BACKGROUND: Lipopolysaccharide (LPS)-induced inflammation of lung tissues triggers irreversible alterations in the lung parenchyma, leading to fibrosis and pulmonary dysfunction. While the molecular and cellular responses of immune and connective tissue cells in the lungs are well characterized, the specific epithelial response remains unclear due to the lack of representative cell models. Recently, we introduced human embryonic stem cell-derived expandable lung epithelial (ELEP) cells as a novel model for studying lung injury and regeneration. METHODS: ELEPs were derived from the CCTL 14 human embryonic stem cell line through activin A-mediated endoderm specification, followed by further induction toward pulmonary epithelium using FGF2 and EGF. ELEPs exhibit a high proliferation rate and express key structural and molecular markers of alveolar progenitors, such as NKX2-1. The effects of Escherichia coli LPS serotype O55:B5 on the phenotype and molecular signaling of ELEPs were analyzed using viability and migration assays, mRNA and protein levels were determined by qRT-PCR, western blotting, and immunofluorescent microscopy. RESULTS: We demonstrated that purified LPS induces features of a hybrid epithelial-to-mesenchymal transition in pluripotent stem cell-derived ELEPs, triggers the unfolded protein response, and upregulates intracellular β-catenin level through retention of E-cadherin within the endoplasmic reticulum. CONCLUSIONS: Human embryonic stem cell-derived ELEPs provide a biologically relevant, non-cancerous lung cell model to investigate molecular responses to inflammatory stimuli and address epithelial plasticity. This approach offers novel insights into the fine molecular processes underlying lung injury and repair.

• MeSH
• Cell Line MeSH
• Antigens, CD metabolism   MeSH
• Endoplasmic Reticulum * metabolism   drug effects   MeSH
• Epithelial-Mesenchymal Transition * drug effects   MeSH
• Epithelial Cells * drug effects   metabolism   cytology   MeSH
• Cadherins * metabolism   MeSH
• Humans MeSH
• Human Embryonic Stem Cells * cytology   MeSH
• Lipopolysaccharides * pharmacology   MeSH
• Lung * cytology   MeSH
• Thyroid Nuclear Factor 1 MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Genová terapie (GT) se postupně stává běžným způsobem léčby. Již není výsadou velkých univerzitních pracovišť, jejichž laboratoře zvládají analytické postupy zaměřené na nukleové kyseliny a jejichž klinické týmy zvládají aplikaci. Původně byla určena pro dědičné choroby, které vzhledem ke svému řídkému výskytu byly označovány jako vzácná onemocnění a GT se dosud uplatňovala jen u dětí, aby působila ještě před rozvojem onemocnění. Nové způsoby léčby začaly být používány i u chorob běžných, jakými jsou např. metabolické poruchy (diabetes), a dokonce u takových, které nás sužují stále častěji, jako nejrůznější malignity a nemoci centrální nervové soustavy (např. Alzheimerova choroba). Cílem genové terapie jsou geny, jejichž změny v podobě patogenních variant (dříve mutací) vyvolávají poruchy fenotypu. Naší snahou je buď jejich vyřazení z funkce (např. u hemoglobinopatií), nebo jejich nahrazení geny s normální funkcí. Ty lze do genomu vnést pomocí některého z vhodných přenašečů (tzv. vektorů), jakými jsou např. viry nebo lipozomy. Proces GT může probíhat přímo v těle pacienta (in vivo), nebo mimo něj na jeho izolovaných buňkách (ex vivo), kterými jsou obvykle indukované pluripotentní kmenové buňky (iPSC – induced pluripotent stem cell). Po úpravě se tyto buňky vracejí do pacientova těla, aby tak naplnily svůj „úděl“. V širším slova smyslu může být GT namířena i na produkt genové transkripce, kterým je messenger RNA (mRNA), nebo konečný produkt realizace genové funkce, jakým jsou funkční bílkoviny (např. u cystické fibrózy). U různých chorob se úspěšně používají uvedené přístupy v závislosti na jejich dostupnosti, která je mimo jiné dána i náklady s GT spojenými nebo přístupností cílové tkáně. Nejen ověřování účinnosti a bezpečnosti GT, ale i ekonomické důvody rozhodují o tom, proč se GT rozvíjí jen pozvolna a proč se jí ujímají většinou jen velké a bohaté instituce. Rozhodující je také to, že celý proces vývoje od výchozích experimentálních prací přes klinické zkoušky až ke konečnému přípravku běžně trvá i dekádu či déle.

