Transgenic manipulation of exogenous and endogenous gene expression in human embryonic stem cells (hESCs) is a powerful approach to decipher the genetic pathways dictating their developmental fate. Presently used genetic tools face limitations including leakiness in inducibility of expression, epigenetic silencing in long-term cell culture, low genomic integration efficiencies, small genetic cargo limit and lack of high-throughput cloning capabilities. To overcome these limitations, I have constructed R4-Integrase and piggyBac transposon genetic vector systems for stable transgene overexpression and knockdown in hESCs. Preliminary functional testing of the piggyBac vector system in HEK 293T and hESCs demonstrated vector inducibility as well as successful overexpression and knockdown of pluripotency factor OCT4. Concurrently, a cost-effective and high efficiency method for chemical transfection of hESCs was developed. Exogenous overexpression and knockdown of transcription factors in hESCs will aid in the elucidation of gene regulatory networks controlling pluripotency and developmental fate.

We used mouse embryonic stem cells (ESCs) as a model to study the mechanisms that regulate stem cell fate. Using gene expression analysis during a time course of differentiation, we identified 281 candidate regulators of ESC fate. To integrate these candidate regulators into the known ESC transcriptional network, we incorporated promoter occupancy data for OCT4, NANOG and SOX2. We used shRNA knockdown studies followed by a high-content fluorescence imaging assay to test the requirement of our predicted regulators in maintaining self-renewal. We further integrated promoter occupancy data for Polycomb group (PcG) proteins, EED and PHC1 to identify 43 transcriptional networks in which we predict that OCT4 and NANOG co-operate with EED and PHC1 to influence the expression of multiple developmental regulators. Next, we turned our focus to the PcG protein PCL2 which we identified as being bound by both OCT4 and NANOG and down-regulated during differentiation. PcG proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs. Using multiple biochemical strategies, we demonstrated that PCL2 associates with Polycomb Repressive Complex 2 (PRC2) in mouse ESCs, a complex that exerts its effect on gene expression through H3K27me3. Although PCL2 was not required for global histone methylation, it was required at specific target regions to maintain proper levels of H3K27me3. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics and defects in differentiation. Integration of global gene expression and promoter occupancy analyses allowed us to identify PCL2 and PRC2 transcriptional targets and draft regulatory networks. We describe the role of PCL2 in both modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation.

Transgenic manipulation of exogenous and endogenous gene expression in human embryonic stem cells (hESCs) is a powerful approach to decipher the genetic pathways dictating their developmental fate. Presently used genetic tools face limitations including leakiness in inducibility of expression, epigenetic silencing in long-term cell culture, low genomic integration efficiencies, small genetic cargo limit and lack of high-throughput cloning capabilities. To overcome these limitations, I have constructed R4-Integrase and piggyBac transposon genetic vector systems for stable transgene overexpression and knockdown in hESCs. Preliminary functional testing of the piggyBac vector system in HEK 293T and hESCs demonstrated vector inducibility as well as successful overexpression and knockdown of pluripotency factor OCT4. Concurrently, a cost-effective and high efficiency method for chemical transfection of hESCs was developed. Exogenous overexpression and knockdown of transcription factors in hESCs will aid in the elucidation of gene regulatory networks controlling pluripotency and developmental fate.

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

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Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

Embryonic stem cells (ESCs) possess certain immunomodulatory properties; the defined components in ESCs, however, are largely unknown. Based on proteomic database, I report here that milk fat globule epidermal growth factor 8 (MFG-E8) is a key component in ESCs to suppress T cell activation and regulate T cell polarization. MFG-E8 is enriched in undifferentiated ESCs while diminishing in differentiated ESCs. Neutralizing ESC-derived MFG-E8 substantially ameliorates the suppressive effects of ESCs on T cell activation and proliferation. Additionally, MFG-E8 in ESCs is capable of up-regulating T regulatory cells. I further prove that MFG-E8 suppresses T cell activation and regulates T cell polarization through inhibiting PKCθ phosphorylation. In vivo teratoma formation assay reveals an increase in ESC engraftments across allogeneic barriers with less immunologic rejection by up-regulation of MFG-E8 expression in ESCs, further validating the immunosuppressive properties of MFG-E8. Identifying an important immunoregulatory component in ESCs will greatly facilitate stem cell-based therapies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22829号 / 医博第4668号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 遊佐 宏介, 教授 斎藤 通紀, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Early embryo development is one of the most sensitive stages to environmental chemicals during the whole life. Prenatal exposures to many environmental chemicals have been shown to impact fetal development and be associated with adverse health outcomes in later life stages. However, the effects of chemical mixture exposure on developing embryos, especially in early developmental stages, have yet to be fully studied. To fulfill this research gap, my thesis was divided into three data chapters and mainly aimed at investigating the effects of a chemical mixture on human early-stage embryo development. In Chapter 2, I chose methylmercury (MeHg) as the main study toxicant to establish procedures for embryotoxicity testing using human embryonic stem cells (hESCs). I then characterized the effects of low doses of MeHg on this stem cell model by screening a set of cell fate decision-related makers and found MeHg is embryotoxic, which is consistent with epidemiological and in vivo findings. In Chapter 3, I studied the embryotoxicity of a chemical mixture that consists of 23 individual environmental chemicals (including MeHg) detected from the maternal blood samples of pregnant women in Nunavik, labelled as Nunavik Contaminant Mixture (NCM), using the same cell model. The effects of NCM exposure on hESCs were compared to MeHg exposure alone. NCM exposure adversely affected cell viability and adhesion, induced apoptosis, disrupted the cell cycle, altered the expression of cytoskeleton and autophagy proteins, and changed the levels of lineage marker gene and protein expressions in a dose-dependent manner. Some distinct effects on hESCs between NCM exposure and MeHg alone exposure were noticed, and the potential interactions among the chemical components within a chemical mixture were indicated. In Chapter 4, I studied the effects of MeHg exposure during the formation of definitive endoderm (DE) cells from hESCs and compared that to MeHg's effects on undifferentiated hESCs. I found that cell specification towards endoderm could be affected by MeHg exposure, mainly through disrupting calcium homeostasis and over-generating reactive oxygen species, leading to increased ribosome biogenesis and protein synthesis. Moreover, MeHg effects are state-dependent; MeHg enhances pluripotency in undifferentiated hESCs, but it promotes differentiation during DE induction. Taken together, this thesis verifies the value of hESCs in testing the embryotoxicity and developmental toxicity of environmental chemicals, enriches the understanding of the toxicity of MeHg and NCM, emphasizes the necessity of evaluating the effects of chemical mixtures and provides new directions in studying environmental chemical toxicity using stem cells. Findings from my thesis could hopefully contribute to predicting the potential effects of prenatal environmental chemical exposures and aid in developing evidence-based public health policy.