Stem cell biology is an exciting field that will lead to significant advancements in science and medicine. We hypothesize that inducing the expression of stem cell genes, using the embryonic stem cell gene nanog, will reprogram cells and dedifferentiate human mesenchymal stem cells into pluripotent stem cells capable of neural differentiation. The aims of initial studies are as follows: Aim 1: Demonstrate that forced expression of the embryonic stem cell gene nanog induces changes in human mesenchymal stem cells to an embryonic stem cell-like phenotype. Aim 2: Demonstrate that induced expression of nanog up-regulates the expression of multiple embryonic stem cell markers and expands the differentiation potential of the stem cells. Aim 3: Demonstrate that these nanog-expressing stem cells have the ability to differentiate along neural lineages in vitro and in vivo, while mock-transfected cells have an extremely limited capacity for transdifferentiation. Alternatively, we hypothesize that embryonic stem cell genes can become activated in malignant gliomas and differentially regulate the subpopulation of cancer stem cells. This study examines the role of embryonic stem cell genes in transformed cells, particularly cancer stem cells. These studies explore has the following objectives: Aim 1: Isolate different sub-populations of cells from tumors and characterize cells with stem cell-like properties. Aim 2: Characterize the expression of embryonic stem cell markers in the sub-population of cancer stem cells. Aim 3: Examine the effects of histone deacetylase inhibitors at inhibiting the growth and reducing the expression of stem cell markers. Our research has demonstrated the potential of the embryonic transcription factor, nanog, at inducing dedifferentiation of human bone marrow mesenchymal stem cells and allowing their recommitment to a neural lineage. Specifically, we used viral and non-viral vectors to induce expression of NANOG, which produced an embryonic stem cell-like morphology in transduced cells. We characterized these cells using real-time PCR and immunohistochemical staining and find an up-regulation of genes responsible for pluripotency and self-renewal. Embryonic stem cell markers including Sox2, Oct4 and TERT were up-regulated following delivery of nanog. The role of nanog in the expression of these markers was further demonstrated in our induced-differentiation method where we transfected embryonic stem cell-like cells, that have been transduced with nanog flanked by two loxP sites, with a vector containing Cre-recominase. We tested the ability of these nanog-transfected cells to undergo neural differentiation in vitro using a neural co-culture system or in vivo following intracranial transplantation. Our next study characterized patient-derived glioblastoma cancer stem cells. We found that cells isolated from serum-free stem cell cultures were enriched for stem cell markers and were more proliferative than the bulk population of cells grown in convention serum-supplemented media. These cancer stem cells expressed embryonic stem cell markers NANOG and OCT4 whereas non-tumor-derived neural stem cells do not. Moreover, the expression of stem cell markers was correlated with enhanced proliferation and could serve as a measure of drug effectiveness. We tested two different histone deacetylase inhibitors, trichostatin A and valproic acid, and found that both inhibited proliferation and significantly reduced expression of stem cell markers in our cancer stem cell lines. These data demonstrate the potential use of stem cell genes as therapeutic markers and supports the hypothesis that cancer stem cells are a major contributor to brain tumor malignancy.

The recent expansion in chemical and manufacturing and innovation has led to a large influx of chemicals to the market, and subsequent release into the environment. Many of these new chemicals, as well as legacy chemicals are untested for their potential developmental toxicity, especially in early embryonic stages. This creates a need for a timely and cost-effective method for screening these chemicals. Furthermore, advances in in vitro methods and human pluripotent cell culturing techniques have revealed some weaknesses in our current animal model-based paradigms. Here we tested an in vitro model for developmental toxicity screening using human embryonic stem cells (hESCs) for environmental chemicals. In this study, hESCs were exposed to three known developmental toxicants that are prevalent in the environment, bisphenol A (BPA), perfluorooctane sulfonate (PFOS), or lead chloride (PbCl₂), at environmentally relevant concentrations of 0-2500 µg/L, 0-2275 µg/L, and 0-6200 µg/L respectively, for 6-days. hESCs were evaluated for dose responses on proliferation level by assaying cell viability, mitochondrial dehydrogenase activity (MDHA), cell confluency, and cell cycle distribution. Differentiation capability was assayed by induction of differentiation into ectoderm, mesoderm, and endoderm; hESCs and differentiated cells were then sequenced for their full transcriptome. Gene expression effects were analyzed by a single cell transcriptome sequencing and analysis of global DNA methylation. Proliferation and methylation effects were tested for all 3 chemicals, while differentiation and single cell sequencing was only tested on PbCl₂. Our results show hESCs were able to identify known and novel proliferation effects of BPA, PFOS, and PbCl₂, reflect differentiation level effects of PbCl₂, and elucidate molecular level drivers of these toxic effects. We also showed that hESCs responded to developmental toxicants at lower doses than in vivo models. In conclusion, our hESC-based model could act as an effective developmental toxicity screening tool for pre- peri- and post-implantation stages of embryo development.

