Smith, Aileen Margaret

2018-03-29T12:20:18Z

2018-03-29T12:20:18Z

2005

Mouse embryonic stem cells (ES cells) are derived from the inner cell mass of 3.5 day blastocysts. ES cells remain totipotent when cultured on embryonic fibroblasts or in the presence of Leukaemia inhibitory factor (LIF). However, under defined culture conditions they are able to differentiate into multiple cell lineages and when reintroduced back into a host blastocyst, possess the remarkable ability to contribute to all adult tissues in the mouse. These unique features of ES cells allow genes to be deleted in mice and when combined with Cre/LoxP technology provide an ideal system for conditional deletion of genes where total 'knockout' would be lethal. One such gene is integrin αᵥ where knockout mice die in utero or perinatally. Integrin αᵥ has been implicated in angiogenesis and cell adhesion as well as in the phagocytosis of dying cells by macrophages and dendritic cells, a process that is important in the resolution of inflammation and tissue remodelling.

The main aim of this thesis was to examine the role of integrin αᵥ in phagocytosis. To do this, two complementary approaches were used. Firstly, ES cells with disrupted integrin αᵥ genes were differentiated into myeloid cells and the effects on phagocytosis by DCs assessed. Secondly Cre was delivered to macrophages cultured from integrin αᵥ targeted mice, to delete integrin αᵥ immediately prior to phagocytosis assays.

This thesis demonstrates that macrophages can be generated from the culture of ES cells. Although low in number these macrophages show similar morphology and surface phentoype to bone marrow derived macrophages. Importantly, these ES macrophages readily phagocytosed latex beads and apoptotic cells. In addition, ES cells produced cells with a dendritic cell phenotype that were capable of apoptotic cell phagocytosis and maturation. Generation of these myeloid cells from ES cells was strongly dependent on serum and the parent ES cell line. Dendritic cells could be generated from integrin αᵥ disrupted ES cells. These DCs retained the ability to phagocytose apoptotic cells suggesting that integrin αᵥ is not essential for this process.

This study also investigates the properties of Cre fused to two transduction proteins. Although Cre retained its recombinase activity as a fusion protein these were unable to translocate into cells. However Cre could be delivered to both primary macrophages and cultured epithelial cells using a replication deficient adenovirus allowing deletion of the targeted genes. Intriguingly, the apoptotic cell phagocytosis was unaffected by lack of integrin av but could no longer be inhibited by the integrin antagonistic peptide RGD.

In conclusion, the capacity of ES cells to differentiate to myeloid cells combined with the ability to deliver Cre to silently targeted myeloid cells provide powerful systems for studying the role of specific genes in phagocytosis. Use of these approaches with integrin αᵥ demonstrates that this gene is not essential for apoptotic cell phagocytosis. However the ability of specific antagonists to inhibit phagocytosis show that integrin αᵥ is intimately involved in this process in normal cells

http://hdl.handle.net/1842/29369

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 17

Already catalogued

Embryonic stem cell differentiation: a novel approach to gene targeting in myeloid cells

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy