To see the other types of publications on this topic, follow the link: Embryonic stem cells.
Dissertations / Theses on the topic 'Embryonic stem cells'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Embryonic stem cells.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Nortjé, Nico. "The moral status of embryonic stem cell research in the South African context /." Link to the online version, 2007. http://hdl.handle.net/10019.1/1372.
Full text
2
Harun, Rosliah. "Derivation of trophoblast stem cells from human embryonic stem cells." Thesis, University of Sheffield, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414643.
Full text
3
Montgomery, Sarah Lynn. "Impedance measurement system for embryonic stem cell and embryoid body cultures." Thesis, Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/24661.
Full text
4
Gilner, Jennifer Bushman Kirby Suzanne Lee. "Enrichment of therapeutic hematopoietic stem cell populations from embryonic stem cells." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1232.
Full textAbstract:
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.<br>Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology and Laboratory Medicine." Discipline: Pathology and Laboratory Medicine; Department/School: Medicine.
5
Jurczak, Daniel. "Stemness in human embryonic stem cells." Thesis, University of Skövde, School of Life Sciences, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-3509.
Full textAbstract:
<p>Stem cells are cells that have a unique ability to divide for an indefinite period. Additionally, they can give rise to a plethora of specialized cell types. The advent of high-throughput technologies made it possible to investigate gene expression on a large scale. This enabled scientists to perform comprehensive gene profiling studies of stem cells. Several authors have suggested that there might be a common set of genes that control the stemness of stem cells. In this study, we suggest that ”stemness” genes that are related to ”stemness” characteristics show a statistically significant down-regulation between undifferentiated and differentiated cells. For this we have analyzed microarray data from five different cell lines and compared their global expression profiles. Common down-regulated transcripts among those data sets were de- rived by using a well-established gene expression analysis procedure called Significance Analysis of Microarrays. Since all three data sets were provided by Cellartis AB, the derived list of common transcripts was subsequently compared with an external study. Moreover, we also performed a comparison with down-regulated genes derived from mouse embryonic stem cells. This was done to determine if there is a common set of stemness genes even across distinct species. Re- sults were further evaluated using a comprehensive data-set from a study by Skottman et al. (2005). All results where compared uti- lizing using a range of false discovery rate threshold values and the results were subsequently used for gene ontology term enrichment. GO terms where utilized to functionally annotate and classify those embryonic stem cell transcripts, that were found to be common in all data-sets and identify over-represented biological processes related to those genes.</p>
6
Nussbaum, Jeannette. "Embryonic stem cells for myocardial infarct repair /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/6312.
Full text
7
Noisa, Parinya. "Characterization of neural progenitor/stem cells derived from human embryonic stem cells." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/5712.
Full textAbstract:
Human embryonic stem cells (hESCs) are able to proliferate indefinitely without losing their ability to differentiate into multiple cell types of all three germ layers. Due to these fascinating properties, hESCs have promise as a robust cell source for regenerative medicine and as an in vitro model for the study of human development. In my PhD study, I have investigated the neural differentiation process of hESCs using our established protocol, identified characteristics associated with each stage of the differentiation and explored possible signalling pathways underlying these dynamic changes. It was found that neural differentiation of hESCs could be divided into 5 stages according to their morphology, marker expression and differentiation potencies: hESCs, neural initiation, neural epithelium/rosette, neuronal progenitor cells and neural progenitor/stem cells (NPSCs) and 4 of these stages have been studied in more detail. At the neural initiation, hESCs firstly lose TRA-1-81 expression but retain SSEA4 expression. This transient cell population shows several similar properties to the primitive ectoderm. After neural-tube like structure/neural rosette formation, neural progenitor cells appear as typical bipolar structures and exhibit several properties of radial glial cells, including gene expression and pro-neuronal differentiation. The neural progenitor cells are able to grow in culture for a long time in the presence of growth factors bFGF and EGF. However, they gradually lose their bipolar morphology to triangular cell type and become pro-glial upon further differentiation. In addition, the state of neural progenitor and stem cells can be distinguished by their differential response to canonical Notch effector, C protein-binding factor 1. It was also found that delta like1 homolog (DLK-1) is temporally upregulated upon initial neural differentiation, but becomes undetectable after the neural progenitor stage. Overexpression of DLK-1 in NPSCs enhances neuronal differentiation in the presence of serum by blocking BMP and Notch pathways. These results show that neural differentiation of hESCs is a dynamic process in which cells go through sequential changes, and the events are reminiscent of the in vivo neurodevelopment process. Moreover, I have characterized stably transfected nestin-GFP reporter hESC lines and found that the cell lines maintained the features of hESCs and the expression of GFP is restricted to the neural lineage after differentiation. Therefore, these reporter lines will be useful for the study of factors that regulate neural differentiation and for the enrichment of neural progenitors from other lineages. Taken together, this study has demonstrated that hESCs are a good in vitro model to study the mechanisms and pathways that are involved in neural differentiation. The availability of hESCs allows us to explore previously inaccessible processes that occur during human embryogenesis, such as gastrulation and neurogenesis.
