Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

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  • NAKANO Rei
    Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan
  • EDAMURA Kazuya
    Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan
  • NAKAYAMA Tomohiro
    Laboratory of Veterinary Radiology, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan
  • TESHIMA Kenji
    Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan
  • ASANO Kazushi
    Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan
  • NARITA Takanori
    Laboratory of Veterinary Biochemistry, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan
  • OKABAYASHI Ken
    Laboratory of Veterinary Biochemistry, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan
  • SUGIYA Hiroshi
    Laboratory of Veterinary Biochemistry, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan

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  • Surgery : Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

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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 Ca2+ mobilization of the cells was evaluated using the Ca2+ indicator Fluo3 to monitor Ca2+ 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 Ca2+ 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.

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