Embryonic stem cells
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The stimulation of myocardium repair is restricted due to the limited understanding of heart regeneration. Interestingly, endogenous opioid peptides such as dynorphins and enkephalins are suggested to support this process. However, the mechanism-whether through the stimulation of the regenerative capacity of cardiac stem cells or through effects on other cell types in the heart-is still not completely understood. Thus, a model of the spontaneous cardiomyogenic differentiation of mouse embryonic stem (mES) cells via the formation of embryoid bodies was used to describe changes in the expression and localization of opioid receptors within cells during the differentiation process and the potential of the selected opioid peptides, dynorphin A and B, and methionin-enkephalins and leucin-enkephalins, to modulate cardiomyogenic differentiation in vitro. The expressions of both κ- and δ-opioid receptors significantly increased during mES cell differentiation. Moreover, their primary colocalization with the nucleus was followed by their growing presence on the cytoplasmic membrane with increasing mES cell differentiation status. Interestingly, dynorphin B enhanced the downregulation gene expression of Oct4 characteristic of the pluripotent phenotype. Further, dynorphin B also increased cardiomyocyte-specific Nkx2.5 gene expression. However, neither dynorphin A nor methionin-enkephalins and leucin-enkephalins exhibited any significant effects on the course of mES cell differentiation. In conclusion, despite the increased expression of opioid receptors and some enhancement of mES cell differentiation by dynorphin B, the overall data do not support the notion that opioid peptides have a significant potential to promote the spontaneous cardiomyogenesis of mES cells in vitro.
- Klíčová slova
- dynorphins, enkephalins, heart regeneration, mouse embryonic stem cells, opioid receptors,
- MeSH
- buněčná diferenciace fyziologie MeSH
- kardiomyocyty cytologie fyziologie MeSH
- myokard cytologie MeSH
- myší embryonální kmenové buňky cytologie metabolismus MeSH
- myši MeSH
- opioidní peptidy metabolismus MeSH
- receptory opiátové metabolismus MeSH
- regenerace fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- opioidní peptidy MeSH
- receptory opiátové MeSH
Chromosomal instability evoked by abnormalities in centrosome numbers has been traditionally considered as a hallmark of aberrant, typically cancerous or senescent cells. We have reported previously that pristine human embryonic stem cells (hESC) suffer from high frequency of supernumerary centrosomes and hence may be prone to undergo abnormal mitotic divisions. We have also unraveled that this phenomenon of multicentrosomal mitoses vanishes with prolonged time in culture and with initiation of differentiation, and it is strongly affected by the culture substratum. In this study, we report for the first time that Cripto-1 protein (teratocarcinoma-derived growth factor 1, epidermal growth factor-Cripto/FRL-1/Cryptic) produced by hESC represents a factor capable of inducing formation of supernumerary centrosomes in cultured hESC. Elimination of Cripto-1 signaling on the other hand restores the normal number of centrosomes in hESC. Linking the secretory phenotype of hESC to the centrosomal metabolism may help to develop better strategies for propagation of stable and safe bioindustrial and clinical grade cultures of hESC. From a broader point of view, it may lead to unravelling Cripto-1 as a micro-environmental factor contributing to adverse cell behaviors in vivo.
- Klíčová slova
- Cripto-1, centrosomes, culture adaptation, embryonic stem cells, multipolar mitoses,
- MeSH
- buněčná diferenciace genetika MeSH
- centrozom * MeSH
- GPI-vázané proteiny antagonisté a inhibitory genetika MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie metabolismus MeSH
- mezibuněčné signální peptidy a proteiny genetika MeSH
- mitóza genetika MeSH
- nádorové proteiny antagonisté a inhibitory genetika MeSH
- signální transdukce genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- GPI-vázané proteiny MeSH
- mezibuněčné signální peptidy a proteiny MeSH
- nádorové proteiny MeSH
- TDGF1 protein, human MeSH Prohlížeč
The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for self-renewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson's disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells.