8
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.
Full text
9
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.
Full text
10
Bowles, K. M. "Generation of haematopoietic cells from human embryonic stem cells." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596829.
Full textAbstract:
Culture of hESCs on murine stromal layers or in stromal free conditions as embryoid bodies results in low levels of haematopoietic cells. Here it is demonstrated that overexpression of the transcription factor HOXB4 considerably augments haematopoetic development of hESCs. Stable HOXB4 expressing hESC clones were generated by lipofection and could be maintained in the undifferentiated state for prolonged passages. Moreover, differentiation of hESCs as embryoid bodies in serum containing medium without the use of additional of cytokines led to sequential expansion of first erythroid then myeloid and monocytic progenitors. These cells retained the capacity to develop into formed blood elements during in vitro culture. Consistent with the development of committed haematopoietic cells the expression of transcription factors known to be critical for haematopoietic development was observed. The successful use of enforced gene expression to promote the differentiation of hESCs into a terminally differentiated tissue is thus demonstrated, thereby revealing an important role for HOXB4 in supporting their in vitro development along the haematopoietic pathway. Once a method for producing significant numbers of haematopoietic cells from hESCs in vitro was established, hESCs were used to study the role of stem cell leukaemia gene (SCL) in human blood and endothelial development. hESC lines were generated in which the expression of SCL, under control of an enhancer previously shown in mice to target expression to blood and endothelial progenitors, was evident through green fluorescent protein (GFP) expression. The enhancer directed GFP expression inhuman K562 cells and some differentiated progeny of HOXB4 transfected hESCs. However a more thorough assessment of GFP positive cells was hindered by problems with transgene silencing.
11
Hayashi, Hideki. "Meningeal cells induce dopaminergic neurons from embryonic stem cells." Kyoto University, 2008. http://hdl.handle.net/2433/124217.
Full text
12
Ericson, Robin J. "Bridging solutions to the religion and science conflict over human embryonic stem cell research." Fairfax, VA : George Mason University, 2007. http://hdl.handle.net/1920/2926.
Full textAbstract:
Thesis (Ph. D.)--George Mason University, 2007.<br>Title from PDF t.p. (viewed Jan. 17, 2008). Thesis director: Richard E. Rubenstein. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Conflict Analysis and Resolution. Vita: p. 228. Includes bibliographical references (p. 222-227). Also available in print.
13
Joannides, Alexis. "Neural differentiation of human embryonic stem cells." Thesis, University of Cambridge, 2009. https://www.repository.cam.ac.uk/handle/1810/252121.
Full textAbstract:
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 <i>in vitro</i> and <i>in vivo.</i> 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 <i>in vitro</i> and <i>in vivo</i> behaviour is characterised with respect to temporal maturation and phenotypic potential.
14
Antonica, Francesco. "Modelling thyroid embryogenesis using embryonic stem cells." Doctoral thesis, Universite Libre de Bruxelles, 2013. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209551.
Full textAbstract:
Congenital hypothyroidism (CH) is the most frequent of the rare endocrine diseases (e.g. Addison's disease, Cushing's syndrome, Congenital adrenal hyperplasia.), which affects 1:2000 – 4000 newborns. If not immediately diagnosed after birth, thyroid hormones deficiency causes severe defects in brain and skeletal development leading to a complex clinical scenario called cretinism. CH can be due to a defective synthesis of thyroid hormones (dyshormonogenesis) or an abnormal embryonic development of the gland. Data obtained using knockout mouse models have shown the pivotal role of four specific transcription factors (NKX2.1, PAX8, FOXE1 and HHEX) for the correct organogenesis or function of the gland. Although mutations in those genes have been identified in few cases of CH patients, the pathogenetic mechanisms remain still elusive in the vast majority of CH cases (95%).<p>For the identification of new genes and molecular events controlling thyroid organogenesis it would be useful to develop an in vitro cellular model to recapitulate thyroid embryogenesis in a dish. Embryonic Stem Cells (ESCs) have recently emerged as system model to recapitulate the embryogenesis of several tissues in vitro.<p>Induced overexpression of defined transcription factors has been shown to have a directing effect on the differentiation of pluripotent stem cells into specific cell types. In this thesis I show that a transient overexpression of the transcription factors NKX2.1 and PAX8 is sufficient to direct the differentiation of murine ESCs into thyroid follicular cells (TFC) and promotes in vitro self- assembly of TFC into three-dimensional follicular structures, when associated to a subsequent thyrotropin (TSH) treatment. Cells differentiated by this protocol showed significant iodide organification activity, a hallmark of thyroid tissue function. Importantly, athyroid mice grafted with mESC-derived thyroid follicles show normalization of plasma T4 levels with concomitant decrease of plasma TSH. In addition, a full normalization of body temperature at 4 weeks after transplantation was observed. Together, these data clearly demonstrate that grafting of our mESC-derived thyroid cells rescues the hypothyroid state and triggers symptomatic recovery along with the normalization of plasma hormone concentrations. The high efficiency of TFC differentiation and follicle morphogenesis in our system will provide an unprecedented opportunity for future studies to decipher regulatory mechanisms involved in embryonic thyroid development, a major research need towards an improved understanding of the molecular mechanisms underlying congenital hypothyroidism, the most common congenital endocrine disorder in humans.<br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished
15
Gordon-Keylock, Sabrina Anne Megan. "Haematopoietic differentiation of murine embryonic stem cells." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/29123.
Full textAbstract:
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 <i>Brachyury-</i>eGFP reporter gene demonstrated that the stromal lines mediated their effects post-<i>Brachyury </i>(mesoderm) induction in the ES cells. In addition, co-culture of sorted ES cell populations confirmed that <i>Brachyury<sup>+</sup>, </i>but not <i>Brachyury<sup>-</sup>, </i>cells gave rise to haematopoietic progenitors in AM co-culture, supporting the notion that ES cell differentiation recapitulated the <i>in vivo</i> 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.
16
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.
Full text
17
Both, Sanne Karijn. "Embryonic stem cells in bone tissue engineering." Enschede : University of Twente [Host], 2008. http://doc.utwente.nl/58765.
Full text
18
Mak, Shiu-kwong Thomas, and 麥肇鑛. "Modeling diabetic cardiomyopathy using embryonic stem cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193562.
Full textAbstract:
Diabetic cardiomyopathy (DCM), a disorder of the heart muscle, is one of the major and most rampant culprits claiming thousands and thousands of lives around the globe every year by interfering with the blood circulation and causing the development of heart failure eventually. The progression of the disease is asymptomatic and having a long latent period, and it is characterized functionally by ventricular dilation, diastolic dysfunction, interstitial fibrosis and cardiomyocytes hypertrophy. It was suggested the pathogenesis of the disease and the related complications are related to the effects of hyperglycemia on cardiomyocytes. So understanding the physiology of both the normal and pathological conditions, and the underlying mechanisms involved are of paramount importance to derive therapies to cope with this disease. However, it is difficult, if not impossible, to study the physiology in vivo using a live sample or to build a cellular model with adult cardiomyocytes due to the insufficient number of the cells harvested. This is not until the emergence of Embryonic Stem Cells (ESCs) that a cellular model with clinical sufficient number of cardiomyocytes could be built for investigation and drug screening.
With a view to mimicking the situation of the Diabetic cardiomyopathy of the Type II Diabetes mellitus (DM) patients, mouse ESCs are used to differentiate into cardiomyocytes using the traditional hanging drop method to produce Embryoid body (EB). The cardiomyocytes were then enriched and plated so that different testing conditions could be applied. The effect of high glucose (HG), Insulin and the combination of high glucose and insulin were then analyzed. This was to show the significance of hyperglycemia, hyperinsulinemia due to insulin resistance and the role of insulin in hyperglycemia on cardiomyocytes respectively.
The results agreed with previous findings that high glucose and insulin alone do induce cells apoptosis while the combination of insulin and glucose did decrease the number of apoptosis and while the co-culture of insulin with High dosage of glucose has shown to reduce the effect of hypertrophy.<br>published_or_final_version<br>Medicine<br>Master<br>Master of Medical Sciences
19
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.
Full textAbstract:
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.
20
Rugg-Gunn, Peter. "Epigenetic status of human embryonic stem cells." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614294.
Full text
21
Karan, Priyanka. "Embryonic stem cells alter cardiomyocyte electrophysiological properties." Thesis, Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/24691.
Full text
22
Lin, Wenyu. "Investigating the immunomodulatory properties of human embryonic stem cell-derived mesenchymal stem cells." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/7060.
Full textAbstract:
The immunosuppressive property of mesenchymal stem cells (MSC) has been utilised to ameliorate autoimmune reactions such as graft-versus-host disease. However, variation exists in primary MSC isolated due to differences in donor age and tissue of origin. Alternatively, human embryonic stem cells (hESC) can be differentiated to homogeneous populations of MSC (hESCMSC), thus providing an unlimited source of MSC for cell therapy. In this study, the immunomodulatory properties of two hESC-MSC lines, hESC-MSC1 and hESC-MSC2, were compared with adult bone marrow-derived MSC (BM-MSC) and neonatal foreskin fibroblast (Fb). hESC-MSC were able to suppress the proliferation of anti-CD3/CD28-stimulated CD4+ T cells in contact and transwell systems. The immunosuppression was demonstrated by both the carboxyfluorescein diacetate succinimidyl ester (CFSE) and [3H]- thymidine proliferation assays. However, hESC-MSC were less potent and twice the number of adherent hESC-MSC (as measured by IC50) compared to BM-MSC and Fb were required to suppress T cell proliferation by 50%. Supernatants collected from transwells of MSC or Fb with T cells were shown to suppress T cell proliferation, suggesting that suppressive factors were only produced in the presence of activated T cells. Among several candidates, endothelial monocyte-activating polypeptide-II (EMAP-II) was identified as a potential suppressive factor. T cells also induced indoleamine-2,3- dioxygenase (IDO) expression in MSC and Fb. IDO led to the depletion of tryptophan, an essential amino acid, and/or the production of tryptophan metabolites (kynurenines), thereby inhibiting T cell proliferation. Interestingly, blocking of IDO with 1-methyltrytophan reversed the suppressive effect, implicating IDO as a potential mediator in T cell suppression. Concomitantly, several candidate suppressive factors in the supernatants have also been identified using antibody arrays. However, their functions require validation. In conclusion, hESC-MSC share similar suppressive properties as BM-MSC and represent a potential cell source for clinical purposes.
23
Vitillo, Loriana. "Focal adhesion kinase regulation of human embryonic stem cells." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/focal-adhesion-kinase-regulation-of-human-embryonic-stem-cells(31052725-50a4-4d34-a1eb-018be57986af).html.
Full textAbstract:
Undifferentiated human embryonic stem cells (hESCs) grow on the extracellular matrix (ECM) substrate fibronectin (FN) in defined feeder-free conditions. The ECM is part of the hESCs pluripotent niche and supports their maintenance, but the contribution to survival remains to be elucidated. Understanding the mechanism of survival is particularly crucial in hESCs, since it affects their expansion in cell culture and ultimately translation of research to the clinic. HESCs bind to FN mainly via alpha5β1- integrin, known to be upstream of important survival cascades in other cell types. However, it is not understood if and how FN/integrin binding supports those molecular pathways in the context of pluripotent hESCs. The aim of this work was to elucidate the survival cascade downstream of the FN/integrin interaction in hESCs. Initially, when hESCs were cultured on a non-integrin activating substrate they initiated an apoptotic response that also occurred when β1-integrin was selectively blocked with antibody, leading the cells to detach from FN. Integrin activation is generally transduced within cells via a complex adhesome of scaffold and kinase proteins, among which the focal adhesion kinase (FAK) plays a key role. Indeed, blocking β1-integrin resulted in dephosphorylation of endogenous FAK in hESCs. When FAK kinase activity was directly inhibited (with small molecule inhibitors), hESCs responded by detaching from FN and activating caspase-3, leading to an increase in apoptosis. Furthermore, flow cytometry analysis showed that the population of hESCs that underwent apoptosis still retained the pluripotency-associated marker NANOG. FAK is a convergent point between growth factor signaling and the PI3K/Akt pathway, with a well-reported role in the maintenance of hESCs. Consistently, FN activated both AKT and its target the ubiquitin ligase MDM2 at the protein levels, while pAkt was reduced after β1-integrin blocking and FAK inhibition. Cell imaging showed that MDM2, which regulates p53 degradation in the nucleus, displayed reduced nuclear localisation after FAK inhibition, opening the possibility for a change in the p53 balance in hESCs. In fact, p53 protein increases after FAK inhibition corresponding also to caspase activation. Further investigation explored if FAK-dependent pathways are also implicated in the maintenance of hESC pluripotency. Inhibition of FAK led the cells that survived apoptosis to lose stem cell morphology, decrease pluripotency-associated markers and change nuclear shape. Moreover, a small pool of FAK was found in the nucleus of hESCs cultured on FN, but decreased after FAK inhibition. FAK was also co- immunoprecipitated with NANOG protein in standard hESC culture while NANOG decreased after sustained FAK inhibition. This data suggests that nuclear roles of FAK could support, together with the cytoplasmic activation of the PI3K cascade, both survival and pluripotency pathways requiring further investigation. In conclusion, the original contribution of this work is to identify in FAK the downstream survival effector of the FN/β1-integrin interaction in hESCs. HESCs survival is maintained by the binding of β1-integrin to FN and activation of FAK kinase and downstream PI3K/Akt, leading to the suppression of p53 and caspase activation. In parallel, promotion of these pathways by FAK is suggested also to support the key pluripotency circuitry, feeding into NANOG. Overall, FAK is proposed here as an important regulator of hESC survival and fate.
