Thesis (Ph.D.)--Purdue University, 2006. / Adviser: Martin Curd. Includes bibliographical references.

Embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo and are characterized by the ability to self-renew and differentiate into all cell types of an adult organism, as demonstrated by their transplantation into embryos in the mouse. Isolation of cells with similar properties from human embryos has permitted the study of human cell differentiation in vitro as might occur during development. As such, human ES cells may be useful to assess and predict the developmental toxicity of environmental compounds capable of epigenetic alterations of the genome and its expression. The first objective of my research was to validate the functional significance to maintenance of an undifferentiated human ES cell state of expressed genes whose epigenetic modification is conserved across diverse lines and/or likely to be deterministic of an embryo stem cell associated epigenetic state. The second goal was to determine the sensitivity and relationship of the expression of these genes to environmental factors known to perturb the epigenome, specifically subcytotoxic exposure to diverse organic and metallic compounds and the availability of atmospheric oxygen. siRNA-mediated knockdown of genes previously identified on the basis of the conserved methylation status of gene associated Cytosine-Guanine islands (i.e. GLIS2, HMGA1, PFDN5, TET1 and JMJD2C) and two related family members (TET2 & 3) resulted in induction of cell differentiation in two independent human ES cell lines (RH1 and H9). Differentiation was reflected by morphological changes, reduction or loss of pluripotency associated markers, qualitative and quantitative reduction in genomic 5-hmC and upregulation of diverse germinal lineage markers. Subcytotoxic exposure of the same human ES cell lines to diverse compounds known to alter the epigenome (i.e. 5-azacytidine, sodium arsenite, cadmium chloride and valproic acid) generally induced downregulation of the aforementioned genes, loss of genomic hydroxymethylation and differentiation when applied under normoxia (20% O2), the exception being valproic acid. The same treatment applied under hypoxia (0.5% O2), did not induce differentiation, with the exception of cadmium chloride. Hypoxia is a general feature of developing embryos prior to the establishment of a maternal/fetal placental interface and fetal cardiovasculature. The protective effect of hypoxia was associated with elevation of ROS, expression of the dioxygenases TET1 and JMJD2C, and genomic hydroxymethylation. This research has demonstrated that genes identified on the basis of a conserved pattern of epigenetic modification function in the maintenance of an undifferentiated human ES cell phenotype. Furthermore, a human ES cell-based toxicology test system has been developed with which one can assess the subcytotoxic effects of compounds known to disrupt the epigenome and affect development by assessing their impact on maintenance of an undifferentiated human ES cell state. This is reflected by alterations in pluripotency markers, epigenetically-defined biomarkers and changes in global 5-hmC levels and the expression of genes responsible for this epigenetic modification (TET1-3). The epigenetically-defined biomarkers of pluripotent human ES cell identity (GLIS2, HMGA1, PFDN5, JMJD2C and TET1) could serve as biomarkers for screenings of compounds at an epigenetic level as their expression has been shown to be altered upon compound exposure along with monitoring the expression of 5-hmC.

