency markers) and revealed that the BDNF/GDNF support affected more the later stages of the NSC differentiation. Among the pathways identified as activated during both spontaneous and BDNF/GDNF differentiation were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. Our follow-up secretome analysis using Luminex multiplex immunoassay revealed significant changes in the secretion of VEGF and IL-6 during NSC differentiation. Our results further demonstrated an increased expression of neuropilin-1 as well as catenin β-1, both known to participate in the regulation of VEGF signaling, and showed that VEGF-A isoform 121 (VEGF121), in particular, induces proliferation and supports survival of differentiating cells.

• Klíčová slova
• SWATH-MS, VEGF, neural differentiation, neural stem cell, proliferation, proteome, secretome,
• 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.

• Klíčová slova
• cell therapy, immunomodulation, neural stem cell differentiation, neural subpopulation, neurodegenerative disease, population-specific protein-expression signature, quantitative mass spectrometry, selected reaction monitoring,
• 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
• Názvy látek
• proteom MeSH