24
Chen, Shi. "Cryopreservation of human embryonic stem cells and hepatocytes." Thesis, University of Oxford, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711665.
Full text
25
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.
Full textAbstract:
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.
26
Faddah, Dina Adel. "Single-cell analyses of cellular reprogramming and embryonic stem cells." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/89941.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.<br>Vita. Cataloged from student-submitted PDF version of thesis.<br>Includes bibliographical references.<br>Three years before the start of this thesis, Yamanaka and Takahashi published a groundbreaking paper entitled "Induced of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." A mere two scientists reprogrammed somatic cells to an embryonic stem-cell like state (termed induced pluripotent stem cells, iPSCs) by simply overexpressing four transcription factors: Oct4, Sox2, c-Myc, and Klf4. During cellular reprogramming, only a small fraction of cells become iPSCs. Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. Using single-cell analysis, we found Esrrb, Utf1, Lin28 and Dppa2 to be predictive markers of reprogramming. We found that single cells exhibit high variation in gene expression early in reprogramming and this heterogeneity decreases are the cell reaches pluripotency. Our results show that a stochastic phase of gene activation is followed by a late hierarchical phase, initiated by activation of the Sox2 locus, leading to the activation of the pluripotency circuitry. Finally, we reprogram cells without Oct4, Klf4, Sox2, c-Myc, and Nanog. Embryonic stem cells (ESCs) are the gold standard comparison for iPSCs. Our investigation of ESCs must continue in parallel to that of iPSCs since we cannot truly understand iPSCs if we do not understand the molecular mechanisms that regulate ESC pluripotency. The homeodomain transcription factor Nanog is a central part of the core pluripotency transcriptional network and plays a critical role in ESC self-renewal. Several reports have suggested that Nanog expression is allelically regulated and that transient downregulation of Nanog in a subset of pluripotent cells predisposes them toward differentiation. Using single-cell gene expression analyses combined with different reporters for the two alleles of Nanog, we show that Nanog is biallelically expressed in ESCs independently of culture condition. We also show that the overall variation in endogenous Nanog expression in ESCs is very similar to that of several other pluripotency markers. Our analysis suggests that reporter-based studies of gene expression in pluripotent cells can be significantly influenced by the gene-targeting strategy and genetic background employed. Our results show that single-cell analysis is essential for deciphering the mechanisms of reprogramming and understanding gene regulation of ESCs, exposing important rarities typically masked by population-based assays.<br>by Dina Adel Faddah.<br>Ph. D.
27
Ngangan, Alyssa V. "Bioactive factors secreted by differentiating embryonic stem cells." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/44913.
Full textAbstract:
Current therapeutic strategies to stimulate endogenous angiogenic processes within injured tissue areas are typically based on introducing exogenous pro-angiogenic molecules or cell populations. Stem cell transplantation for angiogenic therapy aims to deliver populations of cells that secrete angiogenic factors and/or engraft in the new branching vasculature within the damaged tissue. Utilizing stem or progenitor cells has been shown to induce a rather robust angiogenic response despite minimal repopulation of the host vasculature, suggesting that stem cells may provide paracrine factors that transiently induce endogenous angiogenesis of tissues undergoing regeneration. Early differentiating embryonic stem cell (ESC) aggregates, referred to as embryoid bodies (EBs), can undergo vasculogenic differentiation, and also produce extracellular matrix and growth factors that induce proliferation, differentiation, and tissue morphogenesis. Taken together, the ESC extracellular environment may be an effective means by which to manipulate cell behavior. Thus, the objective of this project was to harness morphogens derived from ESCs undergoing differentiation and analyze their bioactive potential. To examine the expression of extracellular factors within EBs, gene expression arrays in conjunction with a variety of analytical tools were utilized to gain an understanding of the importance of extracellular factors in ESC differentiation. Furthermore, the soluble fraction of secreted factors contained within EB-conditioned media was compared to the matrix-associated factors produced by EBs, which led to the development of novel ESC-derived matrices via mechanical acellularization methods. Acellular embryonic stem cell-derived matrices demonstrated the retention of bioactive factors that impacted aspects of angiogenesis. In conclusion, extracellular factors were modulated in response to the progression of EB differentiation and can further be harnessed via acellularization techniques, in order to deliver bioactive ESC-secreted factors in a cell-free manner.
28
Nair, Rekha. "Acellular matrices derived from differentiating embryonic stem cells." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/37170.
Full textAbstract:
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.
29
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.
Full text
30
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.
Full text
31
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.
Full textAbstract:
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.
32
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.
Full textAbstract:
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.
33
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.
Full text
34
Milne, Helen Marie. "Generation of insulin-expressing cells from mouse embryonic stem cells." Thesis, King's College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.417362.
Full text
35
Thomas, Paul Quinton. "Homeobox gene expression in murine embryonic stem cells." Title page, contents and summary only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09pht4608.pdf.
Full textAbstract:
Includes bibliographies. Aims to identify homeobox genes which may have a developmental role during early embryogenesis by the characterization of homeobox gene expression in undifferentiated ES cells, and in a range of differentiated ES cell derivatives.
36
Lee, Yee-ki Carol, and 李綺琪. "Pluripotent stem cells for cardiac regeneration." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46918462.
Full text
37
Meyer, Jason S. "Characterization and therapeutic transplantation of stem cells /." free to MU campus, to others for purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p3164528.
Full text
38
Ismail, Siti N. "Stem cell bioprocessing : the bioengineering of lung epithelium in 3D from embryonic stem cells." Thesis, Imperial College London, 2009. http://hdl.handle.net/10044/1/9013.
Full textAbstract:
Stem cell therapies and tissue engineering strategies are required for the clinical treatment of respiratory diseases. Previous studies have established protocols for the differentiation of airway epithelium from stem cells but have involved costly and laborious culture methods. The aim of this thesis was to achieve efficient and reproducible maintenance and differentiation of embryonic stem cells to airway epithelium, in 2D and 3D culture, by developing appropriate bioprocessing technology. Firstly, the 2D differentiation process of human and murine ES cells into pulmonary epithelial cells was addressed. The main finding in was that the proportion of type II pneumocytes, the major epithelial component of the gas-exchange area of lung, differentiated with this method was higher than that obtained in previous sudies, 33% of resultant cell expressed the specific marker surfactant protein C (SPC) compared with up to 10%. Secondly, the maintenance and differentiation was carried out in 3D. A protocol was devised that maintained undifferentiated human ES cells in culture for more than 200 days encapsulated in alginate without any feeder layer or growth factors. For ES cell differentiation in 3D, a method was devised to provide a relatively cheap and simple means of culture and use medium conditioned by a human pneumocyte tumour cell line (A549). The differentiation of human and murine ES cells into pulmonary epithelial cells, particularly type II pneumocytes, was found to be upregulated by culture in this conditioned medium, with or without embryoid body formation. The third step was to test whether this differentiation protocol was amenable to scale-up and automation in a bioreactor using cell encapsulation. It was possible to show that encapsulated murine ES cells cultured in static, co-culture or rotating wall bioreactor (HARV) systems, differentiate into endoderm and, predominantly, type I and II pneumocytes. Flow cytometry revealed that the mean yield of differentiated type II pneumocytes was around 50% at day 10 of cultivation. The final stage of the work was to design and produce a perfusion system airlift bioreactor to mimic the pulmonary microenvironment in order to achieve large scale production of biologically functional tissue. The results of these studies thus provide new protocols for the maintenance of ES cells and their differentiation towards pulmonary phenotypes that are relatively simple and cheap and can be applied in bioreactor systems that provide for the kind of scale up of differentiated cell production needed for future clinical applications.
39
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.
Full textAbstract:
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.
40
Markoullis, Kyriaki. "Mycoplasma contamination of murine embryonic stem (mES) cells." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-99459.
Full text
41
Randle, Wesley Liam. "Tissue engineering of bone from embryonic stem cells." Thesis, Imperial College London, 2006. http://hdl.handle.net/10044/1/11880.
Full text
42
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.
Full text
43
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.
Full text
44
Meek, Stephen Earl. "Experimental approaches to establish rat embryonic stem cells." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5040.
