Dissertations / Theses on the topic 'Embryonic stem cells Differentiation'
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1
Joannides, Alexis. "Neural differentiation of human embryonic stem cells." Thesis, University of Cambridge, 2009. https://www.repository.cam.ac.uk/handle/1810/252121.
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Human embryonic stem cells (hESCs) are a potential source of defined cell types for studying early human development and application in regenerative medicine. Realising this potential requires a number of challenges to be overcome. The experimental findings reported represent a systematic approach in establishing controlled and standardised conditions for differentiating hESCs down the neural lineage, and characterising neural derivatives both in vitro and in vivo. Human embryonic stem cell cultures were established from two independently-derived liens, H9 and UES9. A novel, efficient method for propagating hESCs is described, avoiding the use of enzymatic products which may lead to karyotypic instability. Controlled neuroectodermal differentiation is demonstrated using a chemically defined system over a period of 16 days, and this process is shown to be dependent on endogenous fibroblast growth factor (FGF) signalling. Neural progenitors generated with this system are subsequently expanded for over 180 days and shown to retain neural stem cell (NSC) identity at the clonal level. Evidence is provided that hESC-derived NSCs follow a developmentally predictable timecourse of neurogenesis followed by gliogenesis, and their in vitro and in vivo behaviour is characterised with respect to temporal maturation and phenotypic potential.
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Gordon-Keylock, Sabrina Anne Megan. "Haematopoietic differentiation of murine embryonic stem cells." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/29123.
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This thesis aimed to determine which subregions of the E10.5 aorta-gonad-mesonephros (AGM) were responsible for the haematopoietic enhancing effects that primary AGM regions had on differentiating ES cells. To this end, a novel co-culture system has been established to test the enhancing effects of a panel of clonal stromal cell lines derived from different subregions of the midgestational AGM. Three stromal cell lines derived from the dorsal aorta and surrounding mesenchyme (AM) subregion of the AGM were able to significantly enhance the frequency of ES cell derived multipotent haematopoietic progenitors. Two stromal cell lines derived from the urogenital ridges (UG) of the AGM did not enhance haematopoietic differentiation of ES cells. The haematopoietic enhancing effects were not retained by extracellular matrices isolated from the AM stromal cell layers and the effects were dependent on direct ES cell-stromal cell contact. Co-culture of an ES cell line carrying a Brachyury-eGFP reporter gene demonstrated that the stromal lines mediated their effects post-Brachyury (mesoderm) induction in the ES cells. In addition, co-culture of sorted ES cell populations confirmed that Brachyury+, but not Brachyury-, cells gave rise to haematopoietic progenitors in AM co-culture, supporting the notion that ES cell differentiation recapitulated the in vivo pattern of lineage specification. Transplantation of co-cultured ES cells into irradiated adult NOD/SCID mouse recipients led to low levels of engraftment in the spleen and bone marrow. Adult bone marrow cells achieved repopulation more readily in the NOD/SCID animal model when transplanted intra-splenically, compared to intravenous injection. This suggests that transplantation of ES-derived haematopoietic cells directly into the haematopoietic niche, by intra-splenic or intra-femoral injection, could facilitate repopulation.
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Lake, Julie-anne. "Differentiation of pluripotential murine embryonic stem cells." Adelaide Thesis (Ph.D.) -- University of Adelaide, Department of Biochemistry, 1996. http://hdl.handle.net/2440/18794.
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Bigdeli, Narmin. "Derivation, characterization and differentiation of feeder-free human embryonic stem cells /." Göteborg : Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine at Sahlgrenska Academy, University of Gothenburg, 2010. http://hdl.handle.net/2077/22353.
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Lu, Xibin, and 盧希彬. "Quantitative characterization of mouse embryonic stem cell state transition." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208049.
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Cho, Sarah K. "Lymphohematopoietic differentiation from embryonic stem cells in vitro." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ63681.pdf.
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Hollands, Peter. "Differentiation and grafting of embryonic haemopoietic stem cells." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.330219.
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Shah, Nadia Nisa. "Human embryonic stem cells : prospects for pancreatic β-cell differentiation." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495052.
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The focus of this thesis was to explore different strategies in trying to generate putative pancreatic β-cells using one of the initial Wisconsin H7 hES cell lines. Prior to this, human pancreas development was assessed during the first trimester of pregnancy in an attempt to determine the spatial and temporal expression of development and mature pancreatic β-cell markers during this period. Spontaneous differentiation of hES cells can be induced by the formation of embryoid bodies (EBs) in suspension culture. EBs began to express markers of pancreatic β-cell development and function at a molecular, protein and functional level upon differentiation over a 3-week period. The constitutive over-expression of the terminal β-cell marker PAX4 enhances this process, whereas karyotypic abnormalities induced in hES cells over prolonged culture can hinder differentiation potential towards pancreatic β-cells. Directed differentiation strategies which mimic mouse pancreas development have led to the elucidation of an in vitro protocol to generate putative definitive endoderm from hES cells through the application of Wnt3a and Activin A in the presence of low serum. Indirect co-culture of this H7 hES cell-derived putative definitive endoderm with mouse islets did not lead to the differentiation of fully functional pancreatic β-cells. The hES cell-derived putative definitive endoderm did however influence the aging mouse islets in a positive manner by allowing the maintenance of insulin secretagogue-induced functional responses which are usually lost in culture. This may prove useful in maintaining viability of human islets during culture to be used for transportation therapies.
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Blyszczuk, Przemyslaw. "Differentiation of embryonic stem cells into pancreatic insulin-producing cells." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=97560032X.
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Kim, Peter Tae Wan. "Directed differentiation of endodermal cells from mouse embryonic stem cells." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/771.
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Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes and chronic liver disease. Various attempts have been made to produce cells that can serve as precursors for pancreas and liver. By using all-trans-retinoic acid, basic fibroblast growth factor, dibutyryl cAMP, and cyclopamine, an attempt has been made to produce definitive endoderm and subsequently cells that can serve as pancreatic and hepatocyte precursors from mouse embryonic stem cells. By using retinoic acid and basic-FGF, in the absence of embryoid body formation, mouse embryonic stem cells were differentiated at different culture periods. Four protocols of varying lengths of culture and reagents and their cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, pancreas and hepatocytes. Inclusion of DBcAMP and extension of culture time resulted in cells that display features of definitive endoderm by expression of Sox 17 and FOXA2 and minimal expression of primitive endoderm and other germ cell layers such as ectoderm and mesoderm. These cells produced insulin and C-peptide and secreted insulin in a glucose responsive manner. However, they seem to lack mature insulin secretion mechanism. There was a production of hepatocyte markers (AFP-2 and transthyretin) but there was insufficient data to assess for convincing production of hepatocytes. In summary, one of the protocols produced cells that displayed characteristics of definitive endoderm and they may serve as pancreatic endocrine precursors.
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Uroić, Daniela Sonja. "Differentiation of embryonic stem cells towards pancreatic β-like cells." Thesis, University of Aberdeen, 2011. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=167694.
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Embryonic stem (ES) cells were used as a model system to understand the signalling events in pancreas development. ES cells were differentiated through known precursor stages towards the tissue of interest in order to recapitulate development in vitro. Thus, protocols directing differentiation of mouse ES cells towards definitive endoderm and pancreatic β-cells were developed. A combination of activin A and bone morphogenic protein 4 resulted in a population of enriched cells expressing genetic markers of definitive endoderm. In vitro differentiation of ES cells into functional pancreatic β-cells has only been partially successful, as it results in cells that are not fully differentiated or functional. This might be due to a lack of cues emanating from surrounding cells present in the developing pancreas. Conditioned media from the mouse MIN6 β-cell line were used on the basis that differentiated β- cells might send out signals affecting the differentiation of the surrounding islet cells. Mouse ES cells were enriched in definitive endoderm and then treated with MIN6 conditioned medium. Gene expression of the β-cell markers Insulin1, Insulin2, and Glucose transporter 2 was significantly increased relative to the untreated control group after 10 days of treatment with conditioned medium. This result was specific for conditioned medium from MIN6 cells as conditioned medium from a kidney-, a neuronal-, and an exocrine pancreatic cell line had no effect. In order to characterise the secreted factor(s) the conditioned medium was subjected to protein precipitation. The pancreatic differentiation factor was present in a protein fraction, suggesting that the factor(s) was proteinaceous. The protein in question was neither proinsulin nor insulin. This knowledge will support the efficient generation of insulin-secreting cells for diabetes therapy.
