title: Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor.
abstract: We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca(2+) mobilization of the cells was evaluated using the Ca(2+) indicator Fluo3 to monitor Ca(2+) influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca(2+) levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.
authors.affiliation: Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan.
authors.forename: Rei
authors.initials: R
authors.lastname: Nakano
authors.forename: Kazuya
authors.initials: K
authors.lastname: Edamura
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nakayama
authors.forename: Kenji
authors.initials: K
authors.lastname: Teshima
authors.forename: Kazushi
authors.initials: K
authors.lastname: Asano
authors.forename: Takanori
authors.initials: T
authors.lastname: Narita
authors.forename: Ken
authors.initials: K
authors.lastname: Okabayashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sugiya
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004285:Dogs; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt