Primary cilia are cellular surface projections enriched in receptors and signaling molecules, acting as signaling hubs that respond to stimuli. Malfunctions in primary cilia have been linked to human diseases, including retinopathies and ocular defects. Here, we focus on TMEM107, a protein localized to the transition zone of primary cilia. TMEM107 mutations were found in patients with Joubert and Meckel-Gruber syndromes. A mouse model lacking Tmem107 exhibited eye defects such as anophthalmia and microphthalmia, affecting retina differentiation. Tmem107 expression during prenatal mouse development correlated with phenotype occurrence, with enhanced expression in differentiating retina and optic stalk. TMEM107 deficiency in retinal organoids resulted in the loss of primary cilia, down-regulation of retina-specific genes, and cyst formation. Knocking out TMEM107 in human ARPE-19 cells prevented primary cilia formation and impaired response to Smoothened agonist treatment because of ectopic activation of the SHH pathway. Our data suggest TMEM107 plays a crucial role in early vertebrate eye development and ciliogenesis in the differentiating retina.

• MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• myši MeSH
• polycystická choroba ledvin * genetika   MeSH
• poruchy ciliární motility * genetika   metabolismus   MeSH
• retina metabolismus   MeSH
• retinopathia pigmentosa * metabolismus   MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Oct4-mediated reprogramming has recently become a novel tool for the generation of various cell types from differentiated somatic cells. Although molecular mechanisms underlying this process are unknown, it is well documented that cells over-expressing Oct4 undergo transition from differentiated state into plastic state. This transition is associated with the acquisition of stem cells properties leading to epigenetically "open" state that is permissive to cell fate switch upon external stimuli. In order to contribute to our understanding of molecular mechanisms driving this process, we characterised human fibroblasts over-expressing Oct4 and performed comprehensive small-RNAseq analysis. Our analyses revealed new interesting aspects of Oct4-mediated cell plasticity induction. Cells over-expressing Oct4 lose their cell identity demonstrated by down-regulation of fibroblast-specific genes and up-regulation of epithelial genes. Interestingly, this process is associated with microRNA expression profile that is similar to microRNA profiles typically found in pluripotent stem cells. We also provide extensive network of microRNA families and clusters allowing us to precisely determine the miRNAome associated with the acquisition of Oct4-induced transient plastic state. Our data expands current knowledge of microRNA and their implications in cell fate alterations and contributing to understanding molecular mechanisms underlying it.

• MeSH
• buněčné linie MeSH
• embryo savčí * MeSH
• fibroblasty cytologie   metabolismus   MeSH
• lidé MeSH
• mikro RNA * biosyntéza   genetika   MeSH
• oktamerní transkripční faktor 3 * biosyntéza   genetika   MeSH
• regulace genové exprese * MeSH
• techniky buněčného přeprogramování * MeSH
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• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Development of neural tube has been extensively modeled in vitro using human pluripotent stem cells (hPSCs) that are able to form radially organized cellular structures called neural rosettes. While a great amount of research has been done using neural rosettes, studies have only inadequately addressed how rosettes are formed and what the molecular mechanisms and pathways involved in their formation are. Here we address this question by detailed analysis of the expression of pluripotency and differentiation-associated proteins during the early onset of differentiation of hPSCs towards neural rosettes. Additionally, we show that the BMP signaling is likely contributing to the formation of the complex cluster of neural rosettes and its inhibition leads to the altered expression of PAX6, SOX2 and SOX1 proteins and the rosette morphology. Finally, we provide evidence that the mechanism of neural rosettes formation in vitro is reminiscent of the process of secondary neurulation rather than that of primary neurulation in vivo. Since secondary neurulation is a largely unexplored process, its understanding will ultimately assist the development of methods to prevent caudal neural tube defects in humans.

• MeSH
• buněčná diferenciace * MeSH
• COUP transkripční faktor II genetika   metabolismus   MeSH
• faktory domény POU genetika   metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neurální trubice cytologie   embryologie   metabolismus   MeSH
• neurulace * MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• transkripční faktor PAX6 genetika   metabolismus   MeSH
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• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tumor necrosis factor-related apoptosis-inducing ligand-TRAIL-is a protein operating as a ligand capable of inducing apoptosis particularly in cancerously transformed cells, while normal healthy cells are typically nonresponsive. We have previously demonstrated that pluripotent human embryonic stem cells (hESC) are also refractory to TRAIL, even though they express all canonical components of the death receptor-induced apoptosis pathway. In this study, we have examined a capacity of DNA damage to provoke sensitivity of hESC to TRAIL. The extent of DNA damage, behavior of molecules involved in apoptosis, and response of hESC to TRAIL were investigated. The exposure of hESC to 1 μM and 2 μM concentrations of cisplatin have led to the formation of 53BP1 and γH2AX foci, indicating the presence of double-strand breaks in DNA, without affecting the expression of proteins contributing to mitochondrial membrane integrity. Interestingly, cisplatin upregulated critical components of the extrinsic apoptotic pathway-initiator caspase 8, effector caspase 3, and the cell death receptors. The observed increase of expression of the extrinsic apoptotic pathway components was sufficient to sensitize hESC to TRAIL-induced apoptosis; immense cell dying accompanied by enhanced PARP cleavage, processing of caspase 8, and full activation of caspase 3 were all observed after the treatment combining cisplatin and TRAIL. Finally, we have demonstrated the central role of caspase 8 in this process, since its downregulation abrogated the sensitizing effect of cisplatin.