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Neilson, Kirstie Jane. "Differentiation of mouse embryonic stem cells into endothelial progenitor cells." Thesis, University of Sheffield, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.500200.
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Nsiah, Barbara Akua. "Fluid shear stress modulation of embryonic stem cell differentiation." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/47552.
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Vascularization of tissue-engineered substitutes is imperative for successful
implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.
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Kleinert, Fanni. "Nuclear architecture in differentiating embryonic stem cells." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/nuclear-architecture-in-differentiating-embryonic-stem-cells(fb45d7d6-69d7-4b63-8785-503eca352131).html.
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Gene expression is regulated at various levels, such as transcription, RNA transport and translation. Additionally, it has been shown that chromatin structure, location and dynamics also have an important role in gene expression control. While active gene regions are strongly associated with an open chromatin structure at the surface of the chromosome territory (CT) and a location in the nuclear interior, inactive gene regions seem to be related with a closed structure within the CT and a position at the nuclear periphery. However, it is still unclear how these features are regulated. Importantly, malfunction of gene regulation can impact on health and longevity. Therefore, the aim of this project was to investigate the correlation of gene expression and chromatin organisation both in single gene loci and the MHC gene cluster. The MHC locus has the highest gene density in mammalian cells and contains genes that can be reprogrammed by pro-inflammatory cytokines. The original goal of this project was to label the MHC locus by the Lac operator/repressor (LacO/LacI) approach in order to study chromatin dynamics in living cells using labelled CTs as reference for genome mobility. The thymidine analogue EdU, that can be used to label CTs, was analysed for its effects on cell cycle progression and survival, and revealed to have a strong negative impact on the cells' well-being. In the end, the LacO/LacI-recognition system for live-cell imaging did not succeed, thus FISH analyses were carried out to study chromatin dynamics in snap-shots. The location and structure of the hybridised gene regions were analysed in response to gene activation and inactivation during ESC differentiation to neuroepithelial progenitors (NPs). Single-gene focused experiments were performed using the cell line specific genes, Oct4 and Sox1, together with Gapdh as a housekeeping gene. Even though, the results showed less changes between the days of differentiation on the Gapdh locus, the gene expression profiles for the cell line specific genes did not match with the hypothesised chromatin organisation (see above). However, investigations on the gene-dense MHC locus showed structural chromatin changes that correlated with the activation of genes in this region. Interestingly, ESC treated with TNFalpha were unable to activate NF-kappaB signalling, probably due to the lack of a functional IKK complex. In summary, this project was focussing on the regulation of gene expression by the chromatin architecture and revealed complex chromatin dynamics that are likely to be affected by the sum of genes in a genome region, rather than a single gene.
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Sargent, Carolyn Yeago. "Effects of hydrodynamic culture on embryonic stem cell differentiation: cardiogenic modulation." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/34710.
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Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.
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Wei, Wenjie, and 魏闻捷. "Calcium signaling in the cardiac differentiation of mouse embryonic stem cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B49617862.
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Intracellular Ca2+ mobilization via secondary messengers modulates multiple cell functions. Cyclic Adenosine 5’-Diphosphate-Ribose (cADPR) is one of the most well recognized endogenous Ca2+ mobilizing messengers. In mammalian, cADPR is mainly formed by CD38, a multi-functional enzyme, from nicotinamide adenine dinucleotide (NAD). It has previously been shown that the cADPR/CD38/Ca2+pathway mediates many cardiac functions, such as regulating the excitation-contraction coupling in cardiac myocytes and modulating the Ca2+ homeostasis during the ischemia injury of the heart. Thus it is reasonable to propose that the cADPR/CD38/Ca2+ pathway plays a role in cardiogenesis. The pluripotent mouse embryonic stem (mES) cells which can be induced to differentiate into all cell types provide an ideal model for studying cardiogenesis. The first part of this dissertation is to determine the role of CD38/cADPR/Ca2+pathwayin the cardiomyogenesis of mES cells. The data showed that CD38 expression was markedly up-regulated during the in vitro embryoid body (EB) differentiation of mouse ES cells, which indicated a regulatory role of CD38 in the differentiation process. Lentivirus mediated shRNA provides a convenient method to knockdown the expression of CD38 in mES cells. Surprisingly, beating clusters appeared earlier and more in CD38 knockdown EBs than that in control EBs. Likewise, the expressions of several cardiac markers were up regulated in CD38 knockdown EBs. In addition, more cardiomyocytes (CMs) existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs than those in control EBs. On the other hand, over-expression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, we showed that CMs derived from the CD38 knock down mES cells possessed the functional properties characteristic of CMs derived fromnormal ES cells. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2cascade during CM differentiation of mES cells, and transiently inhibition of Erk1/2 blocked the enhancive effects of CD38 knockdown on the differentiation of CM from mES cells. Taken together, our data indicate that the CD38/cADPR/Ca2+ signaling pathway suppresses the cardiac differentiation of mES cells.
One of the main goals of the researches on cardiac differentiation of ES cells is to enhance the production of CMs from ES cells, thereby providing sufficient amount of functional intact CMs for the treatment of severe heart disease. Nitric oxide (NO) has been found to be a powerful cardiogenesis inducer of mES cells, in that it can significantly increase the yield of ES-derived CM. The second objective of this dissertation is to explore the mechanism underlying the NO facilitated cardiomyogenesis of mES cells. We found that the NO did induce intracellular Ca2+ increases in mES cells, and this Ca2+ increase was due to internal Ca2+ release from ER through theIP3 pathway. Therefore, the expression of IP3 receptors (IP3Rs) in mES cells were knocked down by lentivirus-mediated shRNAs. Interestingly, only type 3 IP3R (IP3R3) knockdown significantly inhibited the NO induced Ca2+ release in mES cells. Moreover, NO facilitated cardiogensis of mES cells was abolished in IP3R3 knockdown EBs. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO facilitated cardiomyogenesis of mES cells.published_or_final_version
Physiology
Doctoral
Doctor of Philosophy
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Pucci, Fabio. "Short telomeres in embryonic stem cells affect stable differentiation." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/11775.
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Murine embryonic stem cells (ESCs) are self-renewing, pluripotent cells able to differentiate into cells of all three germ layers. Pluripotency and self-renewal are maintained primarily by the core transcriptional factors Nanog, Oct4 and Sox2, but require the cooperation of other factors and coregulators and an efficient telomere maintenance mechanism. In mammals, telomere maintenance is achieved via a telomerase reverse transcriptase (Tert) that acts together with an RNA component (Terc). Maintenance of functional telomeres is essential to allow ESC proliferation, nevertheless if and how it is involved in the achievement and preservation of cell differentiation is still unknown. Here, we used Tert deficient mouse ESCs to elucidate the role of telomere length in differentiation. We found that Tert-/- ESCs with critically short telomeres are delayed, but still capable, to achieve differentiation after leukemia inhibitory factor (LIF) withdrawal and all-trans retinoic acid (ATRA) treatment, but failed to maintain it after LIF re-introduction to the growth medium. Telomere shortening effect on differentiation was accompanied by pluripotency gene dysregulation (e.g. Nanog overexpression), DNA hypomethylation and epigenetic disorders. This phenotype of metastable differentiation could be rescued by telomere lengthening via re-introduction of Tert, depletion of Nanog via short hairpin RNA, or via enforced expression of the de novo DNA methyltransferase 3b. These results reveal an unanticipated role of telomeres in the epigenetic regulation of gene expression and cell fate determination during physiological or pathological processes.
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Watanabe, Kiichi. "Directed differentiation of telencephalic precursors from embryonic stem cells." Kyoto University, 2005. http://hdl.handle.net/2433/144763.
