Development and application of human cortical organoids: A 1q21.1 deletion study

Lunn, Sharna 2022. Development and application of human cortical organoids: A 1q21.1 deletion study. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Cerebral organoids are quickly becoming an essential research model in the study of neurodevelopmental disorders. With the aim of generating resources for investigating such neurodevelopmental disorders, new methodology was devised for immunocytochemistry (ICC) quantification of cerebral organoids. A high throughput ICC analysis pipeline was developed, with post-hoc corrections for apoptotic cell death and creation of a universal constant for normalising morphological features and dead cell count data. In addition to these analysis techniques, revised human induced pluripotent stem cell (hIPSC)-derived cortical organoid (hCO) protocols were trialled with the intention of improving on current guided cerebral organoid models. Each protocol’s hCOs were scrutinised for dorsal forebrain characteristics such as cortical layering and organised neural progenitors, of which varied significantly between protocols. Extended ROCK inhibition during early hCO differentiation proved counterproductive to developing dorsal forebrain identity. With improved analysis methodologies and a characterised hCO protocol, hCOs were generated from patients of the rare, pathogenic copy number variant (CNV), 1q21.1 deletion (1qDel). 1qDel is associated with developmental delay, intellectual disability, schizophrenia and microcephaly. From the onset of hCO differentiation, 1qDel hCOs were microcephalus until neuronal maturation. As the neuroepithelium developed, 1qDel hCOs also exhibited early neurogenesis and disruption of the cell cycle, hypothesised to be a result of repressed NOTCH and Wnt signalling due to 1qDel. Neuronal maturation alleviated these phenotypes, but expansion of ventral forebrain progenitors at Day 30 was suspected to detrimentally affect cortical layering. By two months of age, 1qDel hCOs were predominantly indistinguishable from controls, with the exception of an increase in GABA-ergic presynaptic markers suggesting an excitatory/inhibitory imbalance in neuronal activity. This is the first in vitro example of the frontal lobe-specific microcephaly found in 1qDel patients, as well as providing future insights into CNV-associated cortical dysfunction.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	25 May 2023
Last Modified:	26 May 2023 09:12
URI:	https://orca.cardiff.ac.uk/id/eprint/159954

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