TGF-β/Activin/Nodal 信號通路調節了許多重要的細胞生物學過程,例如,細胞分裂,增殖,分化,遷移和衰老凋亡。此外,它也在胚胎髮育,損傷修復,腫瘤發生,組織纖維化,糖尿病發生及其免疫方面也扮演了重要的角色。TGF-β家族信號分子,包括TGF-β, Activin 和 Nodal,通過結合到它們各自的受體從而啟動它們,而啟動後的受體又可以通絡磷酸化作用進一步激活Smad2 和Smad3 蛋白,激活後的Smad2 和Smad3 蛋白可以和Smad4 蛋白形成複合體,一起從細胞膜轉移到細胞核內調節下游基因的表達。 / 在人的胚胎幹細胞中,TGF-β/Activin/Nodal signaling 做為最關鍵的信號分子,調節了人胚胎幹細胞的自我更新以及胚胎幹細胞多能性的維持。而在小鼠胚胎幹細胞中,該信號通路的功能並沒有清楚的研究。在本論文中,我們分離以及建立了Smad3 突變體的小鼠胚胎幹細胞系(Smad3-/-),該突變體細胞能夠維持正常小鼠胚胎幹細胞的形態,在自我增殖更新方面也沒有缺。此外,幹細胞多能性相關的標記基因以及組織標記基因表達水準也與野生型細胞非常相似,但是,在擬胚體的生長過程中,Smad3 被敲除後導致了組織發育相關的標記基因出現了差異性的表達。與野生型相比,中胚層標記基因（T 和GSC）的表達明顯受到了抑制。另外令人驚奇的是,將Smad3 基因敲除的胚胎幹細胞皮下注射裸鼠後長出了惡性的未完全成熟的畸胎瘤,而野生型的幹細胞則更傾向于長成成熟的良性畸胎瘤。進一步的分析發現,Smad3 功能性缺失後,細胞的增殖速率明顯增加了;紫外(UV)誘變後,相對於野生型,突變體細胞的抗凋亡能力也明顯增強了;並且在分化過程中,突變體細胞的遷移能力也要明顯強于野生型細胞。所有以上的細胞特徵可以解釋為什麼Smad3 基因敲除後會長出惡性的畸胎瘤。 / Microarray 分析結果發現,一個DNA 損傷修復基因Rif1,在Smad3 突變體細胞中呈現出了很高的上調,這個基因的上調現象已經發現是和侵蝕性腫瘤的發生是相關的,而且該基因的上調水準也與乳腺癌的浸潤程度是非常相關的。染色質免疫共沉澱和螢光素酶活性實驗進一步證實了Smad3 可以結合到Rif1 的啟動子領域從而直接抑制該基因的表達。這些實驗進一步說明了Smad3 可能通過下調Rif1 基因的表達,從而抑制了小鼠胚胎幹細胞長出惡性畸胎瘤的發生。 / 總之,我們建立了Smad3-/-基因敲除的小鼠胚胎幹細胞系,並且發現該突變體細胞傾向于長出惡性的畸胎瘤。我們推測,在正常的情況下,Smad3 正是通過抑制了DNA 損傷修復因數基因Rif1 的表達,從而阻止了惡性畸胎瘤的發生。這些研究的結果不僅開闊了我們對於惡性腫瘤發生的認識,而且為我們在幹細胞或誘導多能幹細胞治療應用中防止畸胎瘤的發生提供了新的思路和策略。 / TGF-β/Activin/Nodal signaling controls many important biological procedures in cells, such as cell division, proliferation, differentiation, migration and apoptosis in mammalian cells. It also plays a critical role in embryo development, wound healing, tumorigenesis, tissue fibrosis, diabetes and immunity. TGF-β superfamily ligands, such as TGF-β, Activin and Nodal bind to their respective ligand receptors and activate them, which in turn activate the receptor related SMAD proteins by phosphorylation, including Smad2 and Smad3. Once phosphorylated, they can cooperate with Smad4 and enter nucleus to bind promoter DNA sequence and regulate the target gene expression. / In human embryonic stem (ES) cells, TGF-β/Activin/Nodal signaling has been demonstrated to be the most critical pathways for ES cell self-renewal and maintenance of undifferentiated state. However, in mouse ES cells, its role is yet to be clearly exploited. In this study, we reported the derivation and establishment of mouse Smad3 knockout embryonic stem cell lines (Smad3-/- ES cells). Smad3-/- ES cells maintain normal ES cell morphology and express higher level of mouse ES cell markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1(SSEA1), and display no defect on self-renewal capacity. In addition, both of them show similar expression profiles of pluripotent and lineage marker genes compared to wild type ES cells. However, Smad3 ablation results in transient different expression of germ layer markers during embryoid body (EB) development. The expression of mesoderm lineage marker, like T and GSC, is significantly reduced in the EBs developed by Smad3-/- ES cells compared to EBs formed by wild type ES cells. More interestingly, to investigate the differentiation potential of Smad3-/- ES in vivo, we subcutaneously injected both wild type and Smad3-/- ES cells into nude mice, and observed that Smad3-/- ES cells are prone to grow malignant immature teratomas, while wild type ES cells develop normal mature teratomas. Further characterization of Smad3-/- ES cells demonstrates that depletion of Smad3 increases ES cell proliferation; Smad3-/- ES cells show higher capacity of the anti-apoptosis after UV irradiation and the migration potential of Smad3-/- ES cell differentiated cells is enhanced compared to wild type ES cells in the wound healing assay. Therefore, Smad3-/- ES cells exhibit enhanced malignancy, which may underlie their teratoma malignancy. / Microarray analysis shows that Rif1, a DNA repair factor is highly upregulated in Smad3-/- ES cells. Upregulation of DNA repair factor is found to be associated with invasive tumor. And the expression level of Rif1 is linked to the invasive degree of breast cancer at certain level. Chromatin immunoprecipitation (ChIP) assay and luciferase assay confirm that Smad3 binds to Rif1 promoter region and directly represses its expression; knockdown Rif1 in Smad3-/- ES cells rescues the expression level of Ccnd2 and migration potential to wild type ES cell level. Taken together, all these data suggests that Smad3 may suppress the malignancy of mouse embryonic stem cell formed teratoma through downregulating Rif1 expression in normal condition. / In summary, we reported the establishment of Smad3-/- ES cells and characterization of these cells. We discovered that Smad3-/- ES cells are prone to grow malignant teratomas compared to wild type ES cells. We hypothesized that Smad3 may suppress the malignancy of teratoma through repressing a DNA repair factor, Rif1. This information will not only broaden our general knowledge of malignant teratomas, but also help us to develop strategies to prevent malignant teratoma formation in ES/iPS cell therapy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Peng. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 149-181). / Abstracts also in Chinese.

