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
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• endoteliální buňky metabolismus   MeSH
• histony * metabolismus   MeSH
• indukované pluripotentní kmenové buňky * metabolismus   MeSH
• lidé MeSH
• přeprogramování buněk genetika   MeSH
• sekvenování transkriptomu MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.

• MeSH
• biologické markery MeSH
• buněčná diferenciace genetika   MeSH
• embryoidní tělíska cytologie   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• karyotyp MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• předmléčné zuby cytologie   MeSH
• přeprogramování buněk * MeSH
• transkriptom MeSH
• vývojová regulace genové exprese MeSH
• zubní dřeň cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Reprogramming of non-endocrine pancreatic cells into insulin-producing cells represents a promising therapeutic approach for the restoration of endogenous insulin production in diabetic patients. In this paper, we report that human organoid cells derived from the pancreatic tissue can be reprogrammed into the insulin-producing cells (IPCs) by the combination of in vitro transcribed modified mRNA encoding transcription factor neurogenin 3 and small molecules modulating the epigenetic state and signalling pathways. Upon the reprogramming, IPCs formed 4.6 ± 1.2 % of the total cells and expressed typical markers (insulin, glucokinase, ABCC8, KCNJ11, SLC2A2, SLC30A8) and transcription factors (PDX1, NEUROD1, MAFA, NKX2.2, NKX6.1, PAX4, PAX6) needed for the proper function of pancreatic β-cells. Additionally, we have revealed a positive effect of ALK5 inhibitor RepSox on the overall reprogramming efficiency. However, the reprogrammed IPCs possessed only a partial insulin-secretory capacity, as they were not able to respond to the changes in the extracellular glucose concentration by increasing insulin secretion. Based on the achieved results we conclude that due to the incomplete reprogramming, the IPCs have immature character and only partial properties of native human β-cells.

• MeSH
• antigen AC133 metabolismus   MeSH
• beta-buňky cytologie   účinky léků   MeSH
• dospělí MeSH
• genetická transkripce účinky léků   MeSH
• inzulin biosyntéza   MeSH
• knihovny malých molekul farmakologie   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• organoidy cytologie   MeSH
• přeprogramování buněk účinky léků   genetika   MeSH
• proliferace buněk MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• transkripční faktory bHLH genetika   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The possibility of replacing the originally discovered and widely used DNA reprogramming transcription factors is stimulating enormous effort to identify more effective compounds that would not alter the genetic information. Here, we describe the generation of induced pluripotent stem cells (iPSc) from head-derived primary culture of mouse embryonic cells using small chemical inhibitors of the MEK and TGF-beta pathways without delivery of exogenous transcription factors. These iPSc express standard pluripotency markers and retain their potential to differentiate into cells of all germ layers. Our data indicate that head-derived embryonic neural cells might have the reprogramming potential while neither the same primary cells cultivated over five passages in vitro nor a cell population derived from adult brain possesses this capacity. Our results reveal the potential for small molecules to functionally replace routinely used transcription factors and lift the veil on molecular regulation controlling pluripotency. The conditions described here could provide a platform upon which other genome non integrative and safer reprogramming processes could be developed. This work also shows novel potential for developing embryonic neural cells.

• MeSH
• diferenciační antigeny biosyntéza   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• MAP kinasový signální systém * MeSH
• myši MeSH
• přeprogramování buněk * MeSH
• transformující růstový faktor beta metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus-oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming.

