Clonal In Vitro Analysis of Neurotrophin Receptor p75-Immunofluorescent Cells Reveals Phenotypic Plasticity of Primary Rat Olfactory Ensheathing Cells |
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Authors: | Christian Ebel Gudrun Brandes Christine Radtke Karl Rohn Konstantin Wewetzer |
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Institution: | 1. Department of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str.1, 30625, Hannover, Germany 2. Department of Cell Biology, Hannover Medical School, 30625, Hannover, Germany 3. Department of Plastic, Hand- and Reconstructive Surgery, Hannover Medical School, 30625, Hannover, Germany 4. Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, 30559, Hannover, Germany 5. Center of Systems Neurosciences, University of Veterinary Medicine Hannover, 30559, Hannover, Germany
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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 (p75NTR)-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 p75NTR expression during 2 weeks in vitro and acquired immunoreactivity for Thy-1. The observation that primary OECs from P1 lost expression of p75NTR 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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