Recombinant canine basic fibroblast growth factor-induced differentiation of canine bone marrow mesenchymal stem cells into voltage- and glutamate-responsive neuron-like cells

Abstract

Introduction: Basic fibroblast growth factor (bFGF) is a promising cytokine in regenerative therapy for spinal cord injury. In this study, recombinant canine bFGF (rc-bFGF) was synthesized for clinical use in dogs, and the ability of rc-bFGF to differentiate canine bone marrow mesenchymal stem cells (BMSCs) into functional neurons was investigated. Methods: The rc-bFGF was synthesized using a wheat germ cell-free protein synthesis system. The expression of rc-bFGF mRNA in the purification process was confirmed using a reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was performed to confirm the antigenic property of the purified protein. To verify function of the purified protein, phosphorylation of extracellular signal-regulated kinase (ERK) was examined by in vitro assay using HEK293 cells. To compare the neuronal differentiation capacity of canine BMSCs in response to treatment with rc-bFGF, the cells were divided into the following four groups: control, undifferentiated, rh-bFGF, and rc-bFGF groups. After neuronal induction, the percentage of cells that had changed to a neuron-like morphology and the mRNA expression of neuronal markers were evaluated. Furthermore, to assess the function of the canine BMSCs after neuronal induction, changes in the intracellular Ca2+ concentrations after stimulation with KCl and l-glutamate were examined. Results: The protein synthesized in this study was rc-bFGF and functioned as bFGF, from the results of RT-PCR, western blotting, and the expression of pERK in HEK293 cells. Canine BMSCs acquired a neuron-like morphology and expressed mRNAs of neuronal markers after neuronal induction in the rh-bFGF and the rc-bFGF groups. These results were more marked in the rc-bFGF group than in the other groups. Furthermore, an increase in intracellular Ca2+ concentrations was observed after the stimulation of KCl and l-glutamate in the rc-bFGF group, same as in the rh-bFGF group. Conclusions: A functional rc-bFGF was successfully synthesized, and rc-bFGF induced the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells. Our purified rc-bFGF may contribute, on its own, or in combination with canine BMSCs, to regenerative therapy for spinal cord injury in dogs.

Keywords

• Basic fibroblast growth factor
• Bone marrow
• Dog
• Differentiation
• Neuron
• Mesenchymal stem cell