- Klíčová slova
- Chromatin, Differentiation, Embryonic stem cells, Epigenetics, Nucleus, Pluripotency,
- Publikační typ
- časopisecké články MeSH
Hematopoietic progenitors derived from human embryonic stem cells (hESCs) present both a potential cell source for cell-replacement therapies and an in vitro model for hematopoietic stem cell (HSC) development. Current protocols for the hematopoietic differentiation of hESCs suffer from low efficiency and functional defects in the derived HSCs. Epigenetic mechanisms of HSC development should be addressed to overcome these imperfections. The focus of this review is to summarize the knowledge on the epigenetic regulation of pluripotency and lineage-specific genes with the emphasis on the hematopoietic cell lineage. The potential utilization of this knowledge to improve the generation of HSCs for clinical application is also discussed.
The stability of in vitro cell cultures is an important issue for any clinical, bio-industrial, or pharmacological use. Embryonic stem cells are pluripotent; consequently, they possess the ability to differentiate into all three germ layers and are inherently prone to respond to differentiation stimuli. However, long-term culture inevitably yields clones that are best adapted to the culture conditions, passaging regimes, or differentiation sensitivity. This cellular plasticity is a major obstacle in the development of bio-industrial or clinical-grade cultures. At present, the quality control of cell cultures is limited by the lack of reliable (epi)genetic or molecular markers or by the focus on a particular type of instability such as karyotype abnormalities or adverse phenotypic traits. Therefore, there is an ongoing need for robust, feasible, and sensitive methods of determining or confirming cell status and for revealing potential divergences from the optimal state. We modeled both intrinsic and extrinsic changes in human embryonic stem cell (hESC) states using different experimental strategies and addressed the changes in cell status by intact cell mass spectrometry fingerprinting. The analysis of spectral fingerprints by methods routinely used in analytical chemistry clearly distinguished the morphologically and biochemically similar populations of hESCs and provided a biomarker-independent tool for the quality control of cell culture. Stem Cells Translational Medicine 2018;7:109-114.
- Klíčová slova
- Cell culture, Differentiation, Embryonic stem cells, Technology, Tissue engineering,
- MeSH
- biologické markery analýza MeSH
- buněčné kultury MeSH
- fenotyp MeSH
- kultivované buňky MeSH
- lidé MeSH
- lidské embryonální kmenové buňky fyziologie MeSH
- plasticita buňky fyziologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery 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.
- Klíčová slova
- HMGB1, HMGB2, differentiation, human embryonic stem cells, neuroectodermal cells,
- 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
- práce podpořená grantem MeSH
- Názvy látek
- histony MeSH
- protein HMGB1 MeSH
- protein HMGB2 MeSH
- telomerasa MeSH
Melatonin, a molecule involved in the regulation of circadian rhythms, has protective effects against myocardial injuries. However, its capability to regulate the maturation of cardiac progenitor cells is unclear. Recently, several studies have shown that melatonin inhibits the stabilization of hypoxia-inducible factors (HIFs), important signaling molecules with cardioprotective effects. In this study, by employing differentiating mouse embryonic stem cells, we report that melatonin significantly upregulated the expression of cardiac cell-specific markers (myosin heavy chains six and seven) as well as the percentage of myosin heavy chain-positive cells. Importantly, melatonin decreased HIF-1α stabilization and transcriptional activity and, in contrast, induced HIF-2α stabilization. Interestingly, the deletion of HIF-1α completely inhibited the pro-cardiomyogenic effect of melatonin as well as the melatonin-mediated HIF-2α stabilization. Moreover, melatonin increased Sirt-1 levels in a HIF-1α-dependent manner. Taken together, we provide new evidence of a time-specific inhibition of HIF-1α stabilization as an essential feature of melatonin-induced cardiomyogenesis and unexpected different roles of HIF-1α stabilization during various stages of cardiac development. These results uncover new mechanisms underlying the maturation of cardiac progenitor cells and can help in the development of novel strategies for using melatonin in cardiac regeneration therapy.