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Kyoto University (京都大学)0048
新制・課程博士
博士(医学)
甲第11439号
医博第2862号
新制||医||895(附属図書館)
23082
UT51-2005-D189
京都大学大学院医学研究科脳統御医科学系専攻
(主査)教授 影山 龍一郎, 教授 中辻 憲夫, 教授 山中 伸弥
学位規則第4条第1項該当
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Cope, Emma Louise. "Directed differentiation of human embryonic stem cells to microglial-like cells." Thesis, Cardiff University, 2014. http://orca.cf.ac.uk/72156/.
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Protein aggregations of β-Amyloid (Aβ) and Tau are alone not sufficient to account for all the symptoms and progression of Alzheimer’s Disease (AD), as such there is much emphasis upon the immune component of the disease. The cells of the brain that are capable of initiating an immune response are microglia; these are derivatives of hematopoietic cells and they have the capacity to phagocytose and clear Aβ aggregates or release cytokines such as TNF-a, in a neurotoxic role, promoting apoptosis of the surrounding neurons and hence aiding disease progression. To uncover the precise role of microglia in terms of AD we propose a protocol to enable differentiation of microglia from human embryonic stem cells (hESCs). The protocol we propose is a two-step differentiation procedure i) hESCs to monocytes followed by ii) ES-derived monocytes to microglia. Chapter 3: Exogenous over-expression of PU.1, a transcription factor vital in both the onset of haematopoiesis and the terminal differentiation of monocytes and microglia, was revealed to enable differentiation to a hematopoietic fate. Chapter 4: hESCS were differentiated to monocyte-like cells (CD45+/CD11b+) through culture in the presence of the hematopoietic growth factors; M-CSF and IL-3. Chapter 5: focuses on the differentiation of ES-derived monocytes to microglia. It shows that monocytes cultured in astrocyte conditioned medium give rise to cells that are IBA-1+/Glut5+/CD45low/NG2low/CD80+/CD11c+ and have a ramified microglial phenotype, which upon stimulation with Aβ(1-42) can become activated to the amoeboid phenotype. The development of the protocol for the generation of microglia holds great importance in terms of creating in vitro models for AD research as a whole and can be extended to the differentiation of patient IPSCs that contain mutations in genes associated with innate immunity or SNPs associated with AD risk, disease onset and progression.
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Faial, Caldas Macedo Amaral Tiago. "The role of BRACHYURY in human embryonic stem cell differentiation." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610541.
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Sullivan, Denise D. "Incorporation of bio-inspired microparticles within embryonnic stem cell aggregates for directed differentiation." Thesis, Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54909.
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Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.
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Alaqel, Abdullah. "The directed differentiation of human embryonic stem cells to lung cell lineages." Thesis, University of Bath, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760955.
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Human embryonic stem cells (hESCs) show significant therapeutic potential in treating degenerative disorders. This is in part because of their ability to produce a limitless supply of starting cells and their potential to differentiate into more than 200 different cell types. The aim of the current research was to generate a robust stage wise protocol for the differentiation of hESCs to respiratory epithelial cells. The epithelial cells could then be used either for transplantation studies or, as an in vitro model for drug toxicity testing. In order to achieve this goal, we must identify the key steps in lung development and apply these to the differentiation protocol. In this study, we maintained Shef3 hESCs in their undifferentiated pluripotent state to expand the cells prior to the differentiated towards the definitive endoderm (DE) lineage. I used a two-stage protocol based on culture with a novel glycogen synthase kinase-3 (GSK-3) inhibitor (termed 1m), along with Activin-A. We confirmed the status of the cells by a combination of immunostaining and PCR. We showed loss of the pluripotency markers (Sox2 and Oct3/4) and gain of DE markers (Sox17, FoxA2 and CXCR4). After the induction of DE from hESCs, we then treated the cells with transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) pathway inhibitors (SB431542 and Noggin respectively). This combinatorial treatment resulted in the differentiation into the anterior foregut endoderm (AFE) lineage based on expression of Pax9 and FoxA2 plus the up-regulation of Sox2. Further differentiation of AFE derivatives into more mature epithelial cells, termed lung progenitor cells (LPCs), was achieved following the treatment of AFE cells with a cocktail of trophic factors (BMP4, EGF, bFGF, FGF10, KGF and Wnt3a) yielded a population of NKX2.1-positive and FoxA2-positive cells that potentially corresponded to the lung lineage. Finally, prolonged treatment with FGF10 and FGF2 on LPC derived hESCs induced proximal (CC10, MUC5AC) and distal (SPB, SPC) airway epithelial cells. In addition, we also utilised the ectopic expression of an adenovirus expressing NKX2.1 to promote lung maturation. In conclusion, we have generated a protocol for the differentiation of hESCs into mature lung-like cells. The generation of these cells in vitro could potentially lead to a better in vitro model for toxicity testing and the development of novel therapies for promoting regeneration of lungs in patients with severe lung disorders.
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VanOudenhove, Jennifer J. "Mechanisms Regulating Early Mesendodermal Differentiation of Human Embryonic Stem Cells: A Dissertation." eScholarship@UMMS, 2016. http://escholarship.umassmed.edu/gsbs_diss/849.
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Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events.
To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation.
A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment.
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Kono, Tomoya. "Differentiation of lymphatic endothelial cells from embryonic stem cells on OP9 stromal cells." Kyoto University, 2008. http://hdl.handle.net/2433/135863.
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Cho, Ting-yin. "Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607991.
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Lee, Hyojin. "Directed differentiation and functional characterization of embryonic stem cell-derived motoneurons /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1296098331&sid=4&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Joshi, Ramila Joshi. "Micro-engineering of embryonic stem cells niche to regulate neural cell differentiation." University of Akron / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=akron1544029342969082.
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Sesé, Ballesteros Borja 1983. "The Role of SMYD2 during human embryonic stem cells differentiation." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/128681.
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Embryonic stem (ES) cells are able to differentiate into any cell type, a property called pluripotency, and have unlimited potential for self-renewal. Although the molecular mechanisms responsible for maintaining self-renewal and pluripotency in ES cells are not well known, recent studies have demonstrated the importance of epigenetic mechanisms in maintaining these processes. Histone modifying enzymes play decisive roles in differentiation and development. This study describes that SMYD2 (SET and MYND domain containing protein 2), a histone lysine methyltransferase, is induced during human ES cells differentiation and it is preferentially expressed in somatic cells versus pluripotent cells. Gain and loss-of-function experiments have shown that knockdown of SMYD2 in human ES cells promotes the induction of endodermal markers during differentiation, while overexpression has opposite effects. In vivo experiments in zebrafish revealed that knockdown of smyd2a (a homologue gene of human SMYD2) causes developmental delays and aberrant tail formation. The phenotype of smyd2a-morphant embryos correlates with a low expression of ntl and over induction Nodal-related genes during gastrulation. Finally, SMYD2 is shown to stimulate the activation of BMP signaling pathway and promotes the induction of BMP2-target genes in human ES cells. Overall, these findings suggest that SMYD2 plays a critical role at early stages during development and in human ES cells differentiation.Les cèl·lules mare embrionàries (ES) són capaces de diferenciar-se a qualsevol tipus cel·lular, una propietat coneguda amb el nom de pluripotència, i presenten un potencial il·limitat d’auto-renovació. Tot i que els mecanismes moleculars responsables per al manteniment de la pluripotència i l’auto-renovació en ES encara no es coneixen bé, estudis recents han demostrat la importància dels mecanismes epigenètics en mantenir aquests processos. Els enzims modificadors d´histones juguen un paper decisiu durant la diferenciació i el desenvolupament. Aquest estudi descriu que SMYD2 (SET and MYND domain containing protein 2), una metiltransferasa d’histones, s’indueix durant la diferenciació de cèl·lules ES i s’expressa preferentment en cèl·lules somàtiques envers cèl·lules pluripotents. Experiments de guany i pèrdua de funció mostren que el noqueig de SMYD2 en cèl•lules ES humanes promou la inducció de marcadors d’endoderm durant la diferenciació, mentre que la sobre-expressió té efectes oposats. Experiments in vivo en el peix zebra van revelar que el noqueig de smyd2a (un gen homòleg de SMYD2 humà) causa retard en el desenvolupament i formació aberrant de la cua. El fenotip dels embrions absents de smyd2a es correlaciona amb una baixa expressió de ntl i una sobre-inducció dels gens relatius a Nodal durant la gastrulació. Finalment, SMYD2 estimula l’activació de la via de senyalització BMP i promou la inducció dels gens diana de BMP2 en cèl·lules ES humanes. En general, aquests descobriments suggereixen que SMYD2 juga un paper important durant els estadis primerencs en el desenvolupament embrionari i durant la diferenciació de cèl·lules ES humanes.
