Not that many (any?) will care about this, but since it took up two years of my life I feel justified to put up the abstract for my recently completed MSc project at the University of Otago in Dunedin, NZ, in all its boring glory. If this is of interest to you (hahahaha!……seriously though) let me know. I find it interesting and am always keen to talk about it at length.
BrdU labelling of T progenitor cells in a p53 mutant mouse model: a pilot study
Adult stem cells are evolution’s answer to maintaining tissues with high cellular turnover, providing a pool of protected cells from which multiple lineages can be derived. These cells exist in an array of tissues and organs, but have only been well characterised in specific model tissues, such as the intestinal tract and the central nervous system. So far, the identification of adult stem cells has relied on their known molecular markers, but this presents a problem for their characterisation in novel tissue systems, as they are likely to possess unique and as yet unidentified markers.
A method of identifying stem cells by the relative differences between them and their host tissue would provide a more universal form of characterisation. It is possible to achieve this through the retention of a nuclear label such as BrdU. This tracer gets incorporated into the DNA of all cells undergoing DNA replication, and becomes progressively diluted through each subsequent cell division. As stem cells in general divide less frequently than the cells of the tissue they reside in, they can be identified through their ability to retain this label for a longer period of time. Once an experimental system such as this has been optimised for a particular tissue, it can be used for studying adult stem cell populations and how they are affected by various genetic factors.
The tumour suppressor gene p53 is one of these factors and has recently been shown to play a major role in the homeostatic regulation of adult stem cell populations. Research over the past decade has characterised p53 as a major tumour suppressor gene, with p53 knockout mouse models displaying rapid onset of a spectrum of tumours, particularly lymphoma. Interestingly, separate mouse models where p53 protein function is lost also display an increase in neural and hematopoietic stem cell proliferation. These findings cast light on a potential link between loss of p53 function, stem cell population expansion and tumour development. While this relationship may exist in a variety of tissue types, thymic T cell development and associated malignant transformation into T cell lymphoma presents a valuable model to study this link due to the predominance of T cell lymphoma in p53 knock out mice.
The research covered in this thesis encompasses a pilot study using this model with the aim to investigate the effectiveness of in vivo nuclear labelling to identify early thymic progenitor cells, and analyse the effect of p53 loss-of-function on these identified populations. This was achieved by labelling several mouse litters with BrdU, either as embryos or post natally, and analysing label retention in the thymus over a time course of two months. The results obtained show that label retaining cells (LRCs) present in the thymus after the maximum chase period are consistent with populations of early thymic progenitor cells, the most immature cells present within the thymus. These findings were supported by the observed niche location of the label retaining cells and co-localisation between BrdU label and known T progenitor cell marker CD44 in a subset of these cells.
With this method established, a comparison of label retention between p53 null mutant mice and non-mutant mice was undertaken. While more LRCs were observed in the p53 null mutant, suggestive of an increased progenitor pool size, the results were not statistically significant. This is likely due to a number of factors including the potential that seeding of progenitor cells to the thymus acts independently of bone marrow stem cell population size. Furthermore, the identification of LRCs within a p53 null mutant thymic lymphoma provides evidence for the existence of heterogeneous cell types within this particular tumour, consistent with the prevailing cancer stem cell theory.
The key implication from this research is the establishment of a model system that, with further refinement and expansion, has the potential to further elucidate the link between p53 mediated hematopoietic stem cell expansion and lymphoma development. Lymphoma is one of the most common paediatric cancers and a better understanding of the initial malignant events causative to the disease will lead to more informed diagnosis and improved therapeutic options.
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