Recombinant canine basic fibroblast growth factor-induced differentiation of canine bone marrow mesenchymal stem cells into voltage- and glutamate-responsive neuron-like cells
- PMID: 33426210
- PMCID: PMC7770349
- DOI: 10.1016/j.reth.2020.07.005
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: BMSCs, bone marrow mesenchymal stem cells; Basic fibroblast growth factor; Bone marrow; Differentiation; Dog; EDTA, ethylenediaminetetraacetic acid; ERK, extracellular signal-regulated kinase; FBS, fatal bovine serum; FGFR, basic fibroblast growth factor receptor; GUSB, β-glucuronidase; HEK293, human embryonic kidney cells 293; HRP, horseradish peroxidase; Mesenchymal stem cell; Neuron; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PI3K, phosphatidylinositol 3-kinase; RT-PCR, reverse transcription-polymerase chain reaction; bFGF, basic fibroblast growth factor; cDNA, complementary DNA; mRNA, messenger ribonucleic acid; pERK, phosphorylated extracellular signal-regulated kinase; αMEM, alpha modified eagle minimum essential medium.
© 2020 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.
Conflict of interest statement
None.
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