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Anyfantis, Georgios. "Studies relating to the differentiation of human embryonic stem cells." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/studies-relating-to-the-differentiation-of-human-embryonic-stem-cells(1f8499c7-0f80-4f14-8fe6-e095b7386f7e).html.
Full textAbstract:
Human embryonic stem cells (hESCs) have been a useful tool in the study of the embryo development and could be used by drug developing companies to create disease models and assist in the production of new medicines. One of the models that has been studied before, is the development of the pancreas. Scientists have obtained mixed results so far in the generation of functional pancreatic cells from hESCs. We studied the differentiation potential of hESCs. As purinergic signalling is involved in may physiological processes, including cell proliferation and differentiation, a study of purinergic signalling in hESCs would help us deeper understand the hESC physiology. In order to study the purinergic profile of hESCs we established a culture system that allowed the transfer and attachment of pluripotent hESC colonies on glass coverslips. We then studied the functional purinergic profile of hESCs and found that they do not express functional P2X1 receptors, but they do express functional P2Y6 receptors, which might be implicated in the hESC differentiation. In parallel to these studies, we developed a reporter gene lentivirus, where the mouse Pdx-1 promoter area controlled the expression of a reporter fluorochrome, eGFP. We managed to generate a functional lentivirus, however, further analysis is needed in order to be able to use it in developmental studies. Finally, we tested the hypothesis that glucose affects the differentiation of hESCs towards pancreatic endoderm. Our preliminary results suggested that glucose does affect the differentiation potential of hESCs.
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Jones, Robert. "Proteomic analysis of neural differentiation in mouse embryonic stem cells." Thesis, Bangor University, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412699.
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Murad, Nadia Yousif. "Differentiation of human embryonic stem cells to the pancreatic lineage." Thesis, University of Sheffield, 2008. http://etheses.whiterose.ac.uk/6102/.
Full textAbstract:
Human embryonic stem (hES) cells have great therapeutic potential for the treatment of degenerative conditions such as Parkinson's disease, cardiac failure and type I diabetes. This potential is based on the ability of hES cells in vitro to self-renew and also differentiate to cells of all three germ layers; ectoderm, mesoderm and endoderm. Type I diabetes is due to an autoimmune disease destroying the insulin-secreting cells of the pancreas (β-cells) that regulate plasma glucose concentration. The pancreas develops from the endoderm lineage. 2. To find a cure for type I diabetes based on the use of hES, it is essential to understand the differentiation process of ES cells into the endodermal, β-cell lineage. The aim of this study was to investigate the generation of insulin-secreting cells using hES cells in vitro and to compare sue with those in the developing pancreas of the foetus.
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Burridge, Paul Wesley. "Improving the mesodermal differentiation potential of human embryonic stem cells." Thesis, University of Nottingham, 2008. http://eprints.nottingham.ac.uk/13169/.
Full textAbstract:
Human embryonic stem cells (hESCs) are thought to have enormous potential for use in regenerative medicine, whilst simultaneously allowing us insights into human embryonic development, disease modelling and drug discovery. Differentiation to mesodermal lineages, such as cardiomyocytes and blood, may allow for improved treatment of cardiac and haematopoietic diseases. hESC-derived immune cell types may also allow the circumnavigation of the immune barrier. This thesis aims to test the hypothesis that formation of hESC derivatives is regulated by the same mechanisms and ontology as in vivo embryo development. Therefore, by identifying and facilitating the mechanisms of mesoderm induction, hESC differentiation can be optimised to maximise the production of mesoderm, and, ultimately, mesoderm derivatives. Using a Xenopus laevis animal cap model with simultaneous treatment with activin B or fgf4, together with tall, Im02 and gatal mRNA, resulted in substantial increases in mesodermal, haemangioblast and erythropoietic cell markers. One of the most successful methods for hESC differentiation is by the formation of human embryoid bodies (hEBs). To reduce first the number of variables in current mass culture protocols for hEB formation, such as hEB size, a forced aggregation system was established that produced homogeneous hEBs from defined numbers of cells. This system was then optimised to enhance production beating cardiomyocytes by varying the number of hESCs used for hEB formation and also the number of days in culture. This system was assessed in four hESC lines and demonstrated substantial inter-line variability in cardiomyocyte production (1.6± 1.0% to 9.5±0.9°0). Differentiation was also performed using chemically defined media (CDM) with the addition of actiyin A and FGF2 and resulted in 23.6±3.6% of hESs producing beating cardiomyocytcs. In addition immunohistochemistry was performed to assess the relationship of cells expressing markers for mesoderm, pluripotency, ectoderm, and endoderm to establish a standard spatial and temporal map of hEB differentiation.
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Chatzi, Christina. "Derivation, maintenance and neuronal differentiation of mouse embryonic stem cells." Thesis, University of Aberdeen, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.446228.
Full textAbstract:
This thesis describes successful derivation of 10 novel ES cell lines from C57BL/6J blastocysts. They grow in colonies, express four stem cell markers, display normal karyotype, are able to differentiate in vitro and form chimeras in vivo. Confirmation of germline transmission will enable them to be used for the production of novel mutants on pure C57BL/6J genetic background without backcrossing. The maintenance of stemness of ES cells depends on delicate signalling networks. Spontaneous differentiation is common in ES cell propagation, while its causing factors are largely unknown. The data in this thesis show that ßDC, a dominant negative form of the retinoic acid receptor beta 2, regulates RA-mediated ES cell growth and differentiation. ES cells expressing ßDC are resistant to 100nM RA-induced differentiation in monolayer, while upon 1mM RA induction during aggregation, they differentiate into mesodermal derivatives instead of ectodermal cells. Remarkably, their capacity to participate in normal embryogenesis is not altered by ßDC expression and RA selection. These findings raise the possibility that such a mutant may facilitate long-term maintenance of ES cells. Defective GABAergic signalling is implicated in neurodevelopmental disorders, and brain/spinal cord injuries. As a part of this thesis, a simple differentiation protocol has been developed, which leads to the production of a homogeneous population (93~96%) of GABAergic progenitors from mouse ES cells. Translation of the above technologies to human ES cells may advance the stem cell replacement therapy.
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Whyte, Warren A. (Warren Anthony). "Roles for chromatin regulators during differentiation of embryonic stem cells." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/72821.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Cataloged from PDF version of thesis.
Includes bibliographical references.
Mammalian development involves the process by which a single fertilized egg develops into an adult with over 200 specialized cell types, each with a distinct gene expression pattern controlling its cellular state. As cells differentiate into specialized cell types, changes in the gene expression program occur with associated changes in chromatin. An understanding of the roles for chromatin regulators in the control of gene expression programs during differentiation is fundamental to understanding development. Although it is not yet feasible to elucidate the functions of all chromatin regulators in all vertebrate cells, recent work in embryonic stem (ES) cells has demonstrated that regulatory features of differentiation can be elucidated by focusing on the chromatin regulators involved in the changes in the pluripotent gene expression program as ES cells differentiate. New insights reveal that chromatin regulators of opposing functions share a common set of active genes in ES cells, suggesting a dynamic balance in the control of embryonic stem cell state and differentiation. I describe here the molecular mechanisms by which chromatin regulators contribute to the control of the ES cell state and differentiation, where these regulators play critical roles both in activating new gene expression programs and in silencing old programs.
by Warren A. Whyte.
Ph.D.
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Fujikura, Junji. "Differentiation of embryonic stem cells is induced by GATA factors." Kyoto University, 2003. http://hdl.handle.net/2433/148471.
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Jin, Xin. "Towards differentiation of mouse embryonic stem cells to thymic epithelial progenitor cells." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/12227.
Full textAbstract:
The thymus is the major site for T-cell generation and thus is important for the adaptive immune system. Development of a properly selected, functional T-cell repertoire relies on interactions between developing T cells and a series of functionally distinct thymic stroma cell types including the cortical and medullary thymic epithelial cells (TECs). The thymus is one of the first organs to degenerate in normal healthy ageing. Related to this, there is strong interest in developing protocols for improving thymus function in patients by cell replacement or regenerative therapies. Thymic epithelial progenitor cells (TEPCs) represent a potential source of cells for thymus transplantation. However, the only source of these cells for transplantation is currently fetal thymus tissue. If TEPCs could be generated from pluripotent cells, this could provide an alternative source of cells for transplantation. The work described in this thesis therefore had two central aims (i) to test the stability of thymic epithelial progenitor cells in vivo and (ii) to investigate the possibility of generating TEPCs or TECs from mouse embryonic stem (ES) cells. The forkhead transcription factor, Foxn1, is essential for the development of a functionally mature thymic epithelium, but is not necessary for formation of the thymic primordium or for medullary thymic epithelial sub-lineage specification. By reactivating Foxn1 expression postnatally in mice carrying a revertible hypomorphic allele of Foxn1, Foxn1R, I herein demonstrate that TEPCs that can express only low levels of Foxn1 mRNA can persist postnatally in the thymic rudiment in mice until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression. In parallel with this work, I confirmed the possibility of generating Foxn1-expressing cells from mouse ES cells by using a Foxn1 reporter cell line. As the thymic epithelium has a single origin in the third pharyngeal pouch (3pp) endoderm, I then tested whether or not TEPCs and /or TECs were generated during ES cell differentiation via existing protocols for generating anterior definitive endoderm differentiation cells from mouse ES cells. From this work, I showed that genes expressed in the 3pp and/or TEPC,-including Plet-1, Tbx1, Hoxa3 and Pax9, were induced by differentiation of ES cells using these protocols. I further showed that cells expressing both Plet-1, a marker of foregut endoderm and 3pp, and EpCAM, a marker of proliferating epithelial cells, were induced using a novel protocol (2i ADE) for generating ES cells from ADE. However, gene expression analysis and functional testing suggested that the majority of these cells were non-thymus lineage. I subsequently developed a novel protocol which combined this 2i ADE protocol with co-culturing of the differentiating ES cells with fetal thymic lobes, and demonstrated that this further induced 3pp and TEPC related genes. Finally, I modified the culture conditions in this protocol to conditions predicted to better support TEPC/TEC, and showed that in these conditions, the TEPC-specific markers Foxn1 and IL-7 were induced more strongly than in any other conditions tested. The data presented in this thesis therefore represent an advance towards an optimized protocol for successfully generating TEPCs from ES cells in vitro.
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Doszyn, Olga. "Sex differences in neuronal differentiation of human stem cells." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-384661.
Full textAbstract:
Sexual dimorphism has been long noted in human neurobiology, apparent most notably in sex-biased distribution of multiple neurological disorders or diseases, from autism spectrum disorder to Parkinson's disease. With the advances in molecular biology, genetics and epigenetics have come into focus as key players in sexually dimorphic neural development; and yet, many studies in the field of neuroscience overlook the importance of sex for the human brain. For this project, human embryonic and neural stem cells were chosen for three main reasons. Firstly, they provide an easily obtainable, scalable and physiologically native model for the early stages of development. Secondly, neural stem cells populations are retained within the adult human brain, and are implicated to play a role in cognition and mental illness, and as such are of interest in themselves. Thirdly, stem cell lines are widely used in research, including clinical trials of transplantation treatments, and for this reason should be meticulously examined and characterized. Here, the morphology, behaviour, and expression of selected genes in four stem cell lines, two of female and two of male origin, was examined in side-by-side comparisons prior to and during neuronal differentiation using a variety of methods including light microscopy, time-lapse two-photon microscopy, quantitative real-time PCR and immunocytochemistry. The obtained results have shown previously uncharacterised differences between those cell lines, such as a higher rate of proliferation but a slower rate of neuronal differentiation in male cell cultures compared to female cells cultivated in the same conditions, and a sex-biased expression of several markers of neuronal maturation at late stages of differentiation, as well as diverse patterns of expression of X- and Y-linked genes involved in stem cell proliferation and neural development.
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Taylor, Gillian Catherine Agnes. "H4K16 acetylation during embryonic stem cell differentiation." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8069.
Full textAbstract:
Eukaryote DNA is organised into the more compact nucleosome by wrapping 147bp of DNA around a histone octamer core. The N-terminal tails of the histones protrude through the DNA and can be modified by a variety of enzymes. Acetylation of Histone 4 Lysine 16 (H4K16ac) is an important modification associated with an increase in transcription, and in flies is an important component of the doseage compensation system. It is also unique amongst histone modifications in that it has been directly associated with chromatin decompaction. H4K16ac has been linked to development through its Histone Acetyltransferase, MOF. Deletion of MOF in mice leads to mass chromatin defects, and embryonic lethality prior to the blastocyst stage. I set out to understand the role of H4K16ac in differentiating Embryonic Stem cells (ES cells) and chromatin compaction in vivo. I generated a ChIP-seq profile for H4K16ac in undifferentiated ES cells, and after 3 days of retinoic acid (RA) differentiation. This revealed an association of H4K16ac with the promoters of transcribed genes in pluripotent ES cells, followed by loss H4K16ac on ES cell specific genes and gain of the modification on differentiation specific genes. There were some silent genes in ES cells, however, which were acetylated on their promoters. Through this study I also found that H4K16ac and MOF mark active enhancers in ES cells, along with H3K4me1 and H3K27Ac and p300. H4K16ac did not mark a known regulatory region in limb cells, and it is possible that it marks active enhancers only of ES cells. Furthermore, I looked at the compaction state large regions (>100kb) which lost H4K16ac upon differentiation by FISH, to determine if loss of H4K16ac could predict compaction. The regions selected showed no change in compaction state between UD and D3 cells, meaning that loss of H4K16ac does not directly lead to chromatin compaction in vivo. However loss of H4K16ac may be necessary for any subsequent compaction, or the change in compaction may take place at nucleosomal level. Finally, I attempted both to overexpress and reduce the level of MOF in ES cells. I was unable to manipulate the level of MOF in this cell type in either direction; expression of endogenous MOF was silenced after very little time, and stable MOF shRNA cell lines showed no reduction in levels of MOF. Therefore, potentially, dosage of MOF/H4K16ac in this cell type is critical. This study may help to understand the significance of H4K16ac in ES cell differentiation and chromatin compaction.
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Anderson, Kathryn Gayle Victoria. "Conserved mode of endoderm induction acts to promote context dependent embryonic and extra-embryonic lineage specification." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/16473.
