Exploiting the ability of ES cells to differentiate toward haematopoietic lineages, the project sought to investigate gene expression with the view of identifying novel genes/proteins involved in the early stages of ES cell commitment to haematopoietic lineages. In the culture system used, it has been shown that under standard differentiation conditions 30-50% of embryoid bodies are able to give rise to haematopoietic colonies. This level of haematopoietic commitment was modulated with the addition of chemical inducers of the differentiation environment of embryoid bodies. Using the CFU-A assay to assess haematopoietic commitment and differentiation, we have found that the number of CFU-A colonies can be increased 2-fold after treatment of embryoid bodies with 1% DMSO during the first 48 hours of differentiation, and conversely reduced to below 5% after treatment with 10<SUP>-8</SUP> all <I>trans</I>-retinoic acid (atRA) for the same period. Lineage commitment in untreated, DMSO and atRA embryoid bodies were further investigated using reverse transcription-polymerase chain reaction techniques. The mesoderm marker <I>brachyury</I> was found to be expressed in both DMSO and atRA treated samples to similar extents, suggesting neither compound to be exclusively exerting its effect by enhancing or inhibiting mesoderm commitment. However, the level of transcripts of various haematopoietic markers such as β-globin, PU-1 and CD45 were found to be raised in DMSO treated embryoid bodies compared to either untreated or atRA treated samples. Studies investigating the ability of cells from treated and untreated embryoid bodies, to reconstitute the haematopoietic system of lethally irradiated mice were also carried out. Overall these showed that mice injected with cells from DMSO treated embryoid bodies survived for longer periods of time than those injected with cells from either untreated or atRA treated embryoid bodies. It has been clearly demonstrated that the haematopoietic commitment of ES cells and their differentiated aggregates can be successfully modulated by the addition of chemical inducers. Consequently these effects will prove a useful means of screening for genes and proteins involved in haematopoietic commitment and differentiation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:649651 |
| Date | January 1997 |
| Creators | Doostdar, Lobat |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13690 |
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