Haemopoietic stem cells are capable of generating a variety of mature cells. One of the important steps in stem cell biology involves the commitment of a multipotent precursor to a single lineage. As yet the molecular mechanism for cell fate determination is unknown. This work was focused on commitment of the granulocyte/macrophage (GM) bi-potential precursor towards the macrophage lineage. Published studies have noted that the activity of tyrosine kinase, fyn, correlated with macrophage maturation and increased expression of the macrophage-colony stimulating factor receptor (M-CSFR) in the human myelomonocytic cell line, THP-1. Furthermore, it was also reported that treatment with fyn anti-sense reduced M-CSFR up-regulation and cell adhesion, indicative of macrophage commitment and/or maturation. To investigate whether fyn was involved in the transcriptional activation of c-fms and potentially in macrophage commitment in primary haemopoietic cells I used real-time RT-PCR to determine levels of fyn and c-fms. Examination of fyn RNA levels revealed that it was unlikely to regulate c-fms and suggested that fyn is most likely regulated by c-fms. Having ruled out fyn as a potential positive regulator of macrophage commitment, I subsequently set out to identify other potential regulators. To do so, I examined the expression profiles of single granulocyte/macrophage (CFU-GM), macrophage (CFU-Mac) and neutrophil (Pre-N) precursors using a DNA microarray approach. My single cell microarray data provided a "snap-shot" of the expression profiles of the CFU-GM, CFU-Mac and Pre-N progenitors and revealed the presence of groups of genes whose expression correlated with the developmental stage of the single cells. Furthermore, analysis of the expression profiles identified changes in gene expression due to cell handling and culture treatments. Real-time RT-PCR analysis was used to validate a set of array genes potentially involved in macrophage commitment. For genes expressed at levels greater than 1 copy in lO4 total cDNA molecules a strong correlation was revealed between microarray hybridisation and real-time PCR data. Further analysis of enriched bone marrow precursors and a multi-potent cell line revealed a set of eleven genes as potential regulators of macrophage commitment and five genes as potential early marker of commitment. My results provide a precedent for new experimental studies of the mechanisms underlying monocyte lineage determination and more general mechanisms of commitment that can be extended to different tissues and embryo development.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:488438 |
| Date | January 2004 |
| Creators | Nostro, Maria Cristina |
| Contributors | Brady, Gerard |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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