Full textAbstract:
In mammalian development, endoderm formation occurs in two phases and the fate of these populations is different. In the blastocyst, inner cell mass (ICM) cells generate the primitive endoderm (PrE), which will give rise to the extra-embryonic parietal (PE) and visceral endoderm (VE). Hematopoietically expressed homeobox (Hhex) protein is initially expressed throughout the PrE and subsequently becomes restricted to the anterior visceral endoderm (AVE), one of two important early embryonic signalling centres in the mouse. During gastrulation a second wave of endoderm differentiation occurs, the definitive endoderm (DE), generating the foregut. Immediately following the induction of DE, regional identity is initially established in the anterior region with the expression of Hhex. One of the earliest specification events in this lineage is the specification of anterior fate by Hhex, this time in a second signalling centre, the anterior definitive endoderm (ADE). The ADE is both important for embryonic patterning, and as the precursor population for differentiating to the foregut and its derivatives the thyroid, liver and pancreas. The literature surrounding these early embryonic patterning events is covered in depth in chapter 1. Embryonic stem cells (ESCs) are normal cell lines derived from the mammalian blastocyst at the time that it is making PrE. A number of laboratories have generated protocols to make endoderm from ESCs and in my thesis I define approaches to distinguish between PrE and DE. I generated a new ESC reporter line utilising a gene normally expressed in both the PrE and later in hepatic endoderm; this reporter contains a GFP in the first exon of the Hnf4α locus. This was combined with a second fluorescent reporter containing DSRed in the Hhex locus. This cell line is described and characterised in chapter 3. As Hnf4α is initially expressed in PrE prior to Hhex, but in the DE following Hhex, I was able to use the temporal expression of this reporter to distinguish the induction of PrE from DE. As Activin and Wnt are known to induce endoderm from ESCs, I was then able to ask what sort of endoderm the combination of these two signals induced. In chapter 4 I found that normal ESCs would readily differentiate to iPrE in the presence of Activin and Wnt3a. While this has not been described previously, my analysis suggests that ESC protocols applying these cytokines directly to ESCs have produced PrE. Given that ESCs are derived from the blastocyst, the generation of iPrE from Wnt3a/Activin treatment fits with developmental paradigms. However, Act/Wnt3a is used routinely on Human ESCs (hESCs) and so I attempted to reconcile these observations. HESCs, while derived from the blastocyst, appear to progress developmentally in vitro, to a stage closer to the epiblast, immediately prior to gastrulation. I therefore assessed the effect of Activin and Wnt3a on mouse stem cell lines derived from the epiblast (Epiblast Stem Cells, EpiSCs), that are grown under similar conditions to hESCs. When Wnt3a/Act is applied to these cells I found that they made DE rather than PrE, which I describe in chapter 4. Taken together my observations suggest that Act/Wnt3a are general endoderm inducers that induce context specific differentiation in vitro. The cell type derived in response to this treatment depends on the developmental stage of the starting stem cell culture. During the course of this work, I also observed that PrE was growing under Activin/Wnt3a treatment. As a number of cell culture systems have been established that reflect PE, but not truly bipotent PrE, I investigated the conditions under which PrE can be expanded. In chapter 5 I characterize a new PrE culture system, in which bipotent extra-embryonic endoderm can be expanded indefinitely in culture. I also explore a bit more precisely the nature of the starting cells that initially become exposed to Activin/Wnt3a treatment. Previous work has extensively characterized the existence of a primed population of PrE in ESC culture and in chapter 6 I explore the existence of a primed DE population in EpiSC culture. Taken together, my thesis is the first demonstration that Activin/Wnt3a can induce different endoderm populations in different embryonic stem cell populations. It underlies the notion that the evolutionary origin of both cell types is the same and that the pathways evolved for extra-embryonic development in mammals just exploit the ancient modes of germ layer specification that evolved with gastrulation.
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Chong, Tsz-yat Ian, and 莊子逸. "Inducing the progressive differentiation of hESCs into pancreatic progenitor cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/196433.
Full textAbstract:
Diabetes is a chronic disorder of the pancreas, where a decline in the insulin-producing β-cell population disrupts metabolic homeostasis. Pancreatic transplantation has shown to be effective in circumventing the problem of β-cell insufficiency. However, availability of donor islets remains an obstacle. Although progressive differentiation of embryonic stem cells (ESCs) to pancreatic β-cells is a solution, current protocols are wrought with inefficiencies. It is obvious that to realize ESC differentiation for therapy many steps need to be optimized, and this study describes improvement of Pdx1+pancreatic progenitor derivation, a critical determinant of pancreatic fate.
The compounds melatonin and sPDZD2 have been suggested to act through the
Protein Kinase A (PKA) pathway to exert transcriptional effects, and in particular sPDZD2 stimulates the expression of pancreatic genes in INS-1E rat pancreatic cells. This led to the hypothesis that the PKA-targeting characteristics of said molecules could be exploited for pancreatic specification through post-translational activation ofPdx1. hESCs were first induced to form definitive endoderm before treatment with melatonin and sPDZD2. Pdx1 expression induced by these molecules was then compared with levels triggered by known pancreatic progenitor inducer Indolactam V (ILV). A secondary objective of this study was to assess the endoderm induction potential of small molecules in hESCs, which claim to be potentially useful in differentiation.
In this research, I show that small molecules are noticeably more challenging to use in the hESC context. Between the TGF-β pathwayactivatorsIDE-1 and 2, the latter is more potent at inducing endoderm formation, though it does not surpass the capabilities of Stauprimide, a molecule originally thought to only serve a priming purpose in mESCs.IDE-2 and Stauprimide consistently perform better than Activin A, the near universal factor for endoderm induction. Possible synergy between IDE-2 and Stauprimide was explored, but their combination appears detrimental to Sox17expression. Subsequent pancreatic differentiation was also inefficient, and my results affirm the immaturity of chemically-induced endoderm by contrasting with mainstream means of endoderm induction; levels of endoderm marker expression between the two methods are millions of folds apart. This work exposes the risks of using small molecules, and they necessitate proper characterization before being adopted for differentiation.
Most favorably, both sPDZD2 and melatonin were able to trigger Pdx1 expression in STEMDiffTm derived definitive endoderm; 10 and 30folds respectively, comparable to the known Pdx1 inducer ILV (25 folds). I also reveal concentration-mediated differentiation and proliferative purposes of ILV and sPDZD2, which are highly reminiscent of the signaling mechanisms involved during pancreatic development. Preliminary quantification of Pdx1+ cells suggest that high concentrations of ILV and sPDZD2 favor self-renewal of Pdx1+ progenitors, whilst lower doses elevate Pdx1 expression. Demonstration of Pdx1 at both gene and protein expression levels was encouraging, but it remains uncertain if melatonin and sPDZD2 manipulate PKA signaling to exert Pdx1 promoting effects. My work supports the use of melatonin as a candidate for pancreatic differentiation, and suggests involvement of sPDZD2 in deriving and expanding progenitors during pancreatic organogenesis.published_or_final_version
Biochemistry
Master
Master of Philosophy
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Smith, Aileen M. "Embryonic stem cell differentiation : a novel approach to gene targeting in myeloid cells." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/29369.
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This thesis demonstrates that macrophages can be generated from the culture of ES cells. Although low in number these macrophages show similar morphology and surface phenotype to bone marrow derived macrophages. Importantly, these ES macrophages readily phagocytosed latex beads and apoptotic cells. In additions, ES cells produced cells with a dendritic cell phenotype that were capable of apoptotic cell phagocytosis and maturation. Generation of these myeloid cells from ES cells was strongly dependent on serum and the parent ES cell line. Dendritic cells could be generated from integrin αv disrupted ES cells. These DCs retained the ability to phagocytose apoptotic cells suggesting that integrin αv is not essential for this process. This study also investigates the properties of Cre fused to two transduction proteins. Although Cre retained its recombinase activity as a fusion protein these were unable to translocate into cells. However Cre could be delivered to both primary macrophages and cultured epithelial cells using a replication deficient adenovirus allowing deletion of the targeted genes. Intriguingly, the apoptotic cell phagocytosis was unaffected by lack of integrin αv but could no longer be inhibited by the integrin antagonistic peptide RGD. In conclusion, the capacity of ES cells to differentiate to myeloid cells combined with the ability to deliver Cre to silently targeted myeloid cells provide powerful systems for studying the role of specific genes in phagocytosis. Use of these approaches with integrin αv demonstrates that this gene is not essential for apoptotic cell phagocytosis. However the ability of specific antagonists to inhibit phagocytosis show that integrin αv is intimately involved in this process in normal cells.
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Luo, Zhenling. "MicroRNAs play a role in human embryonic stem cell differentiation into endothelial cells." Thesis, Queen Mary, University of London, 2011. http://qmro.qmul.ac.uk/xmlui/handle/123456789/2425.
Full textAbstract:
The past recent years have seen a surge of evidence demonstrating the regulation of microRNAs (miRNAs) in a myriad of vascular biology events such as cardiogenesis. Nevertheless, the missing miRNA-link in controlling pluripotent human embryonic stem cell (hESC) fate in differentiation towards the endothelial lineage is currently undiscovered. Main objectives in this project are determining which miRNAs are involved in endothelial lineage differentiation from hESCs and the further delineation in their underlying mechanisms. Firstly, undifferentiated hESCs were cultured in differentiating conditions to derive endothelial (ECs) and smooth muscle cells (SMCs). hESC-derived ECs express specific EC markers such as PECAM/CD31, eNOS, and vWF, while hESC-derived SMCs express specific SMC markers such as SMA and SMMHC II. Both hESC-derived cells also displayed functional characteristics upon functional analyses. Next, five potential miRNAs involved in embryonic EC development were determined and selected from the miRNA array expression profile in differentiating hESCs. Using loss- and gain-of-function gene experiments, it was demonstrated that both miR-150 and miR-200c played an important role in EC differentiation from hESCs. However, miR-1915, 141 and 205 did not display such functions. In addition, epithelial-to-mesenchymal transition (EMT)-activator ZEB1/TCF8 was further identified as an important mRNA target for miR-150* and 200c. Importantly, it was also demonstrated that miR-150* and miR-200c were both involved in the vasculogenesis of in vivo chick embryos. These findings may suggest that during hESCs differentiation, an increase of miR-200c expression contribute to the decline or repression of EMT process. Meanwhile, mir-150* also contributes to the differentiation of hESCs, resulting in the formation of more iii differentiated, senescence and less proliferative cells or in this case, mature vascular ECs. These findings illustrate that miR-150* and 200c can regulate the development of ECs from hESCs, providing new targets for modulating vascular formation and creating novel clinical therapies in future cardiovascular disease applications.
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Yin, Xiaoke. "Protein changes associated with embryonic stem cell differentiation to vascular smooth muscle cells." Thesis, Queen Mary, University of London, 2006. http://qmro.qmul.ac.uk/xmlui/handle/123456789/1764.
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Embryonic stem (ES) cells can differentiate into many different cell lines, including vascular smooth muscle cells (SMCs). The aim of this project is to characterize protein changes during this differentiation process. Mouse ES cells are pre-differentiated by withdrawal of the leukemia inhibitory factor-1 from the culture medium. Subsequently, stem cell antigen-1 positive (Sca-1) cells are sorted by magnetic labelling cell sorting with anti-Sca-1 microbeads and cultured in differentiation medium with or without platelet-derived growth factor (PDGF). Protein extracts of ES cells and Sca-1+ cells are separated by two-dimensional electrophoresis. About 300 protein species of each cell lines are analyzed by mass spectrometry. Proteome maps are available online (http:/ /vwvw.v ascular-proteomicsc. om). After stimulation with PDGF for 5 passages, Sca-1+ cells differentiate into SMCs (esSMCs) with 95% staining positive for SMC markers such as smooth muscle a-actin, calponin, and smooth muscle myosin heavy chain. Protein profiles of esSMCs and mouse aortic SMCs are compared using the difference gel electrophoresis approach. esSMCs display decreased expression of myofilaments but increased oxidation of redox-sensitive proteins due to higher levels of reactive oxgen species (ROS). While immunoblotting reveals an upregulation of numerous antioxidants in esSMCs, enzymatic assays demonstrate lower glutathione concentrations compared to aortic SMCs despite a 3-fold increase in glutathione reductase activity. Mitochondrial superoxide measurement revealed the mitochondria-derived superoxide is the main source of ROS in esSMCs and inhibition of electron transport chain complex III by antimycin A showed remarkable increase of ROS in esSMCs. Moreover, depletion of glutathione by diethyl maleate or inhibition of glutathione reductase by carmustine (BCNU) results in a remarkable loss of cell viability in esSMCs compared to aortic SMCs while adding 2-mercaptoethanol increased esSMCs survival. These results indicate that esSMCs require additional antioxidant protection for survival and consequently, treatment with anti-oxidants could be beneficial for tissue repair from ES cells.
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Wang, Nan. "In vitro differentiation of mouse embryonic stem cells into pancreatic insulin-producing cells." Thesis, University of Nottingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508245.
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Murray, Helen. "Role of Grb2 in growth and differentiation of embryonic stem cells." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5894.
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Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo. They exhibit unlimited proliferation in culture and have the ability to differentiate into all three germ layers of the developing organism, a property defined as pluripotency. Previously it was reported that growth factor-bound protein 2 (Grb2) is required for differentiation of the epiblast, the embryonic tissue that harbours the pluripotent founder cells of the foetus. GRB2 is an adapter protein involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in response to extracellular signals. It has also been implicated in the activation of the phosphoinositol-3-kinase (PI3K) pathway in response to fibroblast growth factor (FGF) signaling. The work presented in this thesis examines the role of Grb2 in ES cells and describes previously unreported contributions of this adaptor protein in regulating ES cell growth and differentiation. It has been previously been shown by others that Grb2 deficient (Grb2-/-) cells grow relatively normally in ES growth medium containing serum. However, in serum free conditions (N2B27 medium) in this project, proliferation of Grb2-/- cells is reduced compared with wild type and “restored” Grb2-/- cells stably expressing a Grb2 cDNA mini gene. Under serum free conditions, Grb2-/- cells grow in tight, refractive colonies. Nanog expression was uniformly upregulated, in contrast to the heterogeneous pattern reported in serum-based medium. Colony expansion on the substratum appears to be compromised, although there is no apparent defect in the initial attachment of Grb2-/- cells. Cell cycle analysis indicates that the slower growth of Grb2-/- cells in serum free medium could be due to lengthening of the G1 phase of the ES cell cycle. In an attempt to identify the signalling deficiency responsible for the growth defect of Grb2-/- cells, MAPK activation was restored by two methods, PMA a ligand that bypasses the requirement for Grb2, and Raf-ER, a conditionally regulated component of the MAPK pathway that acts downstream of Grb2 in the MAPK pathway. Although both approaches increased MAPK signalling they were unable to rescue the growth defect. This suggests that MAPK is not required or alone is not sufficient. Inhibition of Glycogen synthase kinase 3 β (GSK3 β ) is known to augment growth of ES cells under MAPK inhibition. Surprisingly, GSK3 β inhibition did not enhance Grb2-/- cell growth. Under GSK3 β inhibition, Grb2-/- ES cells fail to thrive. It is hypothesised that under these conditions cells undergo hyper-self-renewal at the cost of growth. Grb2-/- ES cells are reported to exhibit limited differentiation potential. To examine the potency of Grb2-/- cells, these cells were subjected to embryoid body (EB) and monolayer differentiation. Analysis of EBs showed a loss of Gata4, Gata6 and endoderm marker gene expression. However, markers of ectoderm (Sox1, Pax6, MAP2), the late epiblast/nascent mesoderm (Brachyury) and markers associated with gastrulation (Twist and Snail) were expressed. Outgrowths of morphologically and immunohistochemically identifiable neuronal cells confirmed differentiation of ectodermal cell types, indicating Grb2 is not required for neuronal differentiation. However, beating cardiomyocytes could not be identified in Grb2-/- EBs, though readily found in restored Grb2-/- cells expressing the Grb2 cDNA. This suggests that there is an essential role for Grb2 in the mesoderm/cardiomyocyte differentiation pathway. This may be due to a defect in GATA factor expression since these factors are essential for cardiogenesis. In serum-free monolayer differentiation, Grb2-/- cells formed neuronal cells. Additional inhibition of the MAPK pathway using a small chemical inhibitor failed to prevent this differentiation. However, biochemical analysis of the cells indicates that this occurs when ERK activation is very low, indicating differentiation was not MAPK-independent. Grb2 mediates FGF-MAPK induced exit from the naïve ground state. These data suggest a Grb2-independent pathway can also facilitate this transition. Grb2 is dispensable for differentiation in to some lineages. However as differentiation of Grb2-/- ES cells is restricted, this indicates Grb2 is required for true pluripotency.
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Ridgway, Kimberley Elizabeth. "Factors that influence the chondrogenic differentiation of human embryonic stem cells." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556752.
