The potential application of olfactory ensheathing cells (OECs) to spinal cord injury has been the focus of a lot of research over the past ten years. Currently there are many challenges associated with the use of these cells as a therapy. These include the inherent plasticity of the cells and the fact they are challenging to sustain in culture for prolonged periods due to poor survival and proliferation. They possess unique properties as they are able to support neuronal survival and facilitate the regeneration of severed axons and therefore overcoming these challenges would be a step towards developing a cell therapy for spinal cord injury. Initially rat models were used to determine culture conditions that enhance the protein expression of key OEC markers p75NTR and S100β. An increase in p75NTR expression was achieved by co-culturing the primary rat OECs with a conditionally immortalised immobilised human mucosal fibroblast cell line feeder layer. OECs cultured on feeders were found to adopt a more spindle-like appearance compared with cells cultured on laminin which adopted an enlarged morphology. This morphological change is significant as spindle-shaped OECs are associated with neural regeneration function. Conditioned media collected from the human feeders resulted in an increase in Thy1.1 protein expression, an undesirable marker, with no increase in p75NTR expression and co-culture of primary OECs with mouse feeders (Ms3T3) gave similar results to co-culture with human feeders. It was determined that OECs benefit from the cell to cell contact and not necessarily trophic factors present in the media. The best culture conditions for primary rat OECs were found to be Ms3T3 feeders with DMEM/F12 Glutamax media. Testing with conditionally immortalised human OEC cell lines found contrasting results where an increase in S100β was observed when cells were cultured on laminin and lower levels of expression observed during feeder co-culture. Similarly to primary rat OECs, conditioned media from human mucosal fibroblasts was detrimental to S100β expression. The best culture conditions for human OECs were found to be laminin coated wells with DMEM/F12 Glutamax media. These data sets show that care has to be taken when translating animal models to studies with human cells as the data does not always correlate. Studies continued to characterise optimum culture conditions for the conditionally immortalised human OEC cell line. MACS purification technology was used to remove Thy1 positive cells from the polyclonal population. Although this removal was successful, it was found that the complete removal of Thy1 from the population does not ensure Thy1 is not present in the future population. After 5 days in culture Thy1 was being expressed in the Thy1 negative population. Time point staining determined that Thy1 turns on and off during time in culture and the removal of Triton X from the staining protocol is vital to visualising the presence of this protein. This time point study also revealed p75NTR is not a stable marker for OECs as turns off over time in culture. Further study towards understanding the role Thy1 and p75NTR take in OEC function would be beneficial to development of an OEC cell therapy for spinal cord injury. After the identification of optimum culture conditions for enhanced p75NTR and S100β expression, co-culture with neurons was carried out in order to determine if these conditions would link to an improvement in functional support. Neurite length was measured after 5 days of co-culture and was normalised against the number of neurites and neurons, which is an established method of relating the behaviour of the neurons to functional response after implantation. It was found that conditions that related to higher expression of p75NTR and S100β (laminin coated wells, standard media, shorter time in culture) led to longer and more numerous neurites. From this it can be established that levels of p75NTR and S100β expression are good predictive tools for the extent to which OECs can support neural regeneration in culture. The next step would be to relate the expression of these markers to the myelination of neurons. Thy1 expression was not found to be related to neurite extension and purified populations of negative and positive Thy1 OECs resulted in longer neurites than the original mixed population. This could be due to lateral inhibition but further work is required to confirm this theory. Results described in this thesis have demonstrated that caution needs to be applied when scaling rat studies to human cell work. It has also shown that the method and timing of detecting protein expression can be vital to the results observed. These are key aspects that need to be considered in order to fully characterise cell populations, especially one as variable as OECs. The methods used in this work showed an increase in p75NTR and S100β expression led to longer neurites extended from neurons. Further work should be carried out in order to fully understand the interaction between OECs and neurons and to explain the potential lateral inhibition pattern observed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:747645 |
| Date | January 2018 |
| Creators | Wood, Rachael Claire |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/10047503/ |
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