• Publikační typ
• časopisecké články MeSH

HMGB1 and HMGB2 proteins have been implicated in numerous cellular processes, including proliferation, differentiation, apoptosis, and tumor growth. It is unknown whether they are involved in regulating the typical functions of pluripotent human embryonic stem cells (hESCs) and/or those of the differentiated derivatives of hESCs. Using inducible, stably transfected hESCs capable of shRNA-mediated knockdown of HMGB1 and HMGB2, we provide evidence that downregulation of HMGB1 and/or HMGB2 in undifferentiated hESCs does not affect the stemness of cells and induces only minor changes to the proliferation rate, cell-cycle profile, and apoptosis. After differentiation is induced, however, the downregulation of those proteins has important effects on proliferation, apoptosis, telomerase activity, and the efficiency of differentiation toward the neuroectodermal lineage. Furthermore, those processes are affected only when one, but not both, of the two proteins is downregulated; the knockdown of both HMGB1 and HMGB2 results in a normal phenotype. Those results advance our knowledge of regulation of hESC and human neuroectodermal cell differentiation and illustrate the distinct roles of HMGB1 and HMGB2 during early human development.

• MeSH
• apoptóza genetika   MeSH
• buněčná diferenciace * MeSH
• buněčná sebeobnova genetika   MeSH
• buněčné linie MeSH
• buněčný cyklus genetika   MeSH
• buněčný rodokmen genetika   MeSH
• down regulace genetika   MeSH
• histony metabolismus   MeSH
• lidé MeSH
• lidské embryonální kmenové buňky cytologie   metabolismus   MeSH
• neurální ploténka cytologie   MeSH
• proliferace buněk genetika   MeSH
• protein HMGB1 metabolismus   MeSH
• protein HMGB2 metabolismus   MeSH
• telomerasa metabolismus   MeSH
• transfekce MeSH
• tvar buňky genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

MicroRNA (miRNA) sponges are RNA transcripts containing multiple high-affinity binding sites that associate with and sequester specific miRNAs to prevent them from interacting with their target messenger (m)RNAs. Due to the high specificity of miRNA sponges and strong inhibition of target miRNAs, these molecules have become increasingly applied in miRNA loss-of-function studies. However, improperly designed sponge constructs may sequester off-target miRNAs; thus, it has become increasingly important to develop a tool for miRNA sponge construct design and testing. In this study, we introduce microRNA sponge generator and tester (miRNAsong), a freely available web-based tool for generation and in silico testing of miRNA sponges. This tool generates miRNA sponge constructs for specific miRNAs and miRNA families/clusters and tests them for potential binding to miRNAs in selected organisms. Currently, miRNAsong allows for testing of sponge constructs in 219 species covering 35,828 miRNA sequences. Furthermore, we also provide an example, supplemented with experimental data, of how to use this tool. Using miRNAsong, we designed and tested a sponge for miR-145 inhibition, and cloned the sequence into an inducible lentiviral vector. We found that established cell lines expressing miR-145 sponge strongly inhibited miR-145, thus demonstrating the usability of miRNAsong tool for sponge generation. URL: http://www.med.muni.cz/histology/miRNAsong/.

• MeSH
• HEK293 buňky MeSH
• internet * MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• mikro RNA genetika   metabolismus   MeSH
• počítačová simulace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

MicroRNA (miRNAs) are short noncoding RNA molecules involved in many cellular processes and shown to play a key role in somatic cell induced reprogramming. We performed an array based screening to identify candidates that are differentially expressed between dermal skin fibroblasts (DFs) and induced pluripotent stem cells (iPSCs). We focused our investigations on miR-145 and showed that this candidate is highly expressed in DFs relative to iPSCs and significantly downregulated during reprogramming process. Inhibition of miR-145 in DFs led to the induction of "cellular plasticity" demonstrated by: (a) alteration of cell morphology associated with downregulation of mesenchymal and upregulation of epithelial markers; (b) upregulation of pluripotency-associated genes including SOX2, KLF4, C-MYC; (c) downregulation of miRNA let-7b known to inhibit reprogramming; and (iv) increased efficiency of reprogramming to iPSCs in the presence of reprogramming factors. Together, our results indicate a direct functional link between miR-145 and molecular pathways underlying reprogramming of somatic cells to iPSCs.

• MeSH
• fibroblasty cytologie   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• přeprogramování buněk * genetika   MeSH
• regulace genové exprese MeSH
• reprodukovatelnost výsledků MeSH
• sekvence nukleotidů MeSH
• škára cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH