Ampartzidis, Ioakeim; (2024) Disease modelling and neuronal differentiation using spina bifida hiPSCs. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

During development, the neuroepithelium (NE) forms the neural tube: a crucial structure for brain and spinal cord formation. Neural tube closure (NTC) is partly regulated by apical constriction, controlled by planar cell polarity (PCP) signalling. Failure of apical constriction leads to severe central nervous system (CNS) malformations, including spina bifida (SB). The neural tissue in SB cases is influenced by chemical insults from the uterine environment, contributing to neurodegeneration. The in utero surgery is the gold-standard treatment and can mitigate these effects. In my PhD, I investigated the role of cellular and acellular components in SB using cell-based models. I developed a robust NE differentiation protocol using human induced pluripotent stem cells (hiPSCs). This protocol exhibits conserved cell behaviours, has accessible apical areas, and up-regulates PCP pathway components. Patient-specific mutations in PCP signalling components, such as VANGL2(R353C), result in enlarged apical areas and NE biomechanical dysfunction, validated using laser ablation/recoil assays. To further explore this system, I utilised patient material collected during amniocentesis. Patient-derived hiPSCs, after reprogramming, show inability to form a functional NE or differentiate into neurons. Whole-exome sequencing identified novel gene alterations associated with patient-specific deficiencies. Understanding the effects of the uterine environment on SB progression remains challenging. To that end, I developed a model to assess the impact of amniotic fluid (AF) on neuronal progenitors. Cell-free AF disrupts neuronal growth, alters cell morphology, and gene expression patterns and is not rescued by decellularised neural tissues protein supplementation. Combination of bulk RNA and cytokine analysis revealed a set of cascades which perturbation hopes to improve the neuronal differentiation in vitro. In summary, my research provides insights into the mechanisms underlying SB, identifying apical constriction impairments, patient-specific NE deficiencies, and the impact of AF on neurons. These findings have significant implications for improving the lives of affected individuals.

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