31.
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/m
l). 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.
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