Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.727345
Title: Development of in vitro skeletal disease models using CRISPR/Cas9 genome editing in immortalised mesenchymal stem cells
Author: Carstairs, Alice
ISNI:       0000 0004 6424 256X
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2017
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Abstract:
The emergence of engineered nucleases for genome editing has allowed for greater understanding of human biology in health and disease, particularly through combination with stem cells and differentiation protocols. Mesenchymal stem cells (MSCs) are a multipotent adult stem cell able to differentiate into osteoblasts, chondrocytes and adipocytes. Early mesoderm differentiation pathways are relatively well understood, yet the understanding of how mesoderm transcription factors drive post-natal differentiation is less well studied. Additionally, the impact of skeletal disease on MSCs is often neglected in the furthering of our understanding of pathophysiology and disease phenotypes. To this end, this PhD project aimed to use an immortalised MSC cell line (hTERT MSCs) to develop a methodology suitable for the generation of genetically modified MSCs (GM hTERT MSCs). Firstly, the effects of serum in in vitro cell culture was considered by reducing serum in hTERT MSC culture. This demonstrated in the absence of a nutrient-rich environment hTERT MSCs shift towards a lipid-based metabolism with a consequential increase in osteogenic capabilities. The initial targets of CRISPR/Cas9 were Runx2 and Sox9, two critical transcription factors in the onset of osteogenesis and chondrogenesis respectively. The methodology developed used a fluorescent sorting strategy to maximise the possibility of generating GM-hTERT MSCs and in this way, successful genome editing was demonstrated. Genome editing of Runx2 did not appear to absolve osteogenic potential in the hTERT MSCs and targeting of Sox9 via the CRISPR/Cas9 technology demonstrated an apparent increase in adipogenesis. To demonstrate the disease modelling capabilities of GM-hTERT MSCs, a human disease relevant mutation was created in the FGFR3 gene mimicking the genotype of CATSHL syndrome resulting in a striking phenotype, where cells showed a decreased differentiation ability but an increased proliferative and migratory capacity. These data were developed further through the use of a 3D spheroid model allowing for the study of differentiated MSCs, including GM hTERT-MSCs, in a more in vivo like setting. Together these results demonstrate the potential for expanding our understanding of MSC biology in physiologically relevant in vitro conditions.
Supervisor: Genever, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.727345  DOI: Not available
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