• MeSH
• apoptóza MeSH
• blastocysta cytologie   MeSH
• embryo savčí cytologie   MeSH
• fertilizace in vitro MeSH
• fluorescenční mikroskopie MeSH
• klonování organismů metody   MeSH
• kostní morfogenetický protein 6 farmakologie   MeSH
• lidé MeSH
• oocyty cytologie   MeSH
• ovarium patologie   MeSH
• přeprogramování buněk MeSH
• rekombinantní proteiny metabolismus   MeSH
• skot MeSH
• stanovení celkové genové exprese MeSH
• techniky jaderného přenosu MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Transplantace inzulín produkující tkáně představuje optimální možnost léčby diabetu závislého na inzulínu. Vzhledem k nedostatku lidských orgánových dárců je však tento způsob léčby málo dostupný. Alternativním zdrojem dostupným pro všechny pacienty by mohly být inzulín produkující buňky získané řízenou přeměnou neendokrinních buněk pankreatu. Doposud publikované práce potvrdily, že je možné neendokrinní pankreatické buňky transdiferencovat na inzulín produkující buňky, které jsou po transplantaci schopny vyléčit diabetes. Zásadním problémem dosavadních postupů je však nízká efektivita přeměny na inzulín produkující buňky, nebo nutnost využít postupy genové terapie, které sebou nesou rizika nádorového zvratu. Nedostatky těchto postupů hodláme vyřešit s pomocí nové metody založené na použití modifikovaných molekul mRNA. V předkládaném projektu hodláme ke stimulaci transdiferenciace pankreatických neendokrinních buněk na inzulín produkující beta-buňky využít modifikované molekuly mRNA, které umožní expresi klíčových transkripčních faktorů stimulujících diferenciaci beta-buněk.; Transplantation of the whole pancreas or isolated Langerhans islets are currently the only available therapeutic options that allow to cure insulin-dependent diabetes. However, both of these options are significantly limited due to the lack of available organ donors. Insulin-producing cells (IPCs) derived from non-endocrine pancreatic cells (NEPCs) represent very promising alternative source that could be available for the treatment of all diabetic patients. Published data have confirmed that IPCs derived from NEPCs are able to cure diabetes. However, only a small percentage of pancreatic cells is able to transdifferentiate into IPCs under the standard conditions. Therefore a more efficient but also unsafe method that uses viral vectors has to be applied in order to improve efficiency of that process. New approach of cell reprogramming that is based on the application of synthetic modified mRNA could solve all these issues. Therefore we propose to use synthetic modified mRNAs encoding key transcription factors of beta-cell differentiation for transdifferentiation of NEPCs into IPCs.

• MeSH
• beta-buňky transplantace   MeSH
• diabetes mellitus chirurgie   MeSH
• inzulin terapeutické užití   MeSH
• messenger RNA terapeutické užití   MeSH
• přeprogramování buněk MeSH
• transdiferenciace buněk MeSH
• transkripční faktory MeSH
• transplantace buněk metody   veterinární   MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• diabetologie
• transplantologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Arabidopsis thaliana mutants dysfunctional in the evolutionarily conserved protein complex chromatin assembly factor-1 (CAF-1), which deposits the canonical histone H3 variant H3.1 during DNA synthesis-dependent chromatin assembly, display complex phenotypic changes including meristem and growth alterations, sensitivity to DNA-damaging agents, and reduced fertility. We reported previously that mutants in the FAS1 subunit of CAF-1 progressively lose telomere and 45S rDNA repeats. Here we show that multiple aspects of the fas phenotype are recovered immediately on expression of a reintroduced FAS1 allele, and are clearly independent of the recovery of rDNA copy-numbers and telomeres. In reverted lines, 45S rDNA genes are recovered to diverse levels with a strikingly different representation of their variants, and the typical association of nucleolar organizing region 4 with the nucleolus is perturbed. One of 45S rDNA variants (VAR1), which is silenced in wild-type (WT) plants without mutation history (Col-0 WT), dominates the expression pattern, whereas VAR2 is dominant in Col-0 WT plants. We propose an explanation for the variability of telomere and 45S rDNA repeats associated with CAF-1 function, suggesting that the differences in nuclear partitioning and expression of the rDNA variants in fas mutants and their revertants provide a useful experimental system to study genetic and epigenetic factors in gene dosage compensation.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• chromatin genetika   metabolismus   MeSH
• genom rostlinný genetika   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• ribozomální DNA genetika   metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications.

• MeSH
• buněčná diferenciace genetika   MeSH
• buněčné linie MeSH
• fibroblasty cytologie   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• lidské embryonální kmenové buňky cytologie   metabolismus   MeSH
• metylace DNA * MeSH
• přeprogramování buněk genetika   MeSH
• shluková analýza MeSH
• stanovení celkové genové exprese MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

RNA silencing is a complex of mechanisms that regulate gene expression through small RNA molecules. The microRNA (miRNA) pathway is the most common of these in mammals. Genome-encoded miRNAs suppress translation in a sequence-specific manner and facilitate shifts in gene expression during developmental transitions. Here, we discuss the role of miRNAs in oocyte-to-zygote transition and in the control of pluripotency. Existing data suggest a common principle involving miRNAs in defining pluripotent and differentiated cells. RNA silencing pathways also rapidly evolve, resulting in many unique features of RNA silencing in different taxonomic groups. This is exemplified in the mouse model of oocyte-to-zygote transition, in which the endogenous RNA interference pathway has acquired a novel role in regulating protein-coding genes, while the miRNA pathway has become transiently suppressed.

• MeSH
• fylogeneze MeSH
• lidé MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• mikro RNA klasifikace   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• oocyty cytologie   fyziologie   MeSH
• pluripotentní kmenové buňky cytologie   fyziologie   MeSH
• RNA interference MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• zygota cytologie   fyziologie   MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH