title: Clonal in vitro analysis of neurotrophin receptor p75-immunofluorescent cells reveals phenotypic plasticity of primary rat olfactory ensheathing cells.
abstract: 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.
authors.affiliation: Department of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Ebel
authors.forename: Gudrun
authors.initials: G
authors.lastname: Brandes
authors.forename: Christine
authors.initials: C
authors.lastname: Radtke
authors.forename: Karl
authors.initials: K
authors.lastname: Rohn
authors.forename: Konstantin
authors.initials: K
authors.lastname: Wewetzer
mesh_terms: D000818:Animals; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D020800:Receptor, Nerve Growth Factor / Q000378:metabolism*
chemical_list: D020800:Receptor, Nerve Growth Factor