Gene therapy is gradually becoming a mainstream treatment modality and is no longer the preserve of large university departments whose laboratories master nucleic acid analytical procedures and whose clinical teams manage its administration. It was originally designed for genetic diseases that, because of their prevalence, were a group known as rare diseases. Gene therapy has so far been applied in children to act before the disease development. These new treatments have also begun to be applied for common diseases such as metabolic disorders (e. g. diabetes) and even for those that are increasingly affecting us, such as various malignancies and diseases of the central nervous system (e. g. Alzheimer’s disease). The targets targeted by GT are genes, where pathogenic alterations in the form of pathogenic variants (formerly mutations) induce phenotypic disorders, and our aim is either to knock them out of function (e. g. haemoglobinopathies) or to replace them with genes with normal function, which we introduce into the genome using one of the appropriate vectors, such as viruses or liposomes. The process of GT can take place directly inside the patient's body (in vivo) or outside the body on isolated cells (ex vivo), which are usually stem cells (iPSCs, induced pluripotent stem cell). After treatment, these cells are returned to the patient's body to fulfil their "destiny". In a broader sense, GT can target the product of gene transcription, which is the messenger RNA, or the end product of gene function, such as functional proteins (eg. cystic fibrosis). Any of these approaches have been used successfully in various diseases, depending on their availability, which is determined, among other things, by the costs associated with GT or the accessibility of the target tissue. Ultimately, it is not only the validation of the efficacy and safety of GT, but also economic reasons that determine why GT has been slow to develop and is mostly undertaken only by large and wealthy institutions. Another decisive factor is that from initial experimental work through clinical trials, the whole process of its development normally takes up to a decade.

• MeSH
• Cystic Fibrosis genetics   therapy   MeSH
• alpha 1-Antitrypsin Deficiency genetics   therapy   MeSH
• Muscular Dystrophy, Duchenne genetics   therapy   MeSH
• Genetic Therapy * methods   MeSH
• Huntington Disease genetics   therapy   MeSH
• Hematologic Diseases genetics   therapy   MeSH
• Humans MeSH
• Myotonic Dystrophy genetics   therapy   MeSH
• Neoplasms genetics   therapy   MeSH
• Retinitis Pigmentosa genetics   therapy   MeSH
• Muscular Atrophy, Spinal genetics   therapy   MeSH
• Rare Diseases * genetics   therapy   MeSH
• Check Tag
• Humans MeSH

• MeSH
• Biology MeSH
• Genetics, Medical MeSH
• Molecular Medicine MeSH

• Conspectus
• Lékařské vědy. Lékařství
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• biologie
• NML Publication type
• učebnice vysokých škol

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

• MeSH
• DNA Repair Enzymes genetics   MeSH
• Gene Regulatory Networks MeSH
• Nuclear Proteins genetics   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Neurodevelopmental Disorders * genetics   MeSH
• RNA Splicing MeSH
• RNA Splicing Factors genetics   MeSH
• Spliceosomes * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-β superfamily. On the other hand, the specific roles of TGF-β and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-β (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-β or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-β inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-β and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-β and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-β subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-β (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.

• MeSH
• Cell Differentiation genetics   MeSH
• Zebrafish * genetics   MeSH
• Follicle Stimulating Hormone pharmacology   metabolism   MeSH
• RNA, Messenger genetics   MeSH
• Pyrazoles * MeSH
• Spermatogenesis genetics   MeSH
• Spermatogonia * metabolism   MeSH
• Testis metabolism   MeSH
• Thiosemicarbazones * MeSH
• Transforming Growth Factor beta metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Aim: Human induced pluripotent stem cells (iPSCs) are inefficiently derived from somatic cells by overexpression of defined transcription factors. Overexpression of H2A histone variant macroH2A1.1, but not macroH2A1.2, leads to increased iPSC reprogramming by unclear mechanisms. Materials & methods: Cleavage under targets and tagmentation (CUT&Tag) allows robust epigenomic profiling of a low cell number. We performed an integrative CUT&Tag-RNA-Seq analysis of macroH2A1-dependent orchestration of iPSCs reprogramming using human endothelial cells. Results: We demonstrate wider genome occupancy, predicted transcription factors binding, and gene expression regulated by macroH2A1.1 during reprogramming, compared to macroH2A1.2. MacroH2A1.1, previously associated with neurodegenerative pathologies, specifically activated ectoderm/neural processes. Conclusion: CUT&Tag and RNA-Seq data integration is a powerful tool to investigate the epigenetic mechanisms occurring during cell reprogramming.

• MeSH
• Endothelial Cells metabolism   MeSH
• Histones * metabolism   MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Humans MeSH
• Cellular Reprogramming genetics   MeSH
• RNA-Seq MeSH
• Transcription Factors genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• MeSH
• Cell Differentiation genetics   MeSH
• Embryonic Stem Cells MeSH
• Humans MeSH
• MicroRNAs * genetics   metabolism   MeSH
• Neural Stem Cells * metabolism   MeSH
• Gene Expression Profiling MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Embryonic stem cells and induced pluripotent stem cells provided us with fascinating new knowledge in recent years. Mechanistic insight into intricate regulatory circuitry governing pluripotency stemness and disclosing parallels between pluripotency stemness and cancer instigated numerous studies focusing on roles of pluripotency transcription factors, including Oct4, Sox2, Klf4, Nanog, Sall4 and Tfcp2L1, in cancer. Although generally well substantiated as tumour-promoting factors, oncogenic roles of pluripotency transcription factors and their clinical impacts are revealing themselves as increasingly complex. In certain tumours, both Oct4 and Sox2 behave as genuine oncogenes, and reporter genes driven by composite regulatory elements jointly recognized by both the factors can identify stem-like cells in a proportion of tumours. On the other hand, cancer stem cells seem to be biologically very heterogeneous both among different tumour types and among and even within individual tumours. Pluripotency transcription factors are certainly implicated in cancer stemness, but do not seem to encompass its entire spectrum. Certain cancer stem cells maintain their stemness by biological mechanisms completely different from pluripotency stemness, sometimes even by engaging signalling pathways that promote differentiation of pluripotent stem cells. Moreover, while these signalling pathways may well be antithetical to stemness in pluripotent stem cells, they may cooperate with pluripotency factors in cancer stem cells - a paradigmatic example is provided by the MAPK-AP-1 pathway. Unexpectedly, forced expression of pluripotency transcription factors in cancer cells frequently results in loss of their tumour-initiating ability, their phenotypic reversion and partial epigenetic normalization. Besides the very different signalling contexts operating in pluripotent and cancer stem cells, respectively, the pronounced dose dependency of reprogramming pluripotency factors may also contribute to the frequent loss of tumorigenicity observed in induced pluripotent cancer cells. Finally, contradictory cell-autonomous and non-cell-autonomous effects of various signalling molecules operate during pluripotency (cancer) reprogramming. The effects of pluripotency transcription factors in cancer are thus best explained within the concept of cancer stem cell heterogeneity.

• MeSH
• Cell Differentiation genetics   MeSH
• Embryonic Stem Cells MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Humans MeSH
• Neoplasms * genetics   metabolism   MeSH
• Octamer Transcription Factor-3 genetics   metabolism   MeSH
• Pluripotent Stem Cells * MeSH
• Cellular Reprogramming genetics   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.

• MeSH
• Genome MeSH
• Mouse Embryonic Stem Cells metabolism   MeSH
• Mice MeSH
• Telomere-Binding Proteins * genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Mammals genetics   MeSH
• Telomere * metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP-TEAD respond to cell-cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell-cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP-TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP-TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP-TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype.

• MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Angiomotins metabolism   MeSH
• Cell Differentiation MeSH
• Cell Line MeSH
• Cytoskeleton metabolism   MeSH
• Humans MeSH
• Human Embryonic Stem Cells metabolism   MeSH
• Mesoderm metabolism   MeSH
• YAP-Signaling Proteins genetics   metabolism   MeSH
• Signal Transduction MeSH
• TEA Domain Transcription Factors genetics   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH