somatic cell reprogramming Dotaz Zobrazit nápovědu
Differentiated nuclei can be reprogrammed/remodelled to totipotency after their transfer to enucleated metaphase II (MII) oocytes. The process of reprogramming/remodelling is, however, only partially characterized. It has been shown that the oocyte nucleus (germinal vesicle - GV) components are essential for a successful remodelling of the transferred nucleus by providing the materials for pseudo-nucleus formation. However, the nucleus is a complex structure and exactly what nuclear components are required for a successful nucleus remodelling and reprogramming is unknown. Till date, the only nuclear sub-structure experimentally demonstrated to be essential is the oocyte nucleolus (nucleolus-like body, NLB). In this study, we investigated what other GV components might be necessary for the formation of normal-sized pseudo-pronuclei (PNs). Our results showed that the removal of the GV nuclear envelope with attached chromatin and chromatin-bound factors does not substantially influence the size of the remodelled nuclei in reconstructed cells and that their nuclear envelopes seem to have normal parameters. Rather than the insoluble nuclear lamina, the GV content, which is dissolved in the cytoplasm with the onset of oocyte maturation, influences the characteristics and size of transferred nuclei.
- MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- chromatin metabolismus MeSH
- cytoplazma metabolismus MeSH
- jaderná lamina metabolismus MeSH
- jaderný obal metabolismus MeSH
- messenger RNA metabolismus MeSH
- myši MeSH
- oocyty cytologie metabolismus MeSH
- oogeneze MeSH
- ovariální folikul metabolismus MeSH
- přeprogramování buněk * MeSH
- techniky jaderného přenosu * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
It is now more than nine years since Dolly, the world's first somatic cell cloned mammal was born, and the success of somatic cell nuclear transfer (SCNT) is still disappointingly low. Only about 3-5% of reconstructed embryos develop to term, and it is also evident that even if some clones are born, they are not necessarily fully developed and healthy. Embryonic and neonatal abnormalities of cloned offspring are probably a result of incorrect or incomplete reprogramming of the transferred donor cell nuclei. Such an incomplete reprogramming reflects the extremely low efficiency of SCNT. The key role in the process of reprogramming has been attributed to the enucleated oocyte-cytoplast into which the somatic cell nucleus is transferred. In our chapter, we will discuss the methodological approaches used for the preparation of cytoplasts and their possible reprogramming activities.
- MeSH
- buněčná diferenciace genetika MeSH
- buněčné jádro genetika MeSH
- cytoplazmatické struktury metabolismus MeSH
- embryonální vývoj genetika MeSH
- financování organizované MeSH
- histony genetika MeSH
- klonování organismů metody trendy MeSH
- lidé MeSH
- oocyty metabolismus MeSH
- restrukturace chromatinu genetika MeSH
- techniky jaderného přenosu normy trendy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata 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 foundations of cell reprogramming were laid by Yamanaka and co-workers, who showed that somatic cells can be reprogrammed into pluripotent cells (induced pluripotency). Since this discovery, the field of regenerative medicine has seen advancements. For example, because they can differentiate into multiple cell types, pluripotent stem cells are considered vital components in regenerative medicine aimed at the functional restoration of damaged tissue. Despite years of research, both replacement and restoration of failed organs/ tissues have remained elusive scientific feats. However, with the inception of cell engineering and nuclear reprogramming, useful solutions have been identified to counter the need for compatible and sustainable organs. By combining the science underlying genetic engineering and nuclear reprogramming with regenerative medicine, scientists have engineered cells to make gene and stem cell therapies applicable and effective. These approaches have enabled the targeting of various pathways to reprogramme cells, i.e., make them behave in beneficial ways in a patient-specific manner. Technological advancements have clearly supported the concept and realization of regenerative medicine. Genetic engineering is used for tissue engineering and nuclear reprogramming and has led to advances in regenerative medicine. Targeted therapies and replacement of traumatized , damaged, or aged organs can be realized through genetic engineering. Furthermore, the success of these therapies has been validated through thousands of clinical trials. Scientists are currently evaluating induced tissue-specific stem cells (iTSCs), which may lead to tumour-free applications of pluripotency induction. In this review, we present state-of-the-art genetic engineering that has been used in regenerative medicine. We also focus on ways that genetic engineering and nuclear reprogramming have transformed regenerative medicine and have become unique therapeutic niches.
- MeSH
- genetické inženýrství MeSH
- indukované pluripotentní kmenové buňky metabolismus cytologie MeSH
- lidé MeSH
- přeprogramování buněk * MeSH
- regenerativní lékařství * MeSH
- tkáňové inženýrství MeSH
- vývojová biologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Significance: The architecture of the mitochondrial network and cristae critically impact cell differentiation and identity. Cells undergoing metabolic reprogramming to aerobic glycolysis (Warburg effect), such as immune cells, stem cells, and cancer cells, go through controlled modifications in mitochondrial architecture, which is critical for achieving the resulting cellular phenotype. Recent Advances: Recent studies in immunometabolism have shown that the manipulation of mitochondrial network dynamics and cristae shape directly affects T cell phenotype and macrophage polarization through altering energy metabolism. Similar manipulations also alter the specific metabolic phenotypes that accompany somatic reprogramming, stem cell differentiation, and cancer cells. The modulation of oxidative phosphorylation activity, accompanied by changes in metabolite signaling, reactive oxygen species generation, and adenosine triphosphate levels, is the shared underlying mechanism. Critical Issues: The plasticity of mitochondrial architecture is particularly vital for metabolic reprogramming. Consequently, failure to adapt the appropriate mitochondrial morphology often compromises the differentiation and identity of the cell. Immune, stem, and tumor cells exhibit striking similarities in their coordination of mitochondrial morphology with metabolic pathways. However, although many general unifying principles can be observed, their validity is not absolute, and the mechanistic links thus need to be further explored. Future Directions: Better knowledge of the molecular mechanisms involved and their relationships to both mitochondrial network and cristae morphology will not only further deepen our understanding of energy metabolism but may also contribute to improved therapeutic manipulation of cell viability, differentiation, proliferation, and identity in many different cell types. Antioxid. Redox Signal. 39, 684-707.
The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs.
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
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
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
Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.
- MeSH
- 53BP1 * metabolismus genetika MeSH
- dvouřetězcové zlomy DNA * MeSH
- lidé MeSH
- myši MeSH
- oprava DNA * MeSH
- přeprogramování buněk * MeSH
- protein BRCA1 * metabolismus genetika MeSH
- rekombinační oprava DNA MeSH
- replikace DNA MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
