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. 2019 Oct 21;5(10):e02663.
doi: 10.1016/j.heliyon.2019.e02663. eCollection 2019 Oct.

Simvastatin enhances proliferation and pluripotent gene expression by canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) in vitro

Sirirat Nantavisai  1   2 , Watchareewan Rodprasert  1   2 , Koranis Pathanachai  1   2   3 , Parattakorn Wikran  4 , Podchana Kitcharoenthaworn  4 , Saritpakorn Smithiwong  4 , Suyakarn Archasappawat  4 , Chenphop Sawangmak  1   2   3
Affiliations

Simvastatin enhances proliferation and pluripotent gene expression by canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) in vitro

Sirirat Nantavisai et al. Heliyon. .

Erratum in

Abstract

Establishing the intervention to enhance proliferation and differentiation potential is crucial for the clinical translation of stem cell-based therapy. In this study, the effects of simvastatin on these regards were explored. Canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) were treated with 4 doses of simvastatin, 0.1, 1, 10, and 100 nM. Simvastatin in low-dose range, 0.1 and 1 nM, enhanced dose-dependent cell proliferation at day 5 and 7. Exploration of the mechanisms revealed that simvastatin in low-dose range dose-dependently upregulated sets of cell cycle regulators, Cyclin D1 and Cyclin D2; proliferation marker, Ki-67; and anti-apoptotic gene; Bcl-2. Interestingly, pluripotent markers, Rex1 and Oct4, were dramatically increased upon the low-dose treatment. Contrastingly, treatment with high-dose simvastatin suppressed the expression of those genes. Thus, the results suggested beneficial effects of simvastatin on cBM-MSCs proliferation and expansion. Further study regarding differentiation potential and underlying mechanisms will accelerate the clinical application of the molecule on veterinary stem cell-based therapy.

Keywords: Canine bone marrow-derived mesenchymal stem cells (cBM-MSCs); Cell biology; Cell culture; Cell death; Cytotoxicity; Proliferation; Regenerative medicine; Simvastatin; Stem cell research; Stemness genes.

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Figures

Fig. 1
Fig. 1
Morphology of canine bone marrow-derived mesenchymal stem cells (cBM-MSCs). Morphology of the isolated cBM-MSCs was observed under phase-contrast microscope. Magnifications were 40X (A) and 100X (B), respectively.
Fig. 2
Fig. 2
Relative proliferation of cBM-MSCs upon simvastatin treatment. Proliferative effect of simvastatin was tested. Four simvastatin doses (0.1, 1, 10, and 100 nM) were used, and the MTT assay was explored at day 1, 5, and 7 after treatment. The results were normalized with untreated control group in each day and illustrated as relative cell proliferation. The asterisks indicate statistical difference compared with the control (p-value < 0.05).
Fig. 3
Fig. 3
cBM-MSCs morphology upon simvastatin treatment. Morphology of cBM-MSCs was explored at day 1 and 5 upon simvastatin (0.1, 1, 10, and 100 nM) treatment. Magnification was 40X.
Fig. 4
Fig. 4
Effect of simvastatin on cBM-MSCs pluripotent genes expression in vitro. The effect of simvastatin (0.1, 1, 10, and 100 nM) on cBM-MSCs pluripotent gene expression was analyzed by using RT-qPCR. mRNA expressions of Rex1 and Oct4 were explored at 48 h post-treatment. mRNA expressions of the genes were presented as relative expression by normalizing with reference gene, Gapdh, and the control. The asterisks indicate statistical difference compared with the control (p-value < 0.05).
Fig. 5
Fig. 5
Effects of simvastatin on cBM-MSCs cell cycle-regulated gene expression in vitro. The effect of simvastatin (0.1, 1, 10, and 100 nM) on cBM-MSCs cell cycle-regulated gene expression was analyzed by using RT-qPCR. The mRNA expressions were analyzed at 48 h post-treatment. Cyclin D1, Cyclin D2, Ki-67, and TP53 were explored. mRNA expressions of the genes were presented as relative expression by normalizing with reference gene, Gapdh, and the control. The asterisks indicate statistical difference compared with the control (p-value < 0.05).
Fig. 6
Fig. 6
Effects of simvastatin on cBM-MSCs apoptotic gene expression in vitro. The effect of simvastatin (0.1, 1, 10, and 100 nM) on cBM-MSCs apoptotic gene expression was analyzed by using RT-qPCR. The mRNA expressions were analyzed at 48 h post-treatment. Bcl-2, Bcl-2-L1, Caspase 3, Caspase 8, and Caspase 9 were explored. mRNA expressions of the genes were presented as relative expression by normalizing with reference gene, Gapdh, and the control. The asterisks indicate statistical difference compared with the control (p-value < 0.05).
Fig. 7
Fig. 7
Live/dead and Annexin V-FITC/PI staining of simvastatin-treated cBM-MSCs. The occurrence of apoptotic and necrotic cBM-MSCs upon simvastatin treatment (0.1, 1, 10, and 100 nM) was analyzed using live/dead (NUCLAER-ID® Blue/Red cell viability) (A) and Annexin V-FITC/PI (B) staining at day 7. For live/dead staining, all nuclei and necrotic cells were showed in DAPI and Cy3 panels, respectively. For Annexin V-FITC/PI staining, early apoptosis was showed as FITC-stained cell, while late apoptosis/necrosis was PI-stained cells.
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