Munir, Hafsa (2012)
M.Res. thesis, University of Birmingham.
Mesenchymal stem cells (MSCs) are considered to be an attractive target for cell-replacement therapies due to their extensive self-renewal and multipotent differentiation capabilities. Applying ultrasound to soft tissue lesions and bone fractures has been shown to promote repair of damaged tissue. It was suggested that ultrasound stimulates repair by activating bone-marrow derived MSCs (BMSCs) to begin proliferating and differentiating to replenish the tissue. Therefore, the aim of the study was to analyse the biophysical effects of low-frequency, low-intensity ultrasound on the differentiation and colony forming unit ability of BMSCs. To test this colony forming unit assays were performed on ultrasound treated and untreated cells and cell staining was used to analyse the effects. Furthermore, to determine the intracellular response to ultrasound stimulation, the expression of ultrasound-sensitive genes was also analysed by reverse-transcriptase PCR. The results obtained in this study showed that BMSCs treated with low-frequency, low-intensity ultrasound did not generate a greater number of colonies than untreated cells. Colony size was found to be unchanged by treatment. Furthermore, ultrasound treatment did not appear to enhance the differentiation potential of BMSCs down the osteogenic lineage. This data suggests that low-frequency, low-intensity ultrasound does not appear to activate BMSCs in culture.
Embryonic stem cells (ESCs) are a pluripotent cell population that are able to self-renew and differentiate into cell types of all three germ layers. ESCs are able to differentiate by maintaining developmental genes in a transcriptionally poised state. The Hox genes are developmental regulators which are arranged in clusters. During differentiation, the genes are sequentially expressed according to their position in the cluster. To determine whether changes in histone modifications on Hoxb genes during differentiation reflect changes in their expression, ESCs were differentiated and quantitative PCR was used to determine the patterning of Hoxb1, 5 and 9 expression. Native Chromatin Immunoprecipitation (NChIP) was performed to determine the enrichment of histone modifications. The data showed that changes in histone modifications on Hoxb1 and 5 correlated with the temporal and spatial patterning of expression. Hoxb9 expression did not correlate with its position in the cluster and the levels of histone modifications did not reflect the patterning of expression observed. Also the fold enrichment of marks on the Hoxb genes did not correspond with changes in levels on Hoxa genes. This shows that changes in histone modifications on Hoxb genes reflect their transcriptional status, however but were not predictive of gene expression.
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