Spelling suggestions: "subject:"embryonic step cells"" "subject:"cmbryonic step cells""
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Global Position Effects on the Epigenetics of Variegated Lentiviral Vector Expression in Embryonic Stem CellsKhairandish, Arash 06 January 2011 (has links)
Lentivirus efficiently transduce stem cells, however are notably silenced in embryonic stem cells (ESC). Provirus can be silent, expressing, or variegated when clonal single copy ESCs spawn daughters that revert expression despite containing identical integration sites (IS) indicating epigenetic regulation. In the silent state, variegated provirus are bound by H1 and MeCP2, where H1 compensates for MeCP2 binding in DNA methylation null ESCs, consistent with a model of heterochromatin formation dependent on concentrations of its constituent components. ESC Variegation was hypothesized to result from spreading of nearby heterochromatin. Global IS analysis indicates Variegated IS favour gene deserts, repeat clusters, and LINEs while Expressers prefer gene density with stable modest expression and SINEs. Chromatin data does not support a role for the spread of heterochromatin possibly a consequence of the dynamic/dispersed nature of ESC heterochromatin. Variegation thus may depend on stochastic chromatin regulation by pluripotency factors at proximal genome organizing repeats.
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Acellular matrices derived from differentiating embryonic stem cellsNair, Rekha 10 November 2009 (has links)
Embryonic stem cells (ESCs) can differentiate into all somatic cells, and as such, are a promising cell source for therapeutic applications. In vitro, ESCs spontaneously differentiate via the aggregation of cells into embryoid bodies (EBs), which recapitulate aspects of early embryogenesis and harbor a unique reservoir of cues critical for tissue formation and morphogenesis. Embryonic healing responses employ similar intrinsic machinery used for tissue development, and these morphogenic cues may be captured within the EB microenvironment. Recent studies have shown that when injected into injury or defect models in vivo, ESCs synthesize and secrete extracellular factors that ultimately contribute to repair, suggesting that these molecules may be as important for regenerative therapies as functional differentiation of the cells. The overall objective of this project was to develop novel acellular matrices derived from differentiating ESCs undergoing morphogenesis. The central hypothesis was that embryonic matrices contain complex mixtures of extracellular factors that, when isolated, retain bioactivity and enhance wound healing in an adult environment.
The overall objective was accomplished by: (1) investigating the production of extracellular matrix (ECM) by differentiating ESCs as a function of differentiation time; (2) assessing the ability of solvents to efficiently decellularize EBs; and (3) evaluating the healing response elicited by acellular matrices derived from EBs in a murine dermal wound healing model. Endogenous ECM synthesis by EBs varied with time and was associated with specific differentiation events. Novel techniques were developed to effectively remove cell components from EBs in order to extract complex, bioactive acellular matrices. EB-derived acellular matrices significantly enhanced the healing of excisional dermal wounds in mice, indicating the potency of extracellular factors synthesized by ESCs. All together, these studies demonstrate that acellular matrices derived from ESCs retain morphogenic factors capable of influencing tissue repair. In addition, this work lays the foundation for future studies to further examine the functional role of endogenous matrix molecules on ESC differentiation and to evaluate the utility of a stem cell-derived matrix for a variety of regenerative medicine applications.
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From stem cells to male germ cells: Experimental approaches for the in vitro generation of mouse and human spermatogonial stem cellsMellies, Nadine 29 May 2015 (has links)
No description available.
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Genome-wide Analysis of Ctcf-RNA InteractionsKung, Johnny Tsun-Yi January 2014 (has links)
Ctcf is a "master regulator" of the genome that plays a role in a variety of gene regulatory functions as well as in genome architecture. Evidence from studying the epigenetic process of X-chromosome inactivation suggests that, in certain cases, Ctcf might carry out its functions through interacting with RNA. Using mouse embryonic stem (ES) cells and a modified protocol for UV-crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq), Ctcf is found to interact with a multitude of transcripts genome-wide, both protein-coding mRNA (or noncoding transcripts therein) as well as many long-noncoding RNA (lncRNA). Examples of the latter include both well-characterized species from imprinted loci and previously unannotated transcripts from intergenic space. RNA binding targets of Ctcf are validated by a variety of biochemical methods, and Ctcf is found to interact with RNA through its C-terminal domain, distinct from its DNA-binding zinc-finger domain. Ctcf chromatin immunoprecipitation (ChIP)-seq done in parallel reveals distinct but correlated binding of Ctcf to DNA and RNA. In addition, allelic analysis of Ctcf ChIP pattern reveals significant differences between Ctcf binding to the presumptive inactive and active X chromosomes. Together, the current work reveals a further layer of complexity to Ctcf biology by implicating a role for Ctcf-RNA interactions in its recruitment to genomic binding sites.
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Κλωνοποίηση του γονιδίου της geminin του ποντικού και δημιουργία πλασμιδιακού / Cloning of gemininΚοταντάκη, Πανωραία 29 June 2007 (has links)
Η geminin είναι ένα σχετικά καινούριο μόριο το οποίο διαθέτει καίριο ρόλο κατά την ανάπτυξη και την διαφοροποίηση, εξαιτίας του νευροποιητικού δυναμικού της, της ικανότητάς της να αλληλεπιδρά με μέλη των Hox και polycomb πρωτεϊνών, καθώς επίσης και να δρα σαν ρυθμιστής του κυτταρικού κύκλου, λειτουργώντας ως αναστολέας του παράγοντα αδειοδότησης της αντιγραφής του DNA, CDT1. Στα πλαίσια της εργασίας αυτής, κλωνοποιήσαμε το γονίδιο της geminin στο ποντίκι το οποίο αποτελείται από 7 εξώνια και καλύπτει μία περιοχή γύρω στα 10Kb. Σχεδιάσαμε και κλωνοποιήσαμε ένα πλασμιδιακό όχημα στόχευσης για το γονίδιο της mgeminin και εισήγαμε 3 θέσεις loxP στο γενωμικό locus αυτής, με σκοπό να δημιουργήσουμε υπό συνθήκη ελλειμματικούς ποντικούς για το γονίδιό της. Χρησιμοποιώντας το συγκεκριμένο φορέα στόχευσης αδρανοποιήσαμε το γονίδιο της Geminin σε pc3 (protamine Cre 3) πολυδύναμα κύτταρα ποντικού, δημιουργώντας ετερόζυγους ES κλώνους που φέρουν το «floxed» αλληλόμορφο, καθώς επίσης και το αλληλόμορφο αγρίου τύπου. Το μεταλλαγμένο αλληλόμορφο ελέγχθηκε τόσο με PCR όσο και με ανάλυση κατά Southern για την ορθότητα του ομόλογου ανασυνδυασμού. Ταυτοποιήσαμε 15 ορθά ανασυνδυασμένους ES κλώνους, οι οποίοι μπορούν να χρησιμοποιηθούν για τη δημιουργία υπό συνθήκη knockout ποντικών για το γονίδιο της geminin, μετά από έγχυση σε B6 βλαστοκύστεις και επακόλουθη μεταφορά αυτών σε θετές μητέρες. / Geminin is a novel bifunctional molecule with a pivotal role in the processes of differentiation and cell cycle regulation, due to its neuralizing potential, its ability to interact through Hox and polycomb group members, as well as in the inhibition of cell cycle progression through protein-protein interactions with the licensing factor Cdt1. We have cloned the mouse Geminin gene, which consists of 7 exons and spans approximately a region of 10Kb. We have generated a vector and introduced 3 loxP sites in the Geminin locus, in order to create conditional knockout mice. Using this knockout construct we inactivated the geminin locus in pc3 mouse embryonic stem cells, creating heterozygous ES clones, carrying a “floxed” and a “WT” allele for geminin. The mutant allele in the targeted ES cells has been verified with Southern blotting and PCR for the correct homologous recombination events. We identified 15 correctly targeted ES clones, which can be used for the generation of conditional geminin knockout mice, upon injection into B6 blastocysts and subsequent transfer to foster mothers.
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The brevity of G1 is an intrinsic determinant of naïve pluripotencyCoronado, Diana 19 December 2011 (has links) (PDF)
Pluripotency can be captured and propagated in vitro from the epiblast of the pre-implantation blastocysts in the form of embryonic stem cells (ESCs). ESCs are capable of unlimited proliferation in an undifferentiated state while maintain the potential to differentiate into cells of all three germ layers in the embryo, including the germline. Two key features the ES cell mitotic cycle are (i) a vastly elevated and uniform expression of Cyclin E and Cyclin E/CDK2 complexes throughout the cell cycle and (ii) a short G1 phase characterized by the lack of RB- and p53-dependent checkpoints, and reduced dependency on MAPK signalling. During my PhD project, we explored whether and how the regulation of the cell cycle actively sustains self-renewal of mouse ESCs (mESCs). We demonstrated that: 1/ the G1 phase of mESCs is a phase of increased susceptibility to differentiation inducers. Thus shortening of G1 might shield undifferentiated cells from differentiation inducers and help ESCs to self-renew in the pluripotent state. 2/ Cyclin E opposes differentiation and supports self-renewal of mESCs by two independent mechanisms, one of which being independent of CDK2 activation. 3/ LIF signalling regulates Cyclin E/CDK2 kinase activity therefore accelerating the G1 to S phase transition. Finally, we propose a model in which LIF signalling stimulates the G1 to S phase transition to shield mESCs from undesired differentiation signals and help them to self-renew in the pluripotent state
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The Development of Elastomeric Biodegradable Polyurethane Scaffolds for Cardiac Tissue EngineeringParrag, Ian 01 September 2010 (has links)
In this work, a new polyurethane (PU) chain extender was developed to incorporate a Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases. PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages (Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42°C) with a low soft segment glass transition temperature (Tg < -50°C). Uniaxial tensile testing of PU films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa) and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant difference in mechanical properties.
The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organized fibers and an average fiber diameter of approximately 3.6 µm. In addition, the Phe PU was electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds were investigated.
The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU experienced significant hydrolysis to observe degradation over the course of the experiment.
In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days. Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs improved the organization and differentiation of mESCDCs suggesting these two parameters are important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.
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The Development of Elastomeric Biodegradable Polyurethane Scaffolds for Cardiac Tissue EngineeringParrag, Ian 01 September 2010 (has links)
In this work, a new polyurethane (PU) chain extender was developed to incorporate a Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases. PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages (Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42°C) with a low soft segment glass transition temperature (Tg < -50°C). Uniaxial tensile testing of PU films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa) and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant difference in mechanical properties.
The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organized fibers and an average fiber diameter of approximately 3.6 µm. In addition, the Phe PU was electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds were investigated.
The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU experienced significant hydrolysis to observe degradation over the course of the experiment.
In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days. Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs improved the organization and differentiation of mESCDCs suggesting these two parameters are important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.
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Novel embryonic stem cell-infused scaffold for peripheral neuropathy repairPapreck, Justin Ryan 05 June 2008 (has links)
Peripheral nerve injury in adults often leads to permanent functional loss with or without pain. Traumatic injury or surgery, metabolic injury (diabetic neuropathy), and drug toxicity can lead to neuropathies and all negatively impact the quality of life1-8. Damage to the nervous system is often permanent since neurons in the brain and periphery are post-mitotic and have limited regenerative capacity. Nerve repair involves axon regeneration, a complex and incompletely understood process with repair potential declining with age9-15.
The research and design discussed involves the induction of endogenous repair mechanisms of the peripheral nerve using embryonic stem cells, alginate hydrogel, and the guided support of a biomaterial scaffold composed of PGS. Three different populations of cells are discussed: human embryonic stem cells, neural progenitor cells derived from human embryonic stem cells16, and primary rat bone marrow stromal cells. This study was innovative in that it was the first attempt for use of an elastomeric biomaterial scaffold in an injury model for the purpose of clinical application. This research is significant as it has direct clinical relevance in that it incorporates both functional and neuropathic recovery of patients affected by peripheral nerve damage.
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Characterization of ceramide synthases (Cers) in mammalian cellsPark, Hyejung 13 May 2009 (has links)
This thesis describes the characterization of ceramide (Cer) biosynthesis by mammalian cells. The possibility that Cer undergo developmental changes was explored using mouse embryonic stem cells versus embryoid bodies by analysis of the Cer subspecies by liquid chromatography, electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS) and of the transcript levels for enzymes involved in Cer biosynthesis by qRT-PCR. Cer of embroid bodies had higher proportions of very-long-chain fatty acids, which correlated with the relative expression of mRNA for the respective Cer synthases (CerS) and fatty acyl-CoA elongases, as well as changes in the fatty acyl-CoA's of the cells. Therefore, it is clear that Cer subspecies change during embryogenesis, possibly for functionally important reasons. One CerS isoform, CerS2, was studied further because it has the broadest tissue distribution and a remarkable fatty acyl-CoA specificity, utilizing longer acyl-chain CoAs (C20-C26) in vitro. The fatty acid chain selectivity was refined by analysis of the Cer from livers from CerS2 null mice, which displayed very little Cer with fatty acyl chains with 24 + 2 carbons. Another interesting structural variation was discovered in studies of cells treated with fumonisin B1 (FB1), which inhibits CerS. Under these conditions, cells in culture and animals accumulate substantial amounts of a novel sphingoid base that was identified as 1-deoxysphinganine. This compound arises from utilization of L-alanine instead of L-serine by serine palmitoyltransferase (SPT) based on the inability of LYB cells, which lack SPT, to make 1-deoxysphinganine. In the absence of FB1, 1-deoxysphinganine is primarily acylated to 1-deoxydihydroceramides. These are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation and disease. In summary, cells contain a wide variety of Cer subspecies that are determined by changes in expression of CerS, enzymes that produce co-substrates (such as fatty acyl-CoAs), and the types of amino acids utilized by SPT, the initial enzyme of de novo sphingolipid biosynthesis. One can envision how these changes might impact membranes structure as well as signaling by this family of highly bioactive compounds.
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