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Title: Characterising mesothelial cell cultures derived from the murine omentum
Author: Dauleh, S.
ISNI:       0000 0004 6422 1670
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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Mesothelial cells have been described to possess progenitor characteristics and contribute to regeneration through differentiation. However, it is not clear what the effects of prolonged culture have on the mesothelial cell properties and relative plasticity; as long-term cultures have not yet been established as a result of early senescence. Understanding the effects of time in culture is crucial for the development of novel therapies. In this thesis, we demonstrated that mesothelial cell cultures isolated from murine omentum could be cultured for more than 40 passages and showed relatively stable population doubling times. While initially, the cells did down-regulated the expression of mesothelial markers Wilm’s tumor protein 1 (Wt1) and mesothelin and epithelial genes; their mesenchymal profile was maintained. This along with the increased Snail2 expression suggested the cells were progressing through the mesothelial to mesenchymal (MMT) transdifferentiation programme. TGF-β and more recently EGF are known mediators of MMT. Targeting signalling through these receptors with small molecule inhibitors LY364947 and PD153035, slowed the rate of gap closure, in vitro and increased zonula occludens 1 accumulation at cell-cell contacts. Which seemed to be mediated through the MEK5/ERK5 pathway. Furthermore, siRNA-mediated transient knockdown of Zeb1 and Zeb2 transcription factors also achieved attenuated the rate of migration. Next, we moved onto to accessing the progenitor properties of the mesothelial cells with time in culture. The expression of stem cells markers Bmi1 (Proto-Oncogene, Polycomb Ring Finger), Sox9 ((Sex Determining Region Y)-Box 9) and CD34 were downregulated with repeated passaging. However, the low passage mesothelial cells exhibited clonogenicity and could differentiate into osteoblasts and adipocytes. Finally, in an embryonic kidney rudiment assay, we could show that the mesothelial cells co-existed with the embryonic kidney cells and allowed renal structures to form in their presence. Ultimately, we have demonstrated that the mesothelial cells from the omentum maintain some mesothelial characteristics with prolonged culturing. The cells showed clonogenic and multi-lineage potential and expressed a number of stem cell genes. The MMT programme is complex and could be partly reversed by targeting TGF-βR1 and EGFR tyrosine kinases, which along with transiently silencing the Zeb transcriptional factors seem likely key targets in ameliorating pathological fibrosis.
Supervisor: Wilm, B. W. ; Plagge, A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral