Role Of Sonic Hedgehog Signalling in Human Embryonic Stem Cells And Its Neural Derivatives

Abstract

Human embryonic stem cells (hESC) are pluripotent stem cells that have the unique ability to differentiate into cells of the three germ line lineages. Hence, they have wide potential to be used in cell replacement therapy and drug discovery. To realize the clinical potential of hESC, a deeper understanding of the molecular and cellular mechanism underlying their unique capacity for self-renewal and differentiation is required. This thesis is focused on the role of the Sonic Hedgehog (SHH) signaling pathway, a key pathway essential for the normal development of mammals. By testing the requirement of SHH in undifferentiated hESC cultures, it was revealed that exogenous SHH was not able to maintain the pluripotency or increase the proliferation of hESC. Instead, the SHH pathway was activated upon differentiation and exogenous SHH promoted differentiation to the neuroectoderm lineage. Using a defined neural differentiation protocol, it was found that overexpression of SHH in hESC resulted in a significant increase in neural stem cell marker expression as well as increased proliferation of neuroprogenitors. This demonstrated that SHH enhanced the neural induction and expansion of neuroprogenitors, which resulted in an increased yield of dopaminergic neurons derived from the neuroprogenitors. Transcriptional profiling of overexpressing SHH neuroprogenitors and in silico GLI DNA-binding site analysis identified putative direct and biologically relevant target genes of the SHH pathway. It also revealed an extensive network of genes involved in neural development, neuroprogenitor proliferation, neural specification and axon guidance. Therefore, this thesis contributes to the understanding of SHH signaling in hESC self-renewal and differentiation and provides a comprehensive view of the SHH transcriptional network in hESC-derived neuroprogenitors.