Regeneration of human central nervous system (CNS) neurons is limited due to the inhibitory environment that forms post injury known as the glial scar. Reactive astrocytes within the glial scar produce both inhibitory and permissive extracellular matrix molecules (ECM) into the local environment. Chondroitin sulfate proteoglycans (CSPGs) are a component of the ECM which have been shown in vitro and in vivo to inhibit neurite regeneration. Physiologically relevant in vitro models of the glial scar are essential in developing new therapeutics and understanding the cellular processes that underpin neural regeneration. In this study human pluripotent stem cells were differentiated using the highly potent and stable synthetic retinoid EC23. A concentration dependent profile of the action of EC23 on stem cell differentiation was determined, furthermore, the mechanisms for the enhanced biological activity of EC23 were investigated. This study used the well described small molecule EC23 to form aggregates of neural progenitors which were characterised and used in a two dimension (2D) and three dimension (3D) model of neurite outgrowth. Next, the neurite outgrowth substrate was manipulated to represent the inhibitory ECM of the glial scar using the CSPG Aggrecan. The presence of Aggrecan inhibited neurite development and was used to assess small molecules that could enhance outgrowth in 2D and 3D. Small molecule modulators of; rho-associated protein kinase (ROCK); retinoic acid receptor β2; glycogen synthase kinase 3β and protein tyrosine phosphatase σ were shown to at least partially enhance neurite outgrowth on Aggrecan in 2D and 3D. Furthermore, the bacterial enzyme Chondroitinase ABC was used to cleave chondroitin sulphate glycosaminoglycan side chains (GAG) from Aggrecan to further aid neurite outgrowth in this model. In addition a 2D and 3D co-culture system was developed using the human astroglioma cell line U118MG and human stem cell-derived neural progenitors described previously. This model demonstrated inhibition of neurite outgrowth by U118MG which could be overcome by ROCK inhibition. This thesis describes the development of a novel model of neurite outgrowth using human stem cell-derived neurons. The model described was used to investigate Aggrecan induced neurite inhibition and to investigate pathways involved in neural regeneration.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:600951 |
| Date | January 2014 |
| Creators | Tams, Daniel Mark |
| Publisher | Durham University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.dur.ac.uk/10528/ |
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