Cell type-specific alternative splicing (AS) of pre-mRNA regulated by RNA-binding proteins (RBPs) is widespread, but particularly prominent in the brain, driving gene isoform differences between a diverse range of neuron types. While several AS programs have been shown to be critical to the function of particular neuron types, previous studies have usually been limited to one or a few RBPs and cell types, resulting in a piecemeal understanding of these regulatory patterns. Towards a comprehensive view of the neuron type-specific AS regulatory landscape, we apply current computational and experimental methods to survey neuronal AS, infer its regulation by hundreds of RBPs, and experimentally validate regulatory predictions. In Chapter 1, we examine AS in 133 transcriptomic cell types of mouse cortical neurons defined by single-cell RNA sequencing (scRNA-seq) and define neuron type-specific exons and some of their likely regulators. In Chapter 2, we leverage the rich transcriptomic dynamics of the cortical neuron dataset to systematically infer splicing regulatory network and predict RBP activity on the cell type level. We use the information theory-based method ARACNe to reverse engineer RBP-target regulatory networks and VIPER to infer differential RBP activity across neuron types in a workflow we call Master Regulator analysis of Alternative Splicing (MR-AS). RBP regulons predicted by MR-AS are consistent with high-confidence lists of RBP targets and are supported by motif and CLIP read distribution analyses. Estimation of cell type-specific RBP activity using the predicted regulons shows the expected decreases in RBP KO samples. Chapter 3 focuses on two neuron type-specific AS regulatory programs as case studies, which we validate in vitro using embryonic stem cell (ESC)-derived neuron types. Elavl2 was predicted to drive neurons towards an MGE interneuron-specific AS profile. Elavl2 knockout in ESC-derived MGE interneurons causes modulation of exon inclusion consistent with the predicted regulation of MGE interneuron AS, shifting their splicing profiles towards those of CGE interneurons. We also identified a module of exons that show consistent AS between long- and short-projection neurons across multiple neuronal classes, which are shifted in the expected direction when ESC-derived interneurons are transcriptionally reprogrammed to reflect a long-axon globus pallidus-like neuronal identity. In Chapter 4, we use the RBP regulons to predict RBP activity on a single-cell level and examine its variability, leading us to identify both neuron type-specific AS programs and a neuron type-orthogonal gradient of activity (NTOG). Exons associated with responses to neuronal depolarization and long-term potentiation show a gradient of inclusion across the NTOG, suggesting it may reflect differential activation of activity-dependent AS programs of the assayed neurons. Together, the results described in this thesis demonstrate the validity and broad utility of the inferred AS regulatory networks as a resource for elucidating RBP splicing regulation differences and their functional impact across neuron types.

