Variation in endogenous SHH signalling activity accounts for differential responses of IPSC lines to neural sub-type specification in directed differentiation protocols

Jones, Kimberley 2022. Variation in endogenous SHH signalling activity accounts for differential responses of IPSC lines to neural sub-type specification in directed differentiation protocols. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Available protocols aimed at differentiating human induced pluripotent stem cells (iPSCs) from both control and Huntington’s disease (HD) patients have yielded varying populations of Medium Spiny Neurons (MSNs). This is hugely problematic when trying to identify whether molecular and functional differences are disease attributes independent of individual cell line differences. Furthermore, this limits the ability to screen compounds for drug discovery which selectively target MSNs as well as their potential use in cell replacement therapies. The aim of this thesis was to investigate the inherited inter-line variability in the generation of MSNs following identical patterning cues during iPSC differentiation. Here, endogenous SHH expression and signalling at the neural progenitor stage was identified as the main contributor to the variation in differentiation outcomes. Endogenous SHH signalling resulted in the expression of NKX2.1, a master transcription factor for medial ganglionic eminence (MGE) fate specification and interneuron development. Blockade of canonical SHH signalling during neural patterning stages of in vitro differentiation resulted in a medial- to lateral GE-like switch and consequently an increase in LGE-derived MSNs (~70% DARPP-32/CTIP2). SHH blockade also reduced the variability in differentiation seen between cell lines. Bulk RNA sequencing confirmed SHH signalling as the main contributor to variation. In addition, a novel signalling pathway, namely non-canonical WNT, that may influence inter-line variability was identified. MSN differentiation was further investigated in SHH and NKX2.1 knockout cell lines generated by CRISPR gene editing. Using our standard protocol of BMP and WNT pathway inhibition in the presence of Activin-A, knockout of both SHH and NKX2.1 caused a reduction in MSN differentiations. However, under a ‘default’ cortical differentiation protocol where BMP and WNT signalling was not inhibited, we observed an increase in DARPP-32 protein expression in the NKX2.1 KO lines, however the morphology of the neurons derived appeared to be distinct from that of MSNs. This is the first in-depth investigation into endogenous differentiation bias affecting MSN subtype specification and highlights the need to optimize protocols for individual cell lines, taking into account inherent biases that are determined by gene expression differences present in the pluripotent state and those that arise upon differentiation that influence developmental signalling, neural patterning and fate specification.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	6 April 2023
Last Modified:	27 Apr 2023 11:27
URI:	https://orca.cardiff.ac.uk/id/eprint/158477

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