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Differentiation of Mesenchymal Stem Cells into Cells with a Neuronal Phenotype In Vitro

1Linke S., 2Eisermann E., 2Zander A., 1Richard G., 3Schachner M., 2Lange C., 3Bartsch U.,
1Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Augenheilkunde (Hamburg)
2Einrichtung für Knochenmarktransplantation, Universitätsklinikum Eppendorf (Hamburg)
3Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Institut für Biosynthese neuraler Strukturen (Hamburg)

Purpose: To determine the potential of adult rat bone marrow derived mesenchymal stem cells (MSC) to differentiate into nerve cells in vitro.
Methods: MSCs were harvested from Sprague Dawley rats and expanded in culture as described previously (Haynesworth et al. 1992). In short, after flushing the bone marrow (femur and tibia), cells were transferred to plastic culture dishes containing DMEM-medium and 20% fetal bovine serum (FBS). After the third passage, MSCs were plated at a density of ~2500 cells/cm2 and induced to differentiate in a medium containing 5% FBS, 0,5mM 3-isobutyl 1-methylxanthine (IBMX) and 10µM forskolin or in a medium lacking FBS and containing 2% dimethylsulfoxide (DMSO) and 200µM butylhydroxianisol (BHA). Morphological differentiation was analyzed by phase contrast microscopy. For immunocytochemical analysis differentiated cells were stained with ßIII-tubulin and neurofilament antibodies.
Results: MSCs could be rapidly expanded under our culture conditions. After three passages, the undifferentiated and homogenous (confirmed by FACS-analysis) cell population could be induced to differentiate into cells displaying a neuronal morphology by two distinct differentiation protocols (Woodbury et al., 2000; Deng et al., 2001). Within 45 minutes after initiation of differentiation with DMSO/BHA, undifferentiated, flat MSCs became spherical and extended long neurite-like processes. When cells were maintained for eight hours in differentiation medium, more than 75% of the cells displayed a neuron-like morphology. In comparison, elevated levels of intracellular cAMP, induced by exposure of cells to IBMX and forskolin, resulted in rather slow differentiation of a small fraction of MSCs into neuron-like cells. Interestingly, these morphological changes coincided with increased expression of ß-tubulin III and faint neurofilament immunoreactivity, suggesting neuronal differentiation.
Conclusions: Undifferentiated cells derived from adult rat bone marrow could be induced to differentiate into cells displaying a neuron-like morphology by two distinct differentiation protocols. Experiments are in progress to determine the fate of these cells after transplantation into the pathologically altered mouse retina.