Thesis (Ph. D.)--University of Chicago, Dept. of Pathology, August 2001. / Includes bibliographical references. Also available on the Internet.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Human embryonic stem cells (hESCs) are characterized by unlimited proliferation (self-renewal), capability to differentiate into derivatives of all three germ layers (pluripotency), and abbreviated cell cycle structure. Despite tremendous efforts in identification of important regulators, the complicated molecular mechanisms and essential effectors underlying the distinctive features of hESCs have not yet been fully elucidated. Forkhead box transcription factor M1 (FOXM1) has been demonstrated to be critical for the maintenance of pluripotency in mouse embryonic stem cells (mESCs) and mouse embryonal carcinoma cells (mECCs). The present study hypothesized that FOXM1 is important to the self-renewing capacity and pluripotency of hESCs. The objectives of this study were to characterize FOXM1 expression in undifferentiated and differentiating hESCs, and to study the effect of perturbing FOXM1 expression on pluripotency and proliferation. Undifferentiated VAL3 analyzed by bivariate flow cytometric analysis revealed that FOXM1 expression was regulated in a cell cycle phase-dependent manner, with expression level increased from G1 through S phase, and eventually reached peak levels in G2/M phase. To study the subcellular localization of FOXM1 with respect to cell cycle progression, VAL3 cells were synchronized by nocodazole-mediated cell cycle block, followed by immunocytochemical analysis. The result indicated that FOXM1 underwent nuclear translocation at late-S and early-G2 phase of the cell cycle. When VAL3 spontaneously differentiated as embryoid bodies (EBs), the mRNA expression of FOXM1 displayed profound fluctuation over the differentiation process. Retinoic acid (RA) treatment induced rapid differentiation of VAL3, yet differential expression pattern of FOXM1 was observed for cells grown in different culture media. FOXM1 mRNA expression persisted in differentiating VAL3 cultured in mTeSR. By contrast, RA-driven differentiation of VAL3 cultured in conditioned medium was accompanied by transient depletion and resurgence of FOXM1 protein expression. Differentiation of VAL3 driven by Definitive Endoderm kit did not alter FOXM1 expression, whereas induced differentiation by Bone Morphogenic Protein 4 (BMP4) led to repression of FOXM1. The functional role of FOXM1 in hESCs was investigated with the use of siRNA. Transient knockdown of FOXM1in VAL3 did not induce substantial repression of pluripotent marker (OCT4, SOX2, NANOG) expression nor significant morphological change of colonies, despite upregulation of early differentiation marker SSEA-1. Intriguingly, FOXM1 depletion led to altered cell cycle progression and delay in G2 phase progression, possibly attributed to the downregulation of Cyclin B1 and Cdc25B. Also FOXM1 knockdown impaired VAL3 proliferation, yet no prominent defect in mitosis was observed. In conclusion, the present study reported for the first time the expression and functions of FOXM1 in undifferentiated hESCs. Upon differentiation, FOXM1 expression varied in cells committing to different lineages. Depletion of FOXM1 did not interfere with hESCs pluripotency, but hindered cell cycle progression and cell proliferation, suggesting that FOXM1 is mainly involved in promoting rapid proliferation of hESCs. The functional role and regulatory mechanics of FOXM1 in hESCs cell cycle control and differentiation warrant further investigation. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of 129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 or Day-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues) showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior.

No description available.

Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of 129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 or Day-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues) showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior.

Thesis (M. S.)--Electrical and Computer Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Robert Butera; Committee Member: Pamela Bhatti; Committee Member: Todd McDevitt