Full textAbstract:
The rat has been an established experimental animal model within many areas of biological investigation for over one hundred years due to its size, breeding characteristics, and knowledge of its physiology and behaviour. In recent years its status as a leading biomedical model has been somewhat surpassed by the mouse. This is largely the result of the isolation and application of mouse embryonic stem (ES) cells. Mouse ES cells have the capacity for unlimited self-renew in vitro whilst maintaining pluripotency and germline competence, and most importantly are amenable to sophisticated reverse genetics strategies such as gene targeting, which have provided a route to germ line modification. Thus far, the derivation of rat ES cells has proved elusive. The generation of rat ES cells would therefore facilitate equivalent applications to rat genetics and significantly strengthen the rat as an experimental model system. Previous attempts to derive rat ES cells led to the isolation of rat ES-like cells. However, whilst these cells exhibit extensive self-renew in vitro, it was known that they fail to maintain significant levels of the key functional ES cell marker Oct4 and do not contribute to chimeras. Rather, these cells express the trophectoderm markers Cdx2 and CyclinD3, and have been termed ExS cells due to their probable extra-embryonic nature. In the work described in this thesis, further investigation of ExS cells revealed the absence of expression of the key pluripotency gene Nanog, although the expression pattern of Nanog in the rat embryo was shown to be similar to that of mouse. It was hypothesised that expression of exogenous Oct4 and Nanog or Sox2 genes could facilitate reprogramming of ExS cells into a 'true' ES cell state. Initial work described in this thesis demonstrated that it was possible to introduce transgenes into rat ExS cells and obtain stable transformants with long term transgene expression. On this basis Oct 4, Nanog and Sox2 transgene expression vectors were constructed and stably integrated into ExS cells, and transgene expression verified. However, no reactivation of an endogenous gene expression profile, characteristic of a true ES cell-like state, was observed in any of the transgenic lines produced. Concurrent with work on ExS cells, investigations by others using chemically defined, serum-free medium containing small molecule inhibitors of MEK and GSK3 (called 3i/2i medium) had demonstrated that it was possible to readily isolate mouse ES cells, even from strains known to be refractory to ES cell isolation. Therefore, the ability of this culture system to facilitate rat ES cell derivation was investigated. Rat 3i/2i cell lines were established from ICM outgrowths of Fischer, DA and Sprague Dawley E4.5 rat embryos. These cells maintained expression of Oct4 and Nanog and could generate complex teratomas consisting of all three germ layers. They were distinct from epiblast stem cells (EpiSC) in that they expressed Klf4, Rex1 and Stella and most importantly, they could contribute to the formation of adult chimaeras and demonstrated germline competency. Isolation of these authentic rat ES cells paves the way for gene targeting in the rat, a development that should greatly facilitate new biomedical discoveries.
45
Tilgner, Katarzyna. "Derivation of oocytes from human embryonic stem cells." Thesis, University of Newcastle Upon Tyne, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512211.
Full text
46
Sutha, Ken. "Osteoinductive material derived from differentiating embryonic stem cells." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/51722.
Full textAbstract:
The loss of regenerative capacity of bone, from fetal to adult to aged animals, has been attributed not only to a decline in the function of cells involved in bone formation but also to alterations in the bone microenvironment that occur through development and aging, including extracellular matrix (ECM) composition and growth/trophic factor content. In the development of novel treatments for bone repair, one potential therapeutic goal is the restoration of a more regenerative microenvironment, as found during embryonic development. One approach to creating such a microenvironment is through the use of stem cells. In addition to serving as a differentiated cell source, pluripotent stem cells, such as embryonic stem cells (ESCs), may possess the unique potential to modulate tissue environments via local production of ECM and growth factors. ESC-produced factors may be harnessed and delivered to promote functional tissue regeneration. Such an approach to generate a naturally derived, acelluar therapy has been employed successfully to deliver osteoinductive factors found within adult bone, in the form of demineralized bone matrix (DBM), but the development of treatments derived instead from developing, more regenerative tissues or cells remains attractive. Furthermore, the derivation of regenerative materials from an ESC source also presents the added benefit of eliminating donor to donor variability of adult, cadaveric tissue derived materials, such as DBM. Thus, the objective of this project was to examine the osteoinductive potential harbored within the embryonic microenvironment, in vitro and in vivo. The osteogenic differentiation of mouse ESCs as embryoid bodies (EBs) was evaluated in response to phosphate treatment, in vitro, including osteoinductive growth factor production. The osteoinductivity of EB-derived material (EBM) was then compared to that of adult tissue-derived DBM, in vivo. Phosphate treatment enhanced osteogenic differentiation of EBs. EBM derived from phosphate treated EBs retained bioactive, osteoinductive factors and induced new bone formation, demonstrating that the microenvironment within osteogenic EBs can be harnessed in an acellular material to yield in vivo osteoinductivity. This work not only provides new insights into the dynamic microenvironments of differentiating stem cells but also establishes an approach for the development of an ESC-derived, tissue specific therapy.
47
Coe, Torres Davi. "Understanding H3K36 methyltransferases in mouse embryonic stem cells." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-144706.