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Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases and the third leading cause of death in the US, estimated to increase in magnitude in the future. Current treatment approaches are palliative in nature and restricted to controlling symptoms and reducing the risk of complications. Lung transplantation is an option for certain patients, but this option is limited by the shortage of donor organs and the possibility of rejection and the need for life-long immune-suppression. Therefore, current studies focus on cell based therapies for lung repair and regeneration. In addressing the issue of cell sourcing for such approaches, I tested the hypothesis that the efficiency of directed pulmonary differentiation of mouse embryonic stem cells (mESC) can be enhanced by employing certain micro-environmental cues, found in the developing lung. Such micro-environmental cues will provide appropriate physicochemical signals at the right time during the embryonic development and thus modulate fate decisions of progenitor cells during tissue assembly and maturation. In this study, I explored the effects of matrix stiffness on cell fate decisions in mESC, first into definitive endoderm and then into lung alveolar epithelial cells. I engineered bio-activated polyacrylamide (PA) gels with varying elastic moduli, mimicking those of physiologic tissues, and covalently modified the surfaces with fibronectin to provide optimal stem cell adhesion. My studies demonstrated, for the first time, a biphasic stiffness-dependent enhancement of endodermal differentiation of mESCs, with an optimum at ~ 20 kPa. This effect was qualitatively similar in three different mESC lines. By contrast, increasing matrix stiffness favored mESC differentiation towards a mesodermal phenotype. The enhanced endodermal differentiation of mESCs was abolished in the presence of a specific inhibitor of ROCK, suggesting that this process is mediated through cytoskeletal signaling. The subsequent differentiation of mESC-derived endodermal cells towards pulmonary epithelial cells was no longer dependent on the stiffness of the matrix. In this dissertation I demonstrate for the first time the feasibility of utilizing developmental and physiological / physicochemical cues, such as matrix stiffness, to selectively modulate and enhance mESC differentiation towards endodermal and pulmonary lineages. The impact of the results will be relevant for optimizing cell-based lung therapies and for effectively engineering lung and other endoderm-derived organs. / Bioengineering

Trimethylamine N-oxide (TMAO) is a metabolite derived from dietary choline, betaine, and carnitine via intestinal microbiota metabolism. In several recent studies, TMAO has been shown to directly induce inflammation and reactive oxygen species (ROS) generation in numerous cell types, resulting in cell dysfunction. However, whether TMAO will impact stem cell properties remains unknown. This project aims to explore the potential impact of TMAO on mouse embryonic stem cells (mESCs), which serve as an in vitro model of the early embryo and of other potent stem cell types. Briefly, mESCs were cultured in the absence (0mM) or presence of TMAO under two different sets of treatment conditions: long-term (21 days), low-dose (20µM, 200µM, and 1000µM) treatment or short-term (5 days), high-dose (5mM, 10mM, 15mM) treatment. Under these treatment conditions, mESC viability, proliferation, and stemness were analyzed. mESC properties were not negatively impacted under long-term, low-dose TMAO treatment; however, short-term, high-dose treatment resulted in significant reduction of mESC viability and proliferation. Additionally, mESC stemness was significantly reduced when high-dose treatment was extended to 21 days. To investigate an underlying cause for TMAO-induced loss in mESC stemness, metabolic activity of the mESCs under short-term, high-dose TMAO treatment was measured with a Seahorse XFe96 Analyzer. TMAO treatment significantly decreased the rate of glycolysis, and it increased the rate of compensatory glycolysis upon inhibition of oxidative phosphorylation (OxPHOS). It also significantly increased the rate of OxPHOS, maximal respiratory capacity, and respiratory reserve. These findings indicate that TMAO induced a metabolic switch of mESCs from high glycolytic activity to greater OxPHOS activity to promote mESC differentiation. Additionally, TMAO resulted in increased proton leak, indicating increased oxidative stress, and elucidating a potential underlying mechanism for TMAO-induced loss in mESC stemness. Altogether, these findings indicate that TMAO decreases stem cell potency potentially via modulation of metabolic activity. / Master of Science / Trimethylamine N-oxide (TMAO) is a metabolite that is produced by the bacteria in the gut after the consumption of specific dietary ingredients (e.g., choline, carnitine, betaine). These ingredients are commonly found in meat and dairy products, and thus make up a large part of the average American diet. Recently, it was discovered that high TMAO levels in the bloodstream put people at an increased risk for heart disease, neurodegenerative diseases (e.g., Alzheimer's Disease), diabetes, stroke, and chronic kidney disease. At the cellular level, there is evidence that TMAO increases inflammation and the production of oxygen radicals, which causes cells to lose their function and promotes the onset of disease. TMAO has been well studied in adult cell types; however, no one has investigated whether TMAO will impact cells of the early embryo. This project aims to explore the impact of TMAO on mouse embryonic stem cells (mESCs), which are cells that represent the early stage of embryonic development and are critical for proper development of the final offspring. In addition, mESCs may also help to provide insight into how TMAO impacts other stem cell types, some of which are present throughout the entire human lifespan and play an important role in the body's ability to repair itself and maintain overall health. My project demonstrated that TMAO does not impact the overall health of mESCs under normal conditions, which signifies that TMAO generated by a pregnant mother may not directly impact the early embryonic stage of development. Further studies should be conducted to determine the potential impact of TMAO on late stages of embryonic and fetal development. Next, to simulate diseased conditions, the mESCs were treated with extremely high concentrations of TMAO in order to determine what concentration of TMAO will negatively impact these cells. It was found that at 5mM TMAO, mESCs begin to lose their basic properties and become dysfunctional. They are impaired in their viability, growth, ability to become other cell types, and in their metabolic activity. These mESC properties are shared with several types of adult stem cells, and therefore, these findings help to provide insight into how TMAO may impact stem cells found in the adult body which are exposed to a lifetime of high TMAO levels. In the future, we would like to further explore the impact of TMAO on mESCs at the molecular level as well as examine the direct impact of TMAO on other stem cell types.

