Benzing, Corinne Isabel: In vitro Differentiation of Human Embryonic Stem Cells into Precursors of the Central Nervous System. - Bonn, 2006. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Corinne Isabel Benzing}},
title = {In vitro Differentiation of Human Embryonic Stem Cells into Precursors of the Central Nervous System},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2006,
note = {Human embryonic stem cells with the property of pluripotency and unlimited selfrenewal are a promising tool for basic research and future transplantation strategies. Following the establishment of basic techniques of human ES cell cultivation, 2 different strategies for the generation of human embryonic stem cell (human ES cell)-derived neural precursor cells were established.
Protocols for the neural differentiation of murine embryonic stem cells (murine ES cells) already exist (Okabe et al., 1996; Brüstle et al., 1999). In these strategies, differentiation is induced by aggregation of ES cells to embryoid bodies (EBs). The first part of this project focused on the question whether such a protocol for neural differentiation could be established for human ES cells too. Indeed, modification of several key steps, such as differentiation periods, media formulations and specific coatings permitted to generate an EB-based protocol for human ES cells. This new strategy led to the generation of highly enriched neural precursor cells expressing nestin and the neural markers PSA-NCAM and A2B5. After growth factor withdrawal, the generated neural precursors differentiated into neurons and glia cells, indicating their potential to generate cells of the neuronal and the glial lineage.
In proof-of-principle experiments, it was investigated whether human ES cell-derived neural precursor cells have the potential to integrate into host tissue upon transplantation. For this aim, eGFP-expressing neural precursors were transplanted onto hippocampal slice cultures. Immunohistological analyses revealed that the transplanted cells were able to migrate into the slice and differentiate into neurons. To investigate whether the transplanted cells have the capacity to functionally mature within the host tissue, the cells were analyzed electrophysiologically at the Department of Epileptology (University of Bonn Medical Center). The experiments revealed that only a minority of cells were able to induce action potentials after 3 weeks of engraftment in hippocampal slices.
In an additional set of experiments, the differentiation and migration properties of human ES cell-derived neural precursors upon in vivo transplantation were analyzed. For this purpose, precursor cells were transplanted into the developing brain of pre- and postnatal rats. In both cases, the cells formed clusters, from where single cells migrated Abstract 93 into the host tissues. The transplanted cells differentiated into neurons, as confirmed by the expression of neuron-specific markers.
So far, protocols for the neural differentiation mostly depend on EB-formation, coculture with stromal cells, lineage-selection strategies or mechanical isolation of neural rosettelike structures from differentiated human ES cell cultures. For that reason, the second part of this thesis addresses the question, whether such intermediate steps could be avoided and human ES cells could be directly converted into neurogenic precursors. The newly established direct conversion paradigm consists of an adherent cultivation step, followed by cultivation as neurospheres. In the first step, human ES cells propagated as adherent cultures on extracellular matrix proteins were induced to differentiate into the neural lineage in differentiation media containing fibroblast growth factor-2 (FGF-2). In the second step, the adherent cells were proliferated to form detaching neurospheres. Upon plating, these neurospheres gave rise to a homogenous population of neural precursors capable of generating neurons, astrocytes and oligodendrocytes. In addition to the practical advantage, also a mechanistic knowledge could be gained with the adherently converted human ES cells: The findings suggest that FGF-2 exposure alone suffices to promote neural conversion of adherently growing human ES cell cultures. The results of this study should provide a basis for the efficient generation of neural cell types for analytic and biomedical applications.},

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