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

• Published in: Regenerative therapy Vol. 15; pp. 121 - 128
• Main Authors: Edamura, Kazuya, Takahashi, Yusuke, Fujii, Airi, Masuhiro, Yoshikazu, Narita, Takanori, Seki, Mamiko, Asano, Kazushi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-12-2020; Japanese Society for Regenerative Medicine; Elsevier
• Subjects: Basic fibroblast growth factor; Bone marrow; Differentiation; Dog; Mesenchymal stem cell; Neuron; Original; Mesenchymal stem cell; PBS; HRP; Basic fibroblast growth factor; mRNA; αMEM; EDTA; FGFR; bFGF; HEK293; Neuron; PI3K; RT-PCR; FBS; Bone marrow; cDNA; Differentiation; GUSB; Dog; pERK; BMSCs; PCR; ERK; GUSB, β-glucuronidase; FBS, fatal bovine serum; EDTA, ethylenediaminetetraacetic acid; cDNA, complementary DNA; HEK293, human embryonic kidney cells 293; PBS, phosphate buffered saline; BMSCs, bone marrow mesenchymal stem cells; HRP, horseradish peroxidase; ERK, extracellular signal-regulated kinase; FGFR, basic fibroblast growth factor receptor; pERK, phosphorylated extracellular signal-regulated kinase; bFGF, basic fibroblast growth factor; PCR, polymerase chain reaction; PI3K, phosphatidylinositol 3-kinase; mRNA, messenger ribonucleic acid; RT-PCR, reverse transcription-polymerase chain reaction; αMEM, alpha modified eagle minimum essential medium
• ISSN: 2352-3204; 2352-3204
• DOI: 10.1016/j.reth.2020.07.005

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.
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.
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.
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.
•Functional rc-bFGF was successfully synthesized.•rc-bFGF induced the differentiation of canine BMSCs into neuron-like cells.•rc-bFGF may aid in regenerative therapy of spinal cord injury in dogs.