DNA double-strand breaks and chromosomal aberrations after treatment with N-alkylating agents likely arise as a result of replication fork collision with single-strand breaks generated during base excision repair.

Olfactory ensheathing cells (OECs) have gained wide interest because of their unique regeneration-promoting capacity. However, despite their frequent use in regeneration studies, the characterization of the cells has remained fragmentary. In the present study, we analyzed freshly dissociated neonatal rat OECs at the light and electron microscopic level and studied their fate in vitro using a novel two-step labeling protocol based on antibody internalization. We report the identification and characterization of two distinct OEC populations in situ and in primary cell suspensions that differed in number, p75 NGF receptor expression, and O4 immunoreactivity. The major OEC population in primary cells suspensions did not express p75 but stained positive for the glycolipid O4 (p75-/O4+). During culturing, these cells upregulated p75 expression and lost O4 immunoreactivity. Conversely, the minor OEC population consisted of p75+/O4- OECs that maintained p75 expression in vitro. Interestingly, ultrastructural analysis revealed not only that O4 immunoreactivity of p75- OECs was, in fact, due to O4+ axonal fragments adhering to the cell surface but also that p75- OECs rapidly phagocytosed these fragments in vitro. Taken together, the identification of two distinct OEC populations in the neonatal olfactory bulb that converge into single p75+ phenotype in vitro is reported. The observation that upregulation of p75 receptor expression in vitro was only apparent in those OECs closely associated with O4+ axonal processes may suggest that axonal signalling in vivo negatively regulates p75 receptor expression. The strong phagocytic activity of OECs in vitro may reflect one important aspect of their physiological function.

Clonal in vitro analysis represents a powerful tool for studying cellular differentiation. In the present study, microscope-assisted single cell transfer was combined with immunofluorescence to establish clonal cultures of identified primary rat olfactory ensheathing cells (OECs). During development, OECs originate from the neural crest, a transient population of multipotent cells. Since only neural crest cells have been analyzed at clonal density, it remained unclear whether OECs may retain multipotent features. Neurotrophin receptor p75 (p75(NTR))-immunolabelled rat OECs were seeded at clonal density under visual control using a semiautomated cell selection and transfer device (Quixell™) and emerging clones were analyzed with regard to proliferation and antigenic expression. We demonstrate that OECs from neonatal (P1) and 7 day-old (P7) but not from adult rats formed clones in the presence of OEC- and astrocyte-conditioned media (OEC-CM, A-CM). Cloning efficiency but not in vitro growth of OECs was independent of age but increased upon treatment with OEC-CM. Interestingly, about 75 % of P1 compared to 27 % of P7 OEC clones lost p75(NTR) expression during 2 weeks in vitro and acquired immunoreactivity for Thy-1. The observation that primary OECs from P1 lost expression of p75(NTR) at clonal density and initiated expression of the fibroblast marker Thy-1 may suggest that their developmental potential is greater than previously anticipated. Since microscope-assisted selection of immunofluorescent cells combined with semiautomated transfer guarantees monoclonality in a single step and affords selection of cells according to fluorescent label and/or morphological criteria it may be relevant for a variety of other cell types.

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