During mammalian development a small number of pluripotent cells proliferate and differentiate to give rise to all the mature cell types of the organism. Among the earliest differentiation events is the process of gastrulation, in which pluripotent primitive ectoderm cells form the three germ lineages, mesoderm, ectoderm and endoderm under the control of complex signalling and environmental cues. This process can be modelled using embryonic stem cells, which have proven to respond to embryologically relevant signals during in vitro differentiation and promise to uncover additional insights into the process of germ lineage specification. This thesis describes the differentiation of mouse ES cells to committed cell types via a second intermediate population of pluripotent cells termed Early Primitive Ectoderm-Like (EPL) cells. The similarity of EPL cells to primitive ectoderm and the rapid acquisition of lineage specific markers and loss of pluripotent characteristics upon differentiation of EPL cells suggest they are an excellent model for the cells in the embryo that undergo germ lineage commitment. EPL cells can be differentiated as EPLEBs, which are highly enriched in mesodermal cell types and contain essentially no ectodermal derivatives and no visceral endoderm. Here it is shown that EPLEBs can be generated from EPL cells grown either adherently or in suspension culture provided the cells are reduced to a single cell suspension before reaggregation as EPLEBs. Since EPLEBs are a rich source of mesoderm and contain less non-mesodermal cell types than traditional ESEBs, they were assayed for definitive blood formation, however none was detected. Alternately, EPL cells can be differentiated in the presence of MEDII in aggregates termed EBMs, which are restricted to ectodermal cell fates. Here it is demonstrated that the switch from mesodermal to ectodermal differentiation observed in ELPEBs and EBMs relies on two variables; a mesoderm suppressing activity within MEDII and the pro-mesodermal activity of cell dissociation as undertaken during EPLEB formation. Evidence has been presented that interventions that modulate the epithelial identity of EPL cells are capable of influencing subsequent differentiation such that protection of the epithelial cell state favours ectoderm while disruption favours mesoderm. Staurosporine (SSP) is a kinase inhibitor that has been shown to induce an epithelial to mesenchymal transition in chick neural tube. Here it was added to EPL cells with the result that mesodermal differentiation was enhanced at the expense of ectoderm. DAPT is a potent inhibitor of ƴ-secretase, which cleaves a number of protein targets including the adherens junction component E-cadherin. Addition of DAPT to differentiating EPL cells has the opposite effect to SSP, with an increase in ectodermal differentiation at the expense of medoderm. It is proposed that DAPT is acting by preventing E-cadherin cleavage and thus stabilising the epithelial state. Modulation of epithelial contacts between pluripotent cells represents a novel way to control lineage induction and as such the incorporation of these findings into methodologies for directed differentiation in defined culture conditions is likely to provide improved outcomes in the production of desired cell types. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Indiana University-Purdue University Indianapolis (IUPUI) / SIRT1 (Sirtuin 1) is a founding member of a family of seven proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1-/- mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1-/- mouse embryonic stem (mES) cells in vitro, and hematopoietic progenitors in SIRT1+/+, SIRT1+/-, and SIRT1-/- mice. SIRT1-/- ES cells exhibited markedly delayed/immature formation of blast colony-forming cells (BL-CFCs). When individual blast colonies were analyzed for hematopoietic and endothelial potential, replated SIRT1-/- BL-CFC possessed limited hematopoietic potential, whereas endothelial potential was essentially unaltered. The ability of SIRT1-/- ES cells to form primitive erythroid progenitors was not only delayed but greatly decreased. Moreover, after differentiation of SIRT1-/- mES cells, there were also significant decreases in granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5, decreased β-H1 globin, β-major globin, and Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ES cell commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm the requirement for SIRT1 for normal development of hematopoietic progenitor cells, we assessed embryonic and adult hematopoiesis in SIRT1+/+, SIRT1+/- and SIRT1-/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythroid precursors compared to wild-type (WT) and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow hematopoietic progenitor cells (HPCs) in 5 week and 12 month old mice, which was especially notable at lower (5%) O2 tension. In addition these progenitors survived less well in vitro under conditions of delayed growth factor addition. Taken together, these results demonstrate that SIRT1 plays a role in ES cell hematopoietic differentiation and mouse hematopoiesis.

Master of Science / Department of Anatomy and Physiology / Mark L. Weiss / When mouse embryonic stem cells (ESCs) were grown on mitotically inactivated rat umbilical cord-derived stromal cells (RUCs) in the absence of leukemia inhibitory factor (LIF) and 2-mercaptoethanol (2-MCE), the ESCs showed alkaline phosphatase (AP) staining. ESCs cultured on RUCs maintain expression of the following pluripotency genes, Nanog, Sox2 and Oct4 and grow at a slower rate when compared with ESCs grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). Differences in gene expression for the markers of pluripotency Oct4, Sox2 and Nanog, AP staining and ESC growth rate were also observed after LIF and 2-MCE were removed from the co-cultures. Reverse transcriptase polymerase chain reaction (RT-PCR) suggested differences in Sox2 and Nanog mRNA expression, with both genes being expressed at higher levels in the ESCs cultured on RUCs in the absence of LIF/2-MCE as compared to ESCs cultured on MEFs. Semi-quantitative RT-PCR indicated that Nanog expression was higher when ESCs were grown on RUCs in the absence of LIF and 2-MCE as compared to MEFs in the same treatment conditions. Bisulfite-mediated methylation analysis of the Nanog proximal promoter suggested that the maintenance of Nanog gene expression found in ESCs grown on RUCs after culture for 96 hours in the absence of LIF/2-MCE may be due to prevention of methylation of the CpG dinucleotides in the Nanog proximal promoter as compared to ESCs grown on MEFs. Thus, RUCs may release factors into the medium that maintain the pluripotent state of mouse ESCs in the absence of LIF and 2-MCE.

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