BULBAR and MUCOSAL OECs: FACS and CELL CYCLE

Transplantation of olfactory ensheathing cells (OECs) is a promising route for CNS repair. There have, however, been major discrepancies between the results from different groups. Part of this can be attributed to variations in cell sources and culture protocols. Accurate estimation of the proportions of OECs and their associated fibroblasts (ONFs) and their evolution with time in culture is an essential baseline for establishing the reparative properties of transplants. In this study we compare the evolution of cultures from the superficial layers of the olfactory bulb with tissue from the olfactory mucosa, both whole and split into lamina propria and epithelial layer. In all cases the relationship of antigenicity to morphology depended on plating density. We used FACS based on p75 and Thy1 to provide a robust and objective numerical estimate of the numbers of OECs and ONFs respectively in the cultures. A novel four colour simultaneous antigenic bivariate cell cycle analysis shows that proliferation of OECs is time limited, and is unable to prevent an overall loss of OECs with time. Overall the numbers of OECs in the cultures was inversely correlated with the deposition of fibronectin. Further culture of the cells purified by flow cytometry shows that, whereas the Thy1 population is terminally differentiated, the p75 population from the mucosal samples generates sub-populations with different antigenic phenotypes, including the re-appearance of a sub-population of p75 cells expressing FN. Culturing epithelial samples at high density reveals an unexpected transient stem cell like population of rapidly proliferating p75 positive cells.