Full textAbstract:
Native cartilage does not have the ability to heal effectively; therefore, there is a wide prevalence of articular cartilage disease and injury. Human embryonic stem cells provide unique opportunities for the study of differentiation events in vitro and for use in regenerative medicine therapies. Tissue engineering using these cells represents a promising treatment for cartilage lesions. Previous attempts were qualitative, highly variable and limited mainly to HI and H9 human embryonic stem cell lines. The current study aimed to examine the factors that influence the differentiation of the human embryonic stem cell line KCL002 towards the mesenchymal stem cell lineage and to utilise these cells in a cartilage tissue engineering modality. The studies investigated improving this process by choice of tissue engineering scaffold, coating protein and growth factor combination. KCL002 suggested expression of human embryonic stem cell-specific proteins and genes, including Oct4 and Nanog and by quantitative PCR was shown to contribute to trophectoderm, mesoderm and endoderm. KCL002 differentiation was directed towards the mesenchymal stem cell lineage using multiple protocols. A protocol based on harvesting residual auto differentiated cells led to deriving cells with characteristics of adult tissue-derived mesenchymal stem cells including fibroblastic morphology, plastic adherence, cell surface marker expression, and trilineage multipotential upon adipogenic, chondrogenic and osteogenic stimulation. These human embryonic stem cell-derived mesenchymal stem cells were then applied in a tissue engineering modality. Unique quantitative assays, for both protein and RNA, were used to assess the quality of matrix production by the derived cells to determine the best chondrogenic stimulation conditions. The derived cells seeded onto 3-D fibronectin-coated hyaluronic acid based Hyaff-l l scaffolds in the presence of TGF-β1 and BMP-7 gave the highest matrix production and quality of the conditions tested. In addition, members of the nuclear receptor superfamily were analysed for their stage-specific expression during KCL002 differentiation. Differentially expressed nuclear receptors were targeted with synthetic ligands to chemically control the differentiation of KCL002. A combination ofLE135, a RAR-β and RAR-α antagonist and GW647I, a PPAR-α antagonist, was found to enrich for the mesodermal phenotype in embryoid bodies although results failed to produce notable statistical significance. This small molecule analysis was also investigated for its use in aiding the tissue engineering protocol. In conclusion, this work demonstrated for the first time that it is possible to engineer cartilage from the KCL002 human embryonic stem cell line and this was achieved by improving growth factor combination as well as choice of scaffold and coating protein from the adult tissue-derived mesenchymal stem cell tissue engineering protocol. This is an important lesson for human embryonic stem cell research, not to just use the method to hand in the laboratory but to explore tissue engineering conditions beyond differentiation.
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Qin, Mingde. "Development of new methods for the differentiation of embryonic stem cells." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.498651.
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Sadegh, Cameron. "Directed differentiation of mouse embryonic stem cells into neocortical output neurons." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11064.
Full textAbstract:
During development of the neocortex, many diverse projection neuron subtypes are generated under regulation of cell-extrinsic and cell-intrinsic controls. One broad projection neuron class, corticofugal projection neurons (CFuPN), is the primary output neuron population of the neocortex. CFuPN axons innervate sub-cortical targets including thalamus, striatum, brainstem, and spinal cord.
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Papadopoulos, Angelos. "Molecular mechanisms regulating pluripotency and differentiation of human embryonic stem cells." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8551/.
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Dr James A. Thomson reached a milestone discovery in 1998, as he managed to isolate and in vitro expand embryonic stem cells originating from human blastocysts. Since then, human embryonic stem (hES) cells have served as excellent tools for the understanding of a plethora of events that take place during embryogenesis. A full and comprehensive analysis of the molecular mechanisms that regulate both pluripotency and differentiation procedures will ultimately allow these cells to be utilised for therapeutic purposes. The first part of the present thesis is dedicated to investigating the implication of ADP-Ribosylation Factor 6 (ARF6) in TGFβ signalling. ARF6 is a low molecular weight GTPase involved in various cellular functions. Our preliminary data indicate that ARF6 interacts with SMAD4. Building on that, we uncover novel interactions of ARF6 with proteins SMAD2/3 and the interconnection between nucleotide status and downstream signalling events. The connection between ARF6 and TGFβ signalling led us to hypothesize a role for the GTPase in hES cells. In that system, we characterise the effects of ARF6 activation or knockout on both Activin A and BMP4 signalling. In addition, we uncover a novel role for the GTPase during mesendoderm specification. In the last part of the thesis, we utilise a broad transcriptomic approach to reveal novel candidates that are implicated in early differentiation of hES cells to mesendoderm. The assay has been carried out using a novel culture system, based on the ability of Activin A to preserve pluripotency and BMP4 to initiate differentiation.
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Xin, Mankun, and 信满坤. "The expression and role of ADAMTS9 during differentiation of human embryonic stem cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B4775316X.
Full textAbstract:
A Disintegrin-like And Metalloproteinase with Thrombospondin (TSP)-Type I sequence motifs 9 (ADAMTS9) is widely expressed in mouse and human fetal tissues. ADAMTS9 null mice cannot survive beyond E7.5 and its haploinsufficiency is associated with cardiac and aortic anomalies. This project hypothesized that ADAMTS-9 plays an important role during early embryogenesis. By using human embryonic stem cells (hESCs) as a model, the objectives of this study were to study the expression of ADAMTS9 and to determine the effects of ADAMTS9 perturbation on hESC differentiations.
The expression of ADAMTS9 was compared between undifferentiated and differentiated hESCs. Its mRNA was maintained at similar levels in different passages of normal hESC lines. Interestingly, significantly lower expression was detected in karyotypically abnormal VAL4A when compared to the normal cells (H9 and VAL3). ADAMTS9 immunoreactivity was detected in cells located at the boundary of hESC colonies. The expression of ADAMTS9 was then studied during differentiation of hESC. ADAMTS9 mRNA and protein expression increased time-dependently during the first 24 days? of embryoid body (EB) formation. The expression pattern was similar to that of mesoderm and endoderm markers. Upon more specific lineage differentiation induced by retinoic acid and bone morphogenesis protein 4, ADAMTS9 mRNA expression was significantly increased. The positive correlation of ADAMTS9 with ESC differentiation was also found in mouse system, in which ADAMTS9 was increased time-dependently during mouse EB formation and down-regulated during reprogramming from somatic cells into induced pluripotent cells.
Previous studies have shown that down-regulation of ADAMTS9 in several tumor tissues was attributed to ADAMTS9 hypomethylation. However, the present study demonstrated that the expression of ADAMTS9 was reduced in hESC after treatments with inhibitors of DNA methylation (5-aza-2?deoxycytidine) and histone deacetylase (VPA).
The cellular localization of ADAMTS9 during hESC differentiation was further studied by co-localization of ADAMTS9 with several lineage specific markers. It was found that ADAMTS9 co-localized with mesoderm and endoderm markers. The functional role of ADAMTS9 in hESCs was then studied by transient ADAMTS9 knockdown. ADAMTS9 siRNA significantly decreased the expression level of mesoderm marker, REN. Thus, the role of ADAMTS9 during mesoderm differentiation was followed.
ADAMTS9 was found to be dramatically increased after mesoderm differentiation of hESCs. In mesodermal cells, ADAMTS9 was co-expressed with vascular endothelial markers, VEGF and CD31, but not with pericyte markers, alpha muscle actin. Lentiviral vector encoding ADMATS9 shRNA was used for long term knockdown of ADAMTS9. ADAMTS9 down-regulation had no effect on the proliferation of hESCs. In agreement with the siRNA study, ADAMTS9 shRNA also significantly reduced the expression of REN. Upon mesoderm differentiation, ADAMTS9 knockdown resulted in a decreasing trend of mesoderm marker, CD34.
In conclusion, the present study demonstrated a positive association of ADAMTS9 expression with hESC differentiation. ADAMTS9 was dramatically induced during mesoderm differentiation and its knockdown led to down-regulation of mesoderm markers. Together with the fact that ADAMTS9 expression was associated with endothelial cell markers suggested its possible role during endothelial cells formation. The roles of ADAMTS9 during hESC differentiation and early embryo development warrant further investigation.published_or_final_version
Obstetrics and Gynaecology
Master
Master of Philosophy