Towards differentiation of mouse embryonic stem cells to thymic epithelial progenitor cells
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The thymus is the major site for T-cell generation and thus is important for the adaptive immune system. Development of a properly selected, functional T-cell repertoire relies on interactions between developing T cells and a series of functionally distinct thymic stroma cell types including the cortical and medullary thymic epithelial cells (TECs). The thymus is one of the first organs to degenerate in normal healthy ageing. Related to this, there is strong interest in developing protocols for improving thymus function in patients by cell replacement or regenerative therapies. Thymic epithelial progenitor cells (TEPCs) represent a potential source of cells for thymus transplantation. However, the only source of these cells for transplantation is currently fetal thymus tissue. If TEPCs could be generated from pluripotent cells, this could provide an alternative source of cells for transplantation. The work described in this thesis therefore had two central aims (i) to test the stability of thymic epithelial progenitor cells in vivo and (ii) to investigate the possibility of generating TEPCs or TECs from mouse embryonic stem (ES) cells. The forkhead transcription factor, Foxn1, is essential for the development of a functionally mature thymic epithelium, but is not necessary for formation of the thymic primordium or for medullary thymic epithelial sub-lineage specification. By reactivating Foxn1 expression postnatally in mice carrying a revertible hypomorphic allele of Foxn1, Foxn1R, I herein demonstrate that TEPCs that can express only low levels of Foxn1 mRNA can persist postnatally in the thymic rudiment in mice until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression. In parallel with this work, I confirmed the possibility of generating Foxn1-expressing cells from mouse ES cells by using a Foxn1 reporter cell line. As the thymic epithelium has a single origin in the third pharyngeal pouch (3pp) endoderm, I then tested whether or not TEPCs and /or TECs were generated during ES cell differentiation via existing protocols for generating anterior definitive endoderm differentiation cells from mouse ES cells. From this work, I showed that genes expressed in the 3pp and/or TEPC,-including Plet-1, Tbx1, Hoxa3 and Pax9, were induced by differentiation of ES cells using these protocols. I further showed that cells expressing both Plet-1, a marker of foregut endoderm and 3pp, and EpCAM, a marker of proliferating epithelial cells, were induced using a novel protocol (2i ADE) for generating ES cells from ADE. However, gene expression analysis and functional testing suggested that the majority of these cells were non-thymus lineage. I subsequently developed a novel protocol which combined this 2i ADE protocol with co-culturing of the differentiating ES cells with fetal thymic lobes, and demonstrated that this further induced 3pp and TEPC related genes. Finally, I modified the culture conditions in this protocol to conditions predicted to better support TEPC/TEC, and showed that in these conditions, the TEPC-specific markers Foxn1 and IL-7 were induced more strongly than in any other conditions tested. The data presented in this thesis therefore represent an advance towards an optimized protocol for successfully generating TEPCs from ES cells in vitro.