Semaphorin 3A promotes osteogenic differentiation in human alveolar bone marrow mesenchymal stem cells

Abstract

The aim of the present study was to investigate the role of Semaphorin 3A (Sema3A) in the osteogenic differentiation of human alveolar bone marrow mesenchymal stem cells (hABMMSCs). To investigate whether Sema3A affects hABMMSC proliferation and osteogenic differentiation, a stable Sema3A-overexpression cell line was generated by infection with the pAdCMV-SEMA3A-MCS-EGFP vector. Cell counting kit-8 and clone formation assays were performed to determine the proliferation ability of hABMMSCs, while cell osteogenic differentiation was assayed using Alizarin Red S staining. In addition, reverse transcription-quantitative polymerase chain reaction was employed to detect the mRNA expression level of osteogenesis-associated genes, Runt-related transcription factor 2 (Runx2), osteopontin (Opn) and osteocalcin (Ocn), during the osteogenic differentiation. The results revealed that, compared with the normal control group, the cell morphology of the infected cells was stable and no significant alterations were observed. Overexpression of Sema3A in hABMMSCs significantly increased the cell proliferation ability compared with the control group. Furthermore, the Alizarin Red S staining assay results indicated that the ossification process of hABMMSCs overexpressing Sema3A was evidently faster in comparison with that of the control group cells. Overexpression of Sema3A by pAdCMV-SEMA3A-MCS-EGFP infection also significantly increased the mRNA expression levels of the osteogenic marker genes Runx2, Opn and Ocn. In conclusion, Sema3A was observed to be a key positive regulator in hABMMSC osteogenic differentiation.

Keywords: human alveolar bone marrow mesenchymal stem cells; osteogenic differentiation; semaphorin 3A.

Figure 1.

Overexpression of Sema3A is successfully achieved by vector infection and the cell morphology is examined. Representative overlay images of (A) phase contrast and (B) fluorescence microscopy demonstrated the transduction efficiency of Sema3A for green fluorescent protein using adenoviral transduction. The cell morphology of the (C) untreated control cells, (D) cells infected with pCMV-MCS-EGFP (control vector) and (E) cells infected with pAdCMV-SEMA3A-MCS-EGFP (Sema3A overexpressing vector) was examined under an electron microscope. Experiments were performed in triplicate. Sema3A, semaphorin 3A.

Figure 2.

Sema3A promotes the cell proliferation ability of human alveolar bone marrow mesenchymal stem cells. (A) CCK-8 proliferation assay was conducted, and the OD was measured at 490 nm to determine the cell proliferation. (B) Clone formation assay results. The number of cell clones with >50 cells was counted under a microscope and the cloning efficiency was calculated. Tests were performed in triplicate, and data are represented as the mean ± standard deviation. Sema3A, semaphorin 3A; CCK-8, Cell Counting Kit-8; OD, optical density.

Figure 3.

Alizarin Red S staining of hABMMSCs. At 24 h after third generation log-phase hABMMSCs were infected with the pCMV-MCS-EGFP (Con) or pAdCMV-SEMA3A-MCS-EGFP (Sema3A) vector, the cells were seeded in 6-well plates and grown in osteogenesis-inducing media. Following incubation for 7, 10, 14 and 21 days, Alizarin Red S staining was performed (n=3 per experiment). Sema3A, semaphorin 3A; Con, control; hABMMSCs, human alveolar bone marrow mesenchymal stem cells.

Figure 4.

Relative expression levels of the osteogenesis-associated genes. After infection for 7 and 14 days, reverse transcription-quantitative polymerase chain reaction was performed to detect the mRNA expression levels of osteogenesis-associated genes (Runx2, Opn and Ocn). GAPDH served as an internal control. Experiments were performed in triplicate, and data are represented as the mean ± standard deviation. ***P<0.001, **P<0.01, *P<0.05 vs. control as indicated. Sema3A, semaphorin 3A; Runx2, runt-related transcription factor 2; Opn, osteopontin; Ocn, osteocalcin.