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    Nodal Signalling During Targeted Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    Miller, Duncan

    [Thesis]. Manchester, UK: The University of Manchester; 2012.

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    Abstract

    Targeted differentiation of human embryonic stem cells (hESCs) towards definitive endoderm (DE) is the first step in generating hepatic or pancreatic cell types with potential for clinical application. Characterisation and efficiency of DE differentiation is improving, however the specific effects of the different exogenous growth factors used, and the changing presence and activity of endogenous factors, are still not well understood. One such endogenous factor, the TGFβ ligand Nodal, is known to drive patterning and differentiation of the primitive streak and DE in the developing mouse embryo. The effect of Nodal signalling during hESC DE differentiation is unknown, and the common use of a related exogenous ligand Activin A may also serve to upregulate rather than simply mimic it. In order to explore this, Activin A differentiation of hESCs in defined culture conditions was analysed. The expression of characteristic mesendoderm and DE markers increased during Activin A treatment, which was significantly enhanced by the inclusion of exogenous Wnt3a. A maintained presence of the pluripotency factor Nanog was observed in most cells expressing markers of DE. The levels of Nodal and its co-receptor Cripto, which were raised during the early stage of Activin A treatment, were also marginally enhanced by Wnt3a, and evidence of Nodal endocytosis further suggested an active signalling presence. RNA interference (RNAi) of Nodal negatively affected both pluripotency maintenance during normal pluripotent culture, and the capacity to differentiate towards DE. Use of a Cripto blocking antibody also inhibited differentiation towards DE. The results strongly suggested the presence of Nodal signalling, as well as possible roles for Nanog, Wnt-related signalling, and Nodal signalling during Activin A-mediated DE differentiation. The results contribute to current understanding of how DE differentiation in hESCs is regulated. They also identify clear targets for further investigation, which would lead to improved characterisation and differentiation of DE from hESCs.

    Bibliographic metadata

    Type of resource:
    Content type:
    Form of thesis:
    Type of submission:
    Degree type:
    Doctor of Philosophy
    Degree programme:
    PhD Stem Cell Research
    Publication date:
    Location:
    Manchester, UK
    Total pages:
    250
    Abstract:
    Targeted differentiation of human embryonic stem cells (hESCs) towards definitive endoderm (DE) is the first step in generating hepatic or pancreatic cell types with potential for clinical application. Characterisation and efficiency of DE differentiation is improving, however the specific effects of the different exogenous growth factors used, and the changing presence and activity of endogenous factors, are still not well understood. One such endogenous factor, the TGFβ ligand Nodal, is known to drive patterning and differentiation of the primitive streak and DE in the developing mouse embryo. The effect of Nodal signalling during hESC DE differentiation is unknown, and the common use of a related exogenous ligand Activin A may also serve to upregulate rather than simply mimic it. In order to explore this, Activin A differentiation of hESCs in defined culture conditions was analysed. The expression of characteristic mesendoderm and DE markers increased during Activin A treatment, which was significantly enhanced by the inclusion of exogenous Wnt3a. A maintained presence of the pluripotency factor Nanog was observed in most cells expressing markers of DE. The levels of Nodal and its co-receptor Cripto, which were raised during the early stage of Activin A treatment, were also marginally enhanced by Wnt3a, and evidence of Nodal endocytosis further suggested an active signalling presence. RNA interference (RNAi) of Nodal negatively affected both pluripotency maintenance during normal pluripotent culture, and the capacity to differentiate towards DE. Use of a Cripto blocking antibody also inhibited differentiation towards DE. The results strongly suggested the presence of Nodal signalling, as well as possible roles for Nanog, Wnt-related signalling, and Nodal signalling during Activin A-mediated DE differentiation. The results contribute to current understanding of how DE differentiation in hESCs is regulated. They also identify clear targets for further investigation, which would lead to improved characterisation and differentiation of DE from hESCs.
    Thesis main supervisor(s):
    Thesis advisor(s):
    Language:
    en

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    Record metadata

    Manchester eScholar ID:
    uk-ac-man-scw:183379
    Created by:
    Miller, Duncan
    Created:
    12th December, 2012, 10:54:49
    Last modified by:
    Miller, Duncan
    Last modified:
    9th January, 2019, 09:50:14

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