Previous studies report oxidative stress induced apoptosis and necrosis occur following myocardial infarction. Effects of conditioned medium (CM) prepared from mouse embryonic stem (ES) cells on H2O2 induced apoptosis and necrosis in different cells types is under investigation. Additionally, effects of CM from ES cells primed with TGF-b2 on stress-induced apoptosis and necrosis in H9c2 cells have not been determined. In this study, H20i induced apoptosis was confirmed by trypan blue staining, terminal deoxynucleotide transferase dUTP-mediated nick-end labeling (TUNEL), and apoptotic enzyme labeled immunosorhent assay (ELISA) whereas necrosis was determined by LDH assay. Next, we generated CM from ES cells primed with and without TGF-b2 and determined their effects on H2O2 induced apoptosis and necrosis in H9c2 cells. Apoptosis and necrosis was significantly (P < 0.05) reduced with ES-CM compared with cell culture control. Next, our data showed TGF-B2 primed ES-CM further reduced cell death compared with ES-CM, suggesting increased amounts of cytoprotective released factors from mouse ES cells following TGF-B2 treatment. Furthermore, the treatment of H9c2 cells with TGF-b2 alone did not significantly (P < 0.05) reduce apoptotic cell death. In conclusion, we suggest that factors released from ES cells with and without TGF-B2 treatment contain anti-apoptotic and anti-necrotic factors that inhibit H2O2 induced cell death. Further studies are needed to determine potential additional benefits of the · released factors from TGF-B2 primed ES cells.

Thesis (MPhil)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The progress that has been made over the last decade in the field of medical technology, has made it possible to treat medical conditions today, that was considered incurable before. In the medical field there are three milestones in particular which has led to important new discoveries. These are the charting of the human genome, the development of cloning techniques, and the discovery that stem cells could be used in the treatment of a great number of illnesses, as well as the regeneration of sick or damaged tissue. The development of these therapies are, however, morally controversial. The main reason for this is the fact that in most cases, these techniques involve research on, or the use of, embryonic cells. The reason why many people believe that it is morally problematic to use embryo tissue for research and/or therapeutic purposes, is because embryonic cells have the potential to develop into fully independent human persons. It is, however, also this characteristic of these cells which makes them so suitable for use in stem cell therapies: Because certain types of stem cells, especially stem cells that are found in embryos in an early stage of development, have not yet differentiated into specific cell types, they can be used to repair any cell type in a person with a compatible tissue type. The reason for this is that undifferentiated stem cells have the potential to change into any cell type found in the human body. The question that arises when a decision about the moral acceptability of these therapies has to be made is whether one can say that an existing person who happens to be ill, has a higher moral standing than an embryo. The only way in which the use of embryo tissue could be morally justified, would thus be if it could be proved that the moral standing of an embryo is not equal to the moral standing of a person. The other important consideration that has to be taken into account when the moral acceptability of these therapies has to be taken into account is the fact that it is possible to harvest stem cells from a number of sources. Some of these sources of stem cells are less controversial than others. The discussion of the moral problems arising from the use of embryo tissue for therapeutic purposes, would thus, in this thesis, focus to a large extent on determining what the moral status of the embryo might be. The