- Klíčová slova
- cardiomyogenesis, hypoxia-inducible factor-alpha, melatonin, mouse embryonic stem cells,
- MeSH
- faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus MeSH
- melatonin farmakologie MeSH
- myokard cytologie metabolismus MeSH
- myší embryonální kmenové buňky cytologie metabolismus MeSH
- myši MeSH
- stabilita proteinů účinky léků MeSH
- vývoj svalů účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- faktor 1 indukovatelný hypoxií - podjednotka alfa MeSH
- Hif1a protein, mouse MeSH Prohlížeč
- melatonin MeSH
Embryonic stem cells (ESCs), with their dual capacity to self-renew and differentiate, are commonly used to study differentiation, epigenetic regulation, lineage choices, and more. Using non-directed retroviral integration of a YFP/Cherry exon into mouse ESCs, we generated a library of over 200 endogenously tagged fluorescent fusion proteins and present several proof-of-concept applications of this library. We show the utility of this library to track proteins in living cells; screen for pluripotency-related factors; identify heterogeneously expressing proteins; measure the dynamics of endogenously labeled proteins; track proteins recruited to sites of DNA damage; pull down tagged fluorescent fusion proteins using anti-Cherry antibodies; and test for interaction partners. Thus, this library can be used in a variety of different directions, either exploiting the fluorescent tag for imaging-based techniques or utilizing the fluorescent fusion protein for biochemical pull-down assays, including immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, and more.
- Klíčová slova
- DNA damage, GFP, differentiation, embryonic stem cells, fluorescence, imaging, live imaging, microscopy, pluripotency, protein dynamics,
- MeSH
- buněčná diferenciace genetika MeSH
- exprese genu * MeSH
- genetická heterogenita MeSH
- genová knihovna MeSH
- myší embryonální kmenové buňky cytologie metabolismus MeSH
- myši MeSH
- poškození DNA MeSH
- rekombinantní fúzní proteiny genetika MeSH
- reportérové geny * MeSH
- transportní proteiny MeSH
- vazba proteinů MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- rekombinantní fúzní proteiny MeSH
- transportní proteiny MeSH
BACKGROUND: The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity. METHODS: By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome. RESULTS: These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions. CONCLUSION: Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.
- Klíčová slova
- Differentiation, Epithelium, Foregut endoderm, Lung, hESC,
- MeSH
- buněčná diferenciace MeSH
- epitel MeSH
- lidé MeSH
- lidské embryonální kmenové buňky * MeSH
- plíce metabolismus MeSH
- povrchově aktivní látky metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- povrchově aktivní látky MeSH
Neural stem cells are fundamental to development of the central nervous system (CNS)-as well as its plasticity and regeneration-and represent a potential tool for neuro transplantation therapy and research. This study is focused on examination of the proliferation dynamic and fate of embryonic neural stem cells (eNSCs) under differentiating conditions. In this work, we analyzed eNSCs differentiating alone and in the presence of sonic hedgehog (SHH) or triiodothyronine (T3) which play an important role in the development of the CNS. We found that inhibition of the SHH pathway and activation of the T3 pathway increased cellular health and survival of differentiating eNSCs. In addition, T3 was able to increase the expression of the gene for the receptor smoothened (Smo), which is part of the SHH signaling cascade, while SHH increased the expression of the T3 receptor beta gene (Thrb). This might be the reason why the combination of SHH and T3 increased the expression of the thyroxine 5-deiodinase type III gene (Dio3), which inhibits T3 activity, which in turn affects cellular health and proliferation activity of eNSCs.
- Klíčová slova
- cell differentiation, embryonic neural stem cells, sonic hedgehog, triiodothyronine,
- MeSH
- jodidperoxidasa genetika metabolismus MeSH
- kultivované buňky MeSH
- myší embryonální kmenové buňky cytologie metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nervové kmenové buňky cytologie metabolismus MeSH
- neurogeneze * MeSH
- proteiny hedgehog genetika metabolismus MeSH
- receptor Smoothened genetika metabolismus MeSH
- trijodthyronin metabolismus MeSH
- tyreoidální hormony, receptory beta genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- iodothyronine deiodinase type III MeSH Prohlížeč
- jodidperoxidasa MeSH
- proteiny hedgehog MeSH
- receptor Smoothened MeSH
- Shh protein, mouse MeSH Prohlížeč
- Smo protein, mouse MeSH Prohlížeč
- trijodthyronin MeSH
- tyreoidální hormony, receptory beta MeSH