Full textAbstract:
Methylation of histone 3 (H3) at lysine 36 (K36) has been implicated in several biological processes, such as DNA replication, DNA repair, and transcription. To date, at least eight distinct mammalian enzymes have been described to methylate H3K36 in vitro and/or in vivo. In this work, Set2, Nsd1, and Nsd3 Venus tagged proteins were successfully expressed in mouse embryonic stem cells and, then, analyzed by confocal microscopy, mass spectrometry (MS), and chromatin immunoprecipitation sequencing (ChIP-seq). MS analysis revealed that Setd2, Nsd1, and Nsd3 do not associate in protein complexes with each other. Setd2 was associated with RNA polymerase II subunits and two transcription elongation factors (Supt5 and Supt6), whereas Nsd1 associated with the transcription factor Zfx. In contrast, Nsd3 interacted with multiple protein complexes including Kdm1b and Brd4 complexes.
Interestingly, Nsd1 and Zfx seem to be bound to chromatin during cell division. ChIP-seq analysis of the H3K36 methyltransferases showed different binding profiles at transcribed genes: Nsd1 binds near the transcription start site (TSS), Setd2 loading starts near the TSS and spreads along the gene body, while, Nsd3 is preferentially enriched at the 5’ and 3’ gene regions. Sequential deletion of PWWP and zinger-finger like domains was achieved to study any possible changes in Nsd1 and Nsd3 function. Deletion of either PHD1-4 or PHD5/C5HCH domains decreased Nsd1 recruitment to chromatin. Particularly, the PHD5/C5HCH were identified as the protein-protein interface for Zfx interaction. In agreement, Zfx knockdown also decreased Nsd1 deposition at the Oct4 and Tcl1 promoter regions. Furthermore, Nsd1 depletion reduced bulk histone H3K36me2 and histone H3K36me3 loading at the coding regions of Oct4, Rif1, Brd2, and Ccnd1.
In addition, Nsd1 knockdown led to an increased Zfx deposition at promoters. Our findings suggest Zfx recruits Nsd1 to its target loci, whereas Nsd1 regulates Zfx chromatin release and further contributes to transcription regulation through its H3K36 dimethylase activity. On the other hand, loss of Nsd3’s PHD5/C5HCH or PWWP domains decreased Nsd3 binding to DNA. In addition, we demonstrate that Nsd3 is recruited to target genes in a Brd4-dependent manner. Herein, we provided further insights on how H3K36 methyltransferases are regulated, and how they contribute to changes in the epigenetic landscape in mouse embryonic stem cells.fi
48
Wan, Chen-rei. "Characterization of the cardiogenesis of embryonic stem cells." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/65283.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, February 2011.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 114-127).<br>Cardiovascular diseases persist as the leading cause of mortality worldwide. Stem cell therapy, aimed to restore contractility and proper vasculature, has gained considerable attention as an attractive therapeutic option. However, proper cell differentiation, survival and integration in an infarcted zone remain elusive. This thesis aims to utilize in vitro techniques to obtain a systematic characterization of how individual stimulations can affect the cardiogenesis process of embryonic stem cells. First, a compliant microfluidic system was developed to study the individual and combined effects of culture dimensions and uniaxial cyclic stretch on the differentiation process. A smaller culture dimension, with a characteristic length scale of hundreds of micrometers, dramatically enhanced differentiation partly due to an accumulation of cell-secreted and cardiogenic BMP2. Uniaxial cyclic stretch, on the other hand, inhibited differentiation. With this microfluidic platform and a GFP-reporting differentiation cell line, effects of various external stimuli on differentiation were systematically studied. Next, the effects of collagen I and cell alignment, two biophysical signatures of the adult myocardium, on promoting phenotypic changes of isolated embryonic stem cell derived cardiomyocytes (ESCDMs) were investigated. Effects of collagen I depended on how it was presented to the cells and overlaying collagen gel impeded cell elongation. Binucleation. characteristic of maturing cardiomyocytes, was reduced with soluble collagen supplement and nanoscale topography and was associated with an increase in cytokinesis. Both nanoscale topography and microcontact printing resulted in aligned cardiomyocyte monolayers but produced different morphologies. Lastly, the lessons learned from studying the aforementioned processes were applied to test the utility of ESCDMs as biological actuators. Three proof-of-concept experiments were conducted: ESCDM monolayers were able to contract synchronously as a cell-assemble, force generated by the cell monolayer was estimated to be comparable to that by neonatal myocytes and lastly, the direction of contraction could be controlled with surface patterning. This work advances our understanding on the cardiogenic potential of murine embryonic stem cells and elucidated complex biological questions with well-characterized and controlled tissue engineering techniques.<br>by Chen-rei Wan.<br>Ph.D.
49
Tailor, Jignesh Kishor. "Human neuroepithelial stem cells from the embryonic hindbrain." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607958.
Full text
50
Blair, Kathryn Lee. "Properties of embryonic stem cells from Rattus norvegicus." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610069.
Full text