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.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

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.

miRNAs are short, non-coding RNA molecules that regulate gene expression posttranscriptionally through inhibition of translation and/or mRNA degradation. Mammalian development is a complex series of developmental transitions, which relies on accurate spatial and temporal regulation of gene expression and we are interested in the role that miRNAs may play in these developmental transitions. An initial objective was to establish which, if any, miRNAs were dynamically regulated in a cell model of an early developmental transition, and to establish whether differential expression of any particular miRNA played a functional role in this developmental process. Having established a role for specific miRNAs, further objectives were to assess the reliability of current miRNA-mRNA target identification procedures and to assess the general role of miRNAs in cellular differentiation. In order to explore the roles of miRNAs during an early developmental transition, an embryonic stem (ES) cell model of trophectoderm differentiation was used. In this model system the expression of the key ES cell regulatory gene, Oct4, can be conditionally repressed, which induces the ES cells to differentiate down the trophectoderm lineage. The expression of microRNAs was profiled in this model system by cloning and sequencing of small RNAs. This approach identified miRNAs that were dynamically regulated during differentiation. The expression patterns of differentially regulated miRNAs were confirmed by miRNA northern analysis. The miRNA profiling data showed that mmu-miR-294 and mmu-mir-295 are expressed at similar levels in ES cells and differentiated cells, which disagrees with previous reports that these miRNAs are ES cell specific. Several of the miRNAs with higher expression levels in differentiated cells are encoded within a placental-enriched polycomb group gene, Sfmbt2, suggesting an important role for these miRNAs in extraembryonic development. One of the miRNAs that was expressed at higher levels in ES cells than in differentiated cells, mmu-miR-92a, was shown to play a role in regulation of cell proliferation. Three current methods of identifying miRNA targets were assessed. A sequencebased method using the web-based utility miRecords, which amalgamates results from numerous target prediction databases, was used to generate lists of potential targets of the Sfmbt2 miRNA cluster and of mmu-miR-92a. Amalgamating results from multiple target prediction programs may improve the likelihood that the predicted targets are real. Exemplifying this, the single mmu-miR-92a target that was predicted by six different target prediction programs had been previously experimentally verified. An experimental method of identifying direct miRNA targets, PAR-CLIP, was investigated but proved technically limiting for routine use in the laboratory. A proteome-based experimental method for identifying potential miRNA targets, called SILAC, was successfully used to identify proteins that were differentially expressed in the cell model of trophectoderm differentiation. Differential expression of two of these proteins, CTBP2 and CKB, was confirmed by western analysis. miRecords was then used to assess whether the differentially expressed proteins were likely to be targets of the differentially expressed miRNAs that had been identified in the miRNA profiling analysis. The general role of miRNAs in cell differentiation was investigated using a cell line that does not express miRNAs. This ES cell line is deficient for the miRNAprocessing enzyme DGCR8, which results in loss of expression of mature miRNAs in these cells. Compared to wild type ES cells, miRNA-deficient ES cells expressed normal levels of the ES cell marker genes Oct4 and Sox2 but elevated levels of Nanog. In contrast to wild type ES cells, miRNA-deficient ES cells did not upregulate the mesoderm marker gene Brachyury during embryoid body differentiation and showed reduced upregulation of the endoderm marker gene Gata6. These findings suggest that miRNAs are not required for maintenance of pluripotency, but are essential for proper ES cell differentiation. The results presented in this thesis show that miRNAs are dynamically expressed during a mammalian developmental transition and are involved in regulating early developmental processes. We believe that miRNAs act as an additional level of genetic regulation to ensure canalisation during embryonic development.

Instructive signals, whether external or internal, play critical roles in regulating the developmental potency or ability to self-renew of stem cells. External signals may range from secreted growth factors to extracellular matrix proteins found in the stem cell niche. Internal signals include activated signalling cascades and the eventual transcriptional mechanisms they initiate. In either fashion, stem cells are regulated in a complex temporal and context specific manner in order to maintain or maximise their unique characteristics. Previous experiments suggest that Wnt3a plays a role in maintaining the pluripotent state of mouse embryonic stem (mES) cells. However, in the absence of leukemia inhibitory factor (LIF), Wnt signalling is unable to maintain ES cells in the undifferentiated state. This implies that maintaining the pluripotent state of mES cells is not the primary function of canonical Wnt signalling. To further characterize the role of Wnt3a in pluripotency and lineage specification undifferentiated and differentiated mES cells were induced with Wnt3a. Wnt3a induced the formation of a metastable primitive endoderm state and upon subsequent differentiation, the induction of large quantities of visceral endoderm. Furthermore, we determined that the ability of Wnt3a to induce a metastable primitive endoderm state was mediated by the T-box transcription factor Tbx3. Our data demonstrates a novel role for Wnt3a in promoting the interconversion of undifferentiated mES cells into a pluripotent primitive endoderm state. Aging of skeletal muscle tissue is accompanied by fibrosis, atrophy and remodeling all of which negatively affect muscle performance. Whether this reduction in skeletal muscle competency is directly attributed to a resident adult stem cell population called satellite cells remains largely unknown. Here, we undertook an investigation into how age affects the transcriptional profile of satellite cells and their repopulating ability following transplantation. We determined that as satellite cells age, both their regenerative capacity and ability to colonize the satellite cell niche is reduced. Additionally, we identified satellite cell specific transcriptional profiles that differed with respect to age. Therefore, we conclude that intrinsic factors are an important determinant of satellite cell regenerative capacity during the aging process.

Embryonic stem cells (ESCs) possess hypo-immunogenic properties and have the capacity to modulate allogeneic immune response. ESCs have been shown to reduce immune activation in response to third party antigen presenting cells (APCs) in vitro and have the capacity to promote allograft survival in vivo. Clinical use of live ESCs to treat immunological disorders, however, risks teratoma or ectopic tissue formation. Accordingly, the way lab is studying the immune modulatory potentials of ESC-derived factors and recently, found that dendritic cells (DCs) treated with human ESC extracts are poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. In the present study, I found that ESC-derived extracts directly inhibit T cell proliferation and suppress their activation without inducing cell death. Furthermore, ESC extracts are able to suppress Th1 polarization while increasing the numbers of Foxp3+ CD4+ CD25+ regulatory T cells. Moreover, I found that a protein called Milk fat globule-EGF factor 8 (MFG-E8) appears to be highly expressed in ESCs. Importantly, neutralizing MFG-E8 substantially abrogated the immune suppressive effects of ESC extracts on T cell activation. These findings lead to future studies to further define specific immunomodulatory factors derived from ESCs for potential applications.

SirT1 is a class III histone deacetylase that has beneficial roles in various diseases related to aging such as cancer, diabetes and neurodegenerative disease. Resveratrol is a natural compound that mimics most of the beneficial effects attributed to SirT1. Resveratrol has toxicity towards cancer cells and has been reported to be a direct activator of SirT1. Interestingly, SirT1 over-expression has also been reported to be toxic. We set out to determine if resveratrol toxicity is mediated through activation of SirT1. We have assessed resveratrol toxicity in embryonic stem cells and mouse embryonic fibroblast (MEFs) across different SirT1 genotypes. Our data indicates that SirT1 is not implicated in resveratrol toxicity in either normal or transformed MEFs. Thus, resveratrol toxicity does not appear to be mediated by SirT1.

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.