Master regulators of neuronal maturation in NGN2 reprogrammed iPSC-derived iNeurons
type: CreativeWork
description: Neuronal maturation is an intricate and tightly-controlled process in neurodevelopment, during which neuronal cells acquire their mature molecular, morphological and electrophysiological properties to function as the central components of the nervous system. However, the cell-intrinsic mechanisms and transcriptional programs that govern it remain poorly understood. To address this gap in knowledge, the objective of this thesis was to identify the transcription factors (TFs) that control neuronal maturation and elucidate the pathways through which they exert their function. The present work takes advantage of the recent discovery that Neurogenin 2 (NGN2) overexpression in induced pluripotent stem cells results in their rapid conversion into excitatory induced neurons (iNeurons) that are morphologically complex and electrophysiologically active after 2 weeks in culture. In addition, the inducible control of NGN2 expression from genomic safe harbour sites has provided a deterministic model system, enabling the generation of pure neuronal cultures within days, with minimal variability. Culturing iNeurons together with primary rat glia results in more mature networks with early synchronous burst activity, as measured on multi-electrode arrays (MEAs). Candidate master-regulators of iNeuron maturation were identified using TF motif prediction tools and a dataset combining ATAC-seq and RNA-seq analysis of a granular time-course experiment during NGN2 reprogramming, in the presence and absence of glia. Predicted TFs were found to also be expressed during human cortical development and mutations in them are enriched in neurodevelopmental disorders, suggesting potential functional roles *in vivo*. To simultaneously investigate the function of 25 potential master-regulator TFs, I conducted a pooled CRISPR knock-out (KO) screen with single-cell transcriptome readout (CROP-seq screen) in iNeurons cultured with glia. Differential expression analysis implicated TFs in cellular proc…
abstract: Neuronal maturation is an intricate and tightly-controlled process in neurodevelopment, during which neuronal cells acquire their mature molecular, morphological and electrophysiological properties to function as the central components of the nervous system. However, the cell-intrinsic mechanisms and transcriptional programs that govern it remain poorly understood. To address this gap in knowledge, the objective of this thesis was to identify the transcription factors (TFs) that control neuronal maturation and elucidate the pathways through which they exert their function. The present work takes advantage of the recent discovery that Neurogenin 2 (NGN2) overexpression in induced pluripotent stem cells results in their rapid conversion into excitatory induced neurons (iNeurons) that are morphologically complex and electrophysiologically active after 2 weeks in culture. In addition, the inducible control of NGN2 expression from genomic safe harbour sites has provided a deterministic model system, enabling the generation of pure neuronal cultures within days, with minimal variability. Culturing iNeurons together with primary rat glia results in more mature networks with early synchronous burst activity, as measured on multi-electrode arrays (MEAs). Candidate master-regulators of iNeuron maturation were identified using TF motif prediction tools and a dataset combining ATAC-seq and RNA-seq analysis of a granular time-course experiment during NGN2 reprogramming, in the presence and absence of glia. Predicted TFs were found to also be expressed during human cortical development and mutations in them are enriched in neurodevelopmental disorders, suggesting potential functional roles *in vivo*. To simultaneously investigate the function of 25 potential master-regulator TFs, I conducted a pooled CRISPR knock-out (KO) screen with single-cell transcriptome readout (CROP-seq screen) in iNeurons cultured with glia. Differential expression analysis implicated TFs in cellular proc…
author: Patili, Eleni
language: eng
keywords: iNeurons, iPSCs, master-regulators, neuronal maturation
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ef264d60-af9a-4c16-af60-7dfb6bc89159/download
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2023-09-28
date modified: 2026-07-22T22:24:21Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.108174