Use this URL to cite or link to this record in EThOS:
Title: Characterising the in vitro conversion of mouse fibroblasts into induced neurons using a CRISPR/Cas9 knock-out screen
Author: Carbone, Benedetta
ISNI:       0000 0004 9349 4651
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
As the global phenomenon of population aging has become very prominent in the last few decades, diseases of the aging such as Alzheimer’s and Parkinson’s disease have taken the spotlight as the new challenges of modern medicine. In fact, aging is the biggest risk factor for neurodegeneration. Neurodegenerative diseases are particularly difficult to study, as animal models often fail to recapitulate human disease phenotypes, together with the challenging aspects of monitoring cognitive functions in smaller animals. Thanks to stem cell research, we’re now able to derive large quantities of human neurons in vitro. This has been incredibly helpful for neuroscientific research, but stem cell-derived neurons show cellular and molecular features, such as epigenetic characteristics and metabolic activity, that resemble embryonic neurons and thus fail to recapitulate the damage and oxidative stress typical of aged neurons. In 2010 the first report of in vitro fibroblast-derived neurons opened the way for an alternative source of neurons, that could easily be derived from individual patients, and most importantly maintain the cellular age of the original fibroblasts. This presents then as a better tool to study diseases that affect aged neurons in vitro. The conversion process is nonetheless quite inefficient and leads to a very heterogeneous mix of cell types, in which a limited number of induced neurons would need to be isolated from contaminating fibroblasts, myoblasts and cells of uncharacterized subtypes. In this thesis I describe my efforts to identify the drivers of the reprogramming process that lead to a successful conversion from a fibroblast to a neuron, and the roadblocks that might be hindering it. To achieve this, I decided to take a genetic screening approach, taking advantage of the emerging CRISPR/Cas9 technology that has made genetic knock-outs relatively easy and quick to achieve. By converting Cas9-expessing mouse embryonic fibroblasts (MEFs) into induced neurons (iN) while expressing target gRNAs, I performed a loss-of-function screen to identify genes involved in the conversion process. I first identified a genetic reporter that allows me to isolate the induced neurons from the remaining cell types in the heterogeneous culture. I then selected a subset of genes within the mouse genome as likely candidates to be involved in the cell identity conversion and then proceeded to perform the screen. I identified two genes, Stxbp1 and Sf3a1, as required for iN conversion and survival in the conversion context. Stxbp1 is known to be required for neuronal survival, as its knock-out prevents neurotransmitter release and leads to neuronal death. This thus represents confirmation that iN are a reliable platform to study neuron biology. Sf3a1 is part of the SF3A complex involved in pre-mRNA splicing and has previously been reported to be required for cell identity transitions, but, to the best of my knowledge, had yet not been associated with cell survival in the context of fibroblast to neuron conversion. This work presents a solid platform to genetically engineer and characterize in vitro induced neurons and highlights their relevance to broader neuronal culture systems and general neuron biology.
Supervisor: Kaji, Keisuke ; Kunath, Tilo Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: neurons ; in vitro fibroblast-derived neurons ; CRISPR/Cas9 ; induced neurons ; Stxbp1 ; Sf3a1