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.

Enhancer RNAs (eRNAs) are a family of long noncoding RNAs (lncRNAs) transcribed from enhancer sites by RNA polymerase II (RNAPII) as part of the process of enhancer activation. Normally, eRNAs are typically processed by the Integrator complex and incorporated into the enhancer looping machinery formed by the Mediator and Cohesin complexes. However, some eRNAs escape this processing step and are instead transcribed to produce longer, polyadenylated RNAs. Poly(A)+ eRNAs are usually targeted for exosomal degradation by the Mtr4-containing PAXT complex, but under some conditions can be exported to the cytoplasm. I n this latter option, as shown in Chapter 2 of this thesis, eRNAs that contain ORFs, of which there are a surprising number, are capable of being translated to produce functional proteins. eRNAs can gain ORFs through the process of de-novo gene birth, resulting in novel genes at sites capable of fulfilling the roles of both canonical coding genes and enhancers.This dissertation is divided into three main sections, and an additional fourth section outlining future research directions. In the first section, I review the current research on eRNAs and other lncRNAs including their origins, processing, and functions. I also review current research on proteins encoded by canonically noncoding RNAs, and the process and implications of de-novo gene birth as it relates to the aforementioned topics. In the second section, we aimed to identify translating eRNAs in the human genome using ribosome profiling compared to a database of transcribed enhancers. Using these results, we selected ten large eRNA ORFs to investigate the functions of the proteins they encode, including their subcellular localization and protein interactomes. Finally, we investigated the homologs of these eRNA ORF sequences in other species to determine their level of evolutionary conservation compared to that observed in canonical protein-coding genes. Our findings in the second section provide evidence for novel, highly-basic, arginine-rich proteins encoded by eRNAs and capable of interacting with DNA and RNA, either directly or through interactions with other associated proteins. We also present evidence that the ORFs encoding these proteins appeared relatively recently in human evolution, with most being primate-specific and exhibiting mutation rates associated with purifying selection of coding sequences across their homologs in great apes. In the third section, we present additional results from analysis of published proteomics and RNA-seq results that provide evidence for expression of ORF-containing eRNAs in differentiating stem cells. These results were also confirmed using qRT-PCR of whole-cell RNA samples isolated from these differentiating cells. These results show expression of several eRNAs identified in the second section during the early stages of human embryonic stem cell differentiation into the three germ layers and subsequent mature cell types. These results are also supported by a decrease in Mtr4 protein levels also detected in proteomics results from the same differentiations. These results indicate that expression of ORF-containing eRNAs and the Mtr4-depleted conditions leading to it are present in stem cells during differentiation, and stem cell differentiation is a promising subject of future research on the roles of eRNA-encoded proteins in normal cellular function.