Browsing by Subject "Pluripotent"
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Item Chromatin accessibility and its relationship to pluripotency and the induction of pluripotent stem cells(2015-01) Espland, EricChromatin within stem cells is dynamic and relaxed, allowing transcription and thus lineage specification to occur rapidly. To determine how this property can be used to enhance the generation of induced pluripotent stem cells (iPSC), I examined whether the expression of histone variants or peptidyl-prolyl isomerases (Ppiases) could increase the hyperdynamic, plastic nature of stem cell chromatin and thereby increase the efficiency and speed of reprogramming. I used molecular cloning to generate expression vectors containing the histone variants H3T and H2A.B. I used line-scanning microscopy to measure chromatin dynamics, with initial results suggesting that stem cells are more hyperdynamic in nature than differentiated cells. Although the research on the effect of histone variants and Ppiases on chromatin dynamics and reprogramming was not completed, another study showed that the expression of other histone variants does enhance reprogramming and may also induce an open chromatin structure. If this holds true for the histone variants studied here or Ppiase B (PpiB), this could further enhance the generation of iPSC and make future autologous engraftments of iPSC more feasible.Item Engineering a three-dimensional culture system for the directed differentiation of pluripotent stem cells toward a hepatocyte-like cell fate(2012-11) Owens, Derek JasonBecause stem cells have the ability to self-renew and differentiate into more specialized cell types, they hold enormous potential in the fields of regenerative and personalized medicine as well as providing a model system for studying development in vitro. Stem cells with the capacity to differentiate to hepatocytes, the functional cells of the liver, have potential applications in the pharmaceutical industry in high-throughput drug toxicity screening and in clinical settings in bioartificial liver devices or as candidates for transplantation to treat end-stage liver disease. However, these applications rely on the ability to generate and differentiate the stem cells to functionally mature hepatocytes in a robust and reproducible manner. We have recently optimized a multistage directed differentiation protocol for guiding human embryonic stem (hES) cells and rat multipotent adult progenitor cells (rMAPCs), among other stem cell types, toward a hepatic fate. We also recently showed that rMAPCs, which are isolated from the bone marrow of post-natal rats and exhibit the ability to self-renew and differentiate to all three lineages, can be cultured as three-dimensional aggregates without losing their potency or self-renewal capacity. In this study, we report three-dimensional aggregate-based culture systems that enhance the differentiation of rMAPCs and hES cells to hepatocyte-like cells. rMAPCs were allowed to self-assemble into undifferentiated aggregates before being differentiated via the four-step directed differentiation protocol. Compared to adherent monolayer cultures, differentiation as aggregates resulted in significantly higher expression of liver-specific transcripts, including albumin, and increased secretion of albumin and urea. The differentiated cell aggregates also demonstrated functional activities of primary hepatocytes, as demonstrated by pentoxyresorufin O-dealkylation (PROD) and ethoxyresorufin O-dealkylation (EROD), and ultrastructural features of hepatocytes by electron microscopy. A similar three-dimensional culture system likewise enhanced the differentiation of hES cells. HSF6 cells differentiated as a monolayer culture were dissociated and allowed to self-assemble into three-dimensional spheroids in an extended differentiation culture. Compared cells maintained in the monolayer culture, cells within the spheroids exhibited significantly higher expression levels of liver-enriched transcripts and proteins, including Albuming, PEPCK, and ASGPR-1. Cells in the spheroids demonstrated hepatic functions EROD, PROD, and biliary accumulation of fluorescein diacetate metabolite and ultrastructural characteristics of hepatocytes by electron microscopy. Finally, whole-genome transcriptome analysis was performed to investigate the expression profile of liver-specific sets of genes, including the hepatocyte nuclear factors (HNFs), cytochrome P450s (CYP450s), and UDP-glucuronosyltransferases (UGTs), during differentiation. Cells in the spheroid were shown to have overall increased expression levels of most of the genes in these families, although the expression levels were still lower than in adult liver. The transcriptome analysis was also used to identify genes that change during establishment of the spheroid culture that may play a role in the enhanced differentiation status of the cells; multiple members of the aldo/keto reductase (AKR) and metallothionein (MT) families were found to have much higher expression in spheroids than in monolayer culture. These studies demonstrate the ability of three-dimensional, scalable culture systems to enhance the differentiation of pluripotent stem cells toward a hepatic fate and to maintain the differentiated phenotype for extended culture. With modifications to further enhance the maturity of stem cell-derived hepatocyte-like cells, these systems may facilitate the translation of stem cell generated tissues to technology.Item An Exploration into Human Pluripotency and Its Role in the Development of Squamous Cell Carcinoma in Recessive Dystrophic Epidermolysis Bullosa Patients(2016-01) Ward, EmilyThis thesis discusses 2 projects: 1) generating a methodology for creating a human induced pluripotent stem cell (hiPSC) line receptive to gene editing and 2) exploring the role of Nanog and the epithelial to mesenchymal transition (EMT) proteins in the pathophysiology of squamous cell carcinoma (SCC) of patients with recessive dystrophic epidermolysis bullosa (RDEB).Item Stabilized CD16a Expression on Pluripotent Stem Cell-Derived Natural Killer Cells(2015-12) Blum, RobertHuman embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) have been shown to successfully generate functional hematopoietic cells of multiple lineages. One of the cells that these studies have shown is possible to generate are cytotoxic natural killer (NK) cells. These pluripotent stem cell-derived NK cells could potentially be used as a cell-based therapy to treat a wide range of cancers. A potent method through which NK cells kill their targets is antibody-dependent cell-mediated cytotoxicity (ADCC). During this process an Fc gamma receptor, CD16a, recognizes antibody coated target cells and leads to the stimulation of the NK cell’s cytotoxic pathways. However, upon CD16a activation, a metalloprotease known as ADAM17 has been shown to cleave CD16a leading to a decrease in NK cell ADCC. This project seeks to generate genetically modified NK cells that have CD16a expression stabilized to maximize ADCC. One method to accomplish this is the generation of a non-cleavable version of CD16a. This cDNA construct has been transfected into iPSCs using Sleeping Beauty transposase to stably express CD16a. These cell’s CD16a molecules are resistant to cleavage upon cell activation and are also highly expressed on the surface of the NK cells. Preliminary testing has shown that when combined with antibodies specific to their cognate target ligand, these cells have a greater cytolytic response than untransfected peripheral blood NK cells (PBNK). Also, CRISPR-Cas9 has been employed to knock-out ADAM17. Going forward, comparing the cytotoxicity of these two NK cells will show which is best to employ in the treatment of cancers that express ligands for therapeutic antibodies that have proven refractory to other therapies and adjuvants.