Characterization of bone marrow-derived mesenchymal stem cells from dimethyloxallyl glycine-preconditioned mice: Evaluation of the feasibility of dimethyloxallyl glycine as a mobilization agent

Abstract

The prolyl hydroxylase inhibitor dimethyloxallyl glycine (DMOG) has been increasingly studied with regards to stem cell therapy. Previous studies have demonstrated that endogenous mesenchymal stem cells (MSCs) may be mobilized into peripheral circulation by pharmaceutical preconditioning. In addition, our previous study confirmed that DMOG, as a novel mobilization agent, could induce mouse/rat MSC migration into peripheral blood circulation. Therefore, the present study conducted studies to characterize bone marrow‑derived MSCs (BM‑MSCs) collected from mice following DMOG intraperitoneal injection. The surface antigen immune phenotype, differentiation capability, proliferative ability, migratory capacity and paracrine capacity of the BM‑MSCs collected from DMOG‑preconditioned mice (DBM‑MSCs) or normal saline‑treated mice (NBM‑MSCs) were evaluated by means of flow cytometry, differentiation induction, Cell Counting kit‑8, Transwell assay and enzyme-linked immunosorbent assay, respectively. Compared with NBM‑MSCs, DBM-MSCs displayed a similar immune phenotype and multilineage differentiation capability, reduced proliferative ability and migratory capacity, and similar transforming growth factor and platelet-derived growth factor secretion capacity. These results provide a novel insight into the biological properties of BM‑MSCs from mice preconditioned with DMOG. DBM-MSCs exhibited slightly distinct characteristics to NBM-MSCs; however, they may have therapeutic potential for future stem cell therapy. In addition, the present study suggested that DMOG may be used as a novel mobilization agent in future clinical trials as no adverse effects were observed.

Figure 1

Morphology and immune phenotype of NBM-MSCs and DBM-MSCs. (A) NBM-MSCs and DBM-MSCs from passage 3 were observed under an inverted fluorescence microscope. Scale bar, 500 μm. (B) Surface antigen markers on passage 3 NBM-MSCs and DBM-MSCs were detected by flow cytometry. NBM-MSCs and DBM-MSCs expressed CD44 (93.8 and 97.9%, respectively) and CD90 (93.5 and 96.6%, respectively), but not CD45 (2.59 and 1.70%, respectively). Experiments were performed in triplicate, and there was no significant difference between them (P>0.05). NBM-MSCs, bone marrow-derived mesenchymal stem cells from normal saline-treated mice; DBM-MSCs, bone marrow-derived mesenchymal stem cells from dimethyloxallyl glycine-preconditioned mice; CD, cluster of differentiation.

Figure 2

Multilineage differentiation capacity of NBM-MSCs and DBM-MSCs. (A) NBM-MSCs and DBM-MSCs were cultured in Ad., Os., or Ch. differentiation medium. Adipogenic differentiation was stained with Oil Red O. Osteogenic differentiation was stained with Von Kossa. Chondrogenic differentiation was stained with Toluidine Blue. (Magnification, x100). (B) NBM-MSCs and DBM-MSCs were induced into neuron-like cells. Immunofluorescence staining: Nuclei were dyed blue with DAPI and Nestin expression was dyed green. (Magnification, ×200). NBM-MSCs, bone marrow-derived mesenchymal stem cells from normal saline-treated mice; DBM-MSCs, bone marrow-derived mesenchymal stem cells from dimethyloxallyl glycine-preconditioned mice; Ad., adipogenic; Os., osteogenic; Ch., chondrogenic; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

Cell proliferation was detected using the CCK-8 assay. There were significant differences from day 3 to day 7 between the NBM-MSCs and DBM-MSCs. Data are presented as the mean ± standard deviation. ^*P<0.05. CCK-8, Cell Counting kit-8; BM-MSCs, bone marrow-derived mesenchymal stem cells from normal saline-treated mice; DBM-MSCs, bone marrow-derived mesenchymal stem cells from dimethyloxallyl glycine-preconditioned mice; OD, optical density.

Figure 4

Migratory ability of NBM-MSCs and DBM-MSCs to chemokine SDF-1α. (A) Migration of NBM-MSCs and DBM-MSCs induced by SDF-1α was detected using a Transwell assay. Migrated cells were stained with crystal violet. Scale bar, 100 μm. (B) Quantification of migration results. Data are presented as the mean ± standard deviation. ^*P<0.05. BM-MSCs, bone marrow-derived mesenchymal stem cells from normal saline-treated mice; DBM-MSCs, bone marrow-derived mesenchymal stem cells from dimethyloxallyl glycine-preconditioned mice; SDF-1α, stromal cell-derived factor-1α.

Figure 5

Paracrine capacity of NBM-MSCs and DBM-MSCs. (A) Quantification of TGF concentration in the culture supernatant of NBM-MSCs and DBM-MSCs. There was no significant difference between the expression levels (P>0.05). (B) Quantification of PDGF concentration in the culture supernatant of NBM-MSCs and DBM-MSCs. No significant difference was identified between them (P>0.05). TGF and PDGF concentrations were detected using enzyme-linked immunosorbent assays. Data are presented as the mean ± standard deviation. BM-MSCs, bone marrow-derived mesenchymal stem cells from normal saline-treated mice; DBM-MSCs, bone marrow-derived mesenchymal stem cells from dimethyloxallyl glycine-preconditioned mice; TGF, transforming growth factor; PDGF, platelet-derived growth factor.