Kuijten, MMP; (2016) Characterisation of the olfactory system for cell-based therapies to treat neurodegenerative disease. Doctoral thesis , UCL (University College London).

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Abstract

The olfactory mucosa (OM) is an area of life-long constant regeneration and constitutes a potential cell source for cell-based treatments for neuronal injury, including olfactory ensheathing cells (OECs). Inconsistency in knowledge regarding cell isolation, characteristics and culture methods may influence the efficacy of OEC-based therapies. The aim of the work was to characterise the olfactory mucosa and the bulb to develop a method for isolation of a relatively pure population of OECs as well as to determine a suitable source of OECs. For that purpose immunohistochemical and gene analysis was performed to characterise the OM of postnatal day 16 (young) rat to identify the different cell types in the tissue. The results suggested that ecto-mesenchymal stem cells are present at the junction between the olfactory epithelium and lamina propria (LP) and that in addition, a neural crest (derived) stem cell (NCSC) population was also present at the border of the LP, near the cartilage, and that both cell populations share marker expression with OECs. Furthermore, OM cells were cultured and OECs were isolated using differential adhesion. From these studies it was inferred that this method may result in NCSC contamination in the enriched OEC culture and that a distinctive combination of markers is required to select the OECs. A similar method was used to select for NCSCs using short and long (‘all cells’) trypsination durations. The results suggested the presence of EMSCs in the short trypsination population, whereas the NCSCs were suggested to be present in the ‘all cells’ population. Although differential adhesion may select for certain stem cell populations, the method would not suffice to obtain pure populations. As alternative strategy to select for OECs, EMSCs and NCSCs an attempt at FACS-based cell sorting was made based on the combination of p75, SSEA-1 and ErbB4 expression. Unfortunately, due to low cell viability it was not possible to obtain purified populations. In addition, a comparison was made between the OM of young and adult rat and between the olfactory mucosa and the bulb to determine the most suitable tissue source of OECs. Based on immunohistochemical, gene, Western blotting and flow cytometry analysis, the NCSC population was also suggested to be present in the OM of adult rats and in the olfactory bulb. To obtain insight into neurotrophic ability of the olfactory mucosa, gene expression of six neurotrophic factors in OM tissue and isolated cell populations were studied. From the results it was inferred that BDNF, CNTF, GDNF, NGF, NT3 and NT-4/5 were expressed in both OM tissue and cultured OM cells from young and adult rats, although not to the same extent. These results may suggest that the cells release neurotrophic factors and may be used in neuroprotection strategies to treat neuronal injury. When comparing gene expression of the six factors in cultured OM cell populations to BMSCs, the results may indicate that their neurotrophic potential is not higher compared to BMSCs. The knowledge obtained from the characterisation of the olfactory system may be of use to develop new or improve existing treatment strategies for neuronal injury.

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