Directed differentiation of mouse embryonic stem cells for transplantation in Huntington's disease

Precious, Sophie Victoria 2009. Directed differentiation of mouse embryonic stem cells for transplantation in Huntington's disease. PhD Thesis, Cardiff University.

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Abstract

The research presented in this thesis focused on neural precursors derived from mouse ES cells (also called neural embryoid bodies, NEBs) in particular attempting to direct their differentiation towards specific telencephalic precursors, with a view to providing an alternative donor cell source to primary foetal tissue for the generation of DARPP-32 positive medium spiny neurons (MSNs) for neural transplantation in Huntington's disease. Neural induction of mouse ES cells was addressed, with further directed differentiation by addition of factors known to be important in the neural tube for development of specific telencephalic precursors. Subsequent neuronal differentiation of precursors was assessed both in vitro and in vivo. Chapter 3 characterised the neural and forebrain features of NEBs up to day 8 in neural induction medium, using a reporter cell line, in which LacZ was tagged to the early forebrain marker, Foxgl. Addition of FGF2 but not the Wnt inhibitor DKK-1, increased the forebrain population within the overall cell population. Day 8 NEBs, subjected to neuronal differentiation conditions, yielded p-III-tubulin positive neurons but no DARPP-32 expression. In Chapter 4, NEBs cultured for either 8 or 16 days were compared for expression of neural markers and then transplanted into the QA lesioned rat striatum to assess their potential to generate mature phenotypes in vivo. Day 8 NEBs generated teratomas by 2 weeks post transplantation. At 6 weeks post-transplantation, day 16 NEBs generated heterogeneous grafts with graft-derived neurons and glia, but no graft-derived DARPP-32 expression. Expression of dorso-ventral telencephalon markers in NEBs was investigated in Chapter 5, drawing comparisons to expression in the mouse (El4) developing telencephalon. The effects of the SHH agonist, purmorphamine, and the SHH antagonist, cyclopamine, were used in an attempt to enrich the forebrain precursors generated in Chapter 3 for ventral telencephalic-like precursors. Although gene expression changes were demonstrated, no distinct dorso-ventral patterns were shown. In vitro neuronal differentiation of day 16 NEBs was analysed in Chapter 6, in particular looking for expression of the MSN marker DARPP-32. Cultures of cells from the lateral ganglionic eminence (LGE) (the origin of MSNs) were used to determine if certain factors could increase the yield of DARPP-32 positive neurons. Addition of BDNF yielded the highest proportion of DARPP-32 from LGE. However, when this condition was applied to neuronal differentiation cultures of day 16 NEBs there was no effect on the expression of GAB A, DARPP-32 or FoxPl, all markers of MSNs. In Chapter 7, day 16 NEBs, with and without addition of purmorphamine, were transplanted into the QA lesioned mouse striatum. All resulting grafts were small, but analysis revealed densities of neuronal and striatal markers in day 16 NEB-derived grafts to be similar to E14-derived grafts.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Medicine
Subjects:	Q Science > QH Natural history > QH426 Genetics R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
ISBN:	9781303217937
Date of First Compliant Deposit:	30 March 2016
Last Modified:	10 Oct 2017 15:25
URI:	https://orca.cardiff.ac.uk/id/eprint/54958

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