different positions with respect to the moral standing of the embryo will be discussed in the light of arguments for, as well as against the use of embryo tissue for therapeutic purposes. An explanation will also be given of therapies for which the use of embryo tissue might be needed, at present as well as in the future. The potential gains for people suffering from certain conditions, could possibly serve as a justification for destroying embryos for therapeutic uses. The main purpose of this thesis is to be able to give morally justifiable reasons for the therapeutic use of embryo tissue. The specific conditions that would have to be met to make these therapies morally justifiable will also be explained. / AFRIKAANSE OPSOMMING: Die vordering wat die afgelope dekade gemaak is ten opsigte van mediese tegnologie, het tot gevolg gehad dat dit vandag moontlik is om siektetoestande te behandel wat voorheen as ongeneeslik beskou is. Daar is veral drie belangrike mylpale wat in die mediese veld tot belangrike nuwe ontdekkings gelei het, naamlik die kartering van die menslike genoom, die ontwikkeling van kloningstegnieke, en die ontdekking dat stamselle gebruik kan word vir die behandeling van 'n groot aantal siektetoestande, asook die regenereering van siek of beskadigde weefsel. Die ontwikkeling van hierdie terapieë is egter moreel kontroversieel. Die rede hiervoor is dat hierdie tegnieke in die meeste gevalle navorsing op, of die gebruik van embrionale selle behels. Die rede waarom baie mense van mening is dat dit moreel problematies is om embrionale weefsel vir navorsing enlofterapeutiese doeleindes te gebruik, is omdat embrionale selle die potensiaal het om te ontwikkel tot volwaardige persone. Dit is egter ook hierdie eienskap van hierdie selle wat hulle so geskik maak vir terapeutiese doeleindes: Omdat sekere tipes stamselle, veral stamselle wat verkry word van embrio's wat in 'n vroeë stadium van ontwikkeling verkeer, nog nie gedifferensieer is wat seltipe betrefnie, kan hulle gebruik word om enige seltipe in die liggaam van 'n persoon met 'n verenigbare weefseltipe te herstel. Die rede hiervoor is dat ongedifferensieerde stamselle die potensiaal het om in enige seltipe wat in die menslike liggaam voorkom, te verander. Die vraag wat ontstaan wanneer daar besluit moet word oor die morele aanvaarbaarheid van hierdie terapieë, is of daar gesê kan word dat 'n reeds bestaande persoon wat siek is, 'n hoër morele status sou hê as 'n embrio. Die enigste manier waarop die gebruik van embrionale selle moreel regverdigbaar sou wees, sou dus wees indien daar bewys kan word dat die morele status van 'n embrio nie gelykstaande is aan die morele status van 'n persoon nie. Die ander belangrike oorweging wat in ag geneem moet word wanneer die morele aanvaarbaarheid van hierdie terapieë beoordeel moet word, is dat dit moontlik is om stamselle te verkry uit 'n verskeidenheid bronne. Sommige van hierdie bronne van stamselle is moreel minder kontroversieel as ander. Die bespreking van die morele problematiek rondom die gebruik van embrionale weefsel VIr terapeutiese doeleindes in hierdie tesis, sal dus tot 'n groot mate fokus op die bepaling van die morele status van die embrio. Die verskillende standpunte oor die morele status van die embrio sal bespreek word in die lig van argumente vir, sowel as teen die gebruik van embrionale weefsel vir terapeutiese doeleindes. Daar salook 'n verduideliking gegee word van watter tipe terapieë waarvoor die gebruik van embrionale weefsel nodig sou wees, tans en in die toekoms moontlik sou wees. Die potensiële baat wat siek persone uit hierdie terapieë sou kon vind, sou moontlik ook as 'n regverdiging vir die vernietiging van embrio's vir terapeutiese doeleindes kon dien. Die uiteindelike doel van hierdie tesis is om moreel regverdigbare redes te kan gee vir die terapeutiese gebruik van embrionale weefsel. Die spesifieke voorwaardes wat nagekom sou moes word om hierdie terapieë moreel regverdigbaar te maak, salook verduidelik word.