Cellular processes in the induction of embryonic stem cell differentiation into neural crest cell derivatives

Fielding, Stuart John (2015) Cellular processes in the induction of embryonic stem cell differentiation into neural crest cell derivatives. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Neural crest induction and migration is the culmination of an intricate network of signalling from both the ectoderm and underlying mesoderm. Due to the transient nature of the neural crest, little is known about the specific interactions of growth factors and morphogens required for correct patterning. Neural crest stem cells were differentiated from embryonic stem cells in serum-free feeder-free culture conditions. Cells were further differentiated into functional peripheral neurons, expressing the synaptic protein neurexin3, able to spontaneously generate action potentials and showing responses to the neuroactive compounds GABA and NMDA. Microarray analyses and subsequent RT-qPCR experiments revealed that gene expression of the key pluripotency associated factor Oct-4 was initially downregulated upon differentiation before expression levels increased as cells developed into a neural crest phenotype. Further experiments indicated an intrinsic role for bone morphogenetic protein-4 (BMP-4) in mediating this resurgence of expression. In addition to BMP-4 influenced expression of Nanog, members of the developmental pluripotency associated family of genes and important adhesion molecules with roles in the epithelial to mesenchymal transition of the neural crest. Global gene expression profiling of differentiating functional neurons offers new insights into BMP-4 mediated patterning of the neural crest and peripheral nervous system during development. This study implicates BMP-4 as a key factor in neural crest differentiation, with a role in diverse cellular processes including proliferation, fate determination and cell migration. The role of BMP-4 in mediating expression of pluripotency and adhesion factors highlights a potential role in oncogenesis; neural crest cells share many phenotypic traits with cancer cells. Finally, the generation of functional peripheral neurons in feeder free culture conditions offers a reliable method for the recapitulation of these tissues and the possibility of use in future tissue replacement therapy.