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. 2020 Aug 1:15:121-128.
doi: 10.1016/j.reth.2020.07.005. eCollection 2020 Dec.

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

Affiliations

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

Kazuya Edamura et al. Regen Ther. .

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.

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Conflict of interest statement

None.

Figures

Fig. 1
Fig. 1
The expression of rc-bFGF mRNA in the purification process of rc-bFGF by the cell-free protein synthesis system. Lane 1–6: the results of RT-PCR in reaction solution after synthesis of canine bFGF. Lane 7-12: the results of RT-PCR in reaction solution without synthesis of canine bFGF (negative control). Lane 13–18: the results of PCR in the pET28a plasmid for synthesis by E. coli (positive control).
Fig. 2
Fig. 2
Western blotting of purified protein. Band formation was observed at almost same position as rc-bFGF synthesized using E. coli (arrows). Lane 1: reaction solution after synthesis of canine bFGF. Lane 2: reaction solution without synthesis of canine bFGF (negative control). Lane 3: rc-bFGF synthesized using E. coli, Lane 4: commercially available rh-bFGF (positive control).
Fig. 3
Fig. 3
Phosphorylation of ERK in HEK293 cells after stimulation by purified protein. pERK expression was observed after stimulation of HEK293 cells by our purified protein, as it was after stimulation by commercially available rh-bFGF. Lane 1: extract from HEK293 cells after no stimulation of rc-bFGF or rh-bFGF (negative control). Lane 2: extract from HEK293 cells after stimulation by 5 ng/mL of rc-bFGF. Lane 3: extract from HEK293 cells after stimulation by 15 ng/mL of rc-bFGF. Lane 4: rc-bFGF synthesized solution only. Lane 5: extract from HEK293 cells after stimulation by 5 ng/mL of commercially available rh-bFGF (positive control). Lane 6: blotting buffer.
Fig. 4
Fig. 4
The morphologies of canine BMSCs at 10 days of neuronal induction. Canine BMSCs were changed to a neuron-like morphology after neuronal induction in the rh-bFGF and the rc-bFGF groups (arrows). A: control group, B: undifferentiated group, C: rh-bFGF group, and D: rc-bFGF group.
Fig. 5
Fig. 5
The percentage of canine BMSCs that had changed to a neuron-like morphology at 10 days of neuronal induction. Error bars show mean ± standard error (n = 6 in each group). Asterisks indicate statistical difference among the groups (P < 0.05). Control; control group, Undifferent; undifferentiated group, rh-bFGF; rh-bFGF group, rc-bFGF; rc-bFGF group.
Fig. 6
Fig. 6
The mRNA expression of neuronal markers pre- and post-neuronal induction. A: NEFL, B: NEFH, C: MAP2, and D: TUBB3. Error bars show mean ± standard error (n = 6 in each group). Asterisks indicate statistical difference between pre- and post-neuronal induction (P < 0.05). Control; control group, Undifferent; undifferentiated group, rh-bFGF; rh-bFGF group, rc-bFGF; rc-bFGF group.
Fig. 7
Fig. 7
Relative fluorescence intensity of intracellular Ca2+ concentration after the stimulation by 50 mM KCl (A, B, C, D) or 100 μM l-glutamate (E, F, G, H). A and E: Control group, B and F: Undifferentiated group, C and G: rh-bFGF group, D and H: rc-bFGF group.

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