Mitchell, Jamie Samuel; (2021) Investigating directed differentiation strategies in hiPSCs to model cell type-specific vulnerability in ALS. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The concept of vulnerability is highly relevant to neurodegenerative diseases, whereby specific subsets of neurons display marked and devastating disease-related pathologies, but neighbouring cells may not. Amyotrophic lateral sclerosis (ALS) provides a perfect example, where spinal pathology manifests in lower motor neurons (MNs), with neighbouring cells remaining relatively unaffected, at least until late-stage disease. Interestingly, spinal MNs display selective vulnerability, with larger and more heavily myelinated alpha motor neurons degenerating the earliest. Additionally, the role of cell types surrounding MNs in contributing to ALS pathogenesis have become more evident over recent years. This includes non-cell-autonomous toxicity mediated by astrocytes and the denervation of Renshaw interneurons (INs) from MNs. Subsequent elucidation of mechanisms underlying cell type-specific vulnerability in ALS would drastically improve our understanding of ALS, the spectrum of cell types affected and provide alternative and tractable cellular targets for therapeutic intervention. The advent of human induced pluripotent stem cells (hiPSCs) has revolutionised disease modelling, providing a virtually inexhaustible source of patient-specific material. As a consequence, a variety of cell types has been generated using ontogeny-driven directed differentiation strategies. However, there is a pressing need for deeper phenotyping and further refinement of differentiation strategies, in order to generate more enriched and disease-relevant populations. With this in mind, I employed an established hiPSC-derived MN protocol and manipulated extrinsic signalling cues during two distinct developmental phases; patterning and terminal differentiation. In this manner, I was able to induce post-mitotic motor columnar diversity, resulting in the specification of lateral motor column phenotype; highly susceptible to degeneration in ALS. Separately, I was able to generate hiPSC-derived dorsal spinal INs arising from dI4-6, which subserve pain, temperature, itch and touch sensations (dI4&5) or indeed those regulating left-right coordination (dI6). Importantly, these INs retain the same axial identity, but are positioned more dorsally in the absence of sonic hedgehog signalling. Lastly, hiPSC-derived INs and MNs were investigated in a valosin-containing protein (VCP) mutant model of ALS. This revealed key differences relating to cell type vulnerability between MNs and INs, including RNA binding protein mislocalisation and alternative splicing events. Overall, the results of this PhD present novel ontogeny-driven directed differentiation strategies of hiPSC-derived cell types and a robust platform for modelling mechanisms of selective vulnerability in ALS. The experiments contained herein also demonstrate that iPSC models can capture neuronal subtype-selective vulnerability.

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