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    Regulation of embryonic stem cell pluripotency and differentiation by notch signaling

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    Name:
    Noggle_Scott_PhD_2004.pdf
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    Authors
    Noggle, Scott A.
    Issue Date
    2004-05
    URI

    http://hdl.handle.net/10675.2/623273
    
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    Abstract
    Mammalian development prior to implantation is involved with establishing the support tissues needed for.interaction with the maternal blood stream. Molecular studies of these processes are in early stages. Embryonic stem cells (ESCs) derived from the inner cell mass of preimplantation embryos offer a window into the molecular biology· of preimplantation development. While many studies have explored the potential for differentiation into germ layer derivatives, there are few examples that have explored the ability of ESCs to differentiate into extraembryonic lineages. I have used two ESC model systems to study the molecular basis of extraembryonic lineage development. Human ESC (HESC) lines derived from human blastocysts seem to require cell-cell interactions to be maintained in an undifferentiated state. However, the passaging techniques currently in use can result in heterogeneous cultures as assayed by pluripotency markers. Here I show that there is spontaneous differentiation of extraembryonic cell types resembling primitive endoderm in HESC cultures. The Notch signaling pathway has been shown to control many developmental processes. The Notch pathway is present and can be activated in undifferentiated HESCs. However, the pathway may not be activated in undifferentiated HESCs and may instead be involved in the differentiation of primitive endoderm. Notch expression is regulated upon differentiation but constitutive signaling could not induce differentiation. Notch is activated in the primitive endoderm population of HESC-derived embryoid bodies. Inhibition of y-secretase reduces the proportion of differentiating HESCs. Thus, inhibition of y-secretase mediated Notch signaling may improve the homogeneity of HESCs and suggests a molecular mechanism that controls early human development. In the mouse ESC system, primitive endoderm can be efficiently removed from mouse embryoid bodies by hypotonic lysis to isolate primitive ectoderm-like cells. Lysed embryoid bodies maintain a primitive ectoderm marker in a defined serum-free medium and under defined conditions may form neurectoderm. This technique may allow isolation of an alternate pluripotent cell type in vitro and has implications for homogeneous differentiation of embryonic cell types for cell therapy applications.
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    School of Graduate Studies
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