Comparison on senescence-related properties and osteogenic differentiation capacity of bone-derived mesenchymal stem cells from mice in different ages

Abstract

Objective To explore the age-related biological properties of bone-derived mesenchymal stem cells (BMSCs) from mice of different age groups and their osteogenic differentiation induced by bone morphogenetic protein 2 (BMP2). Methods Eight C57BL/6J mice were divided into a young group (4 weeks old, weighing 10~15 g, n=4) and an old group (12 months old, weighing 20~25 g, n=4), with half male and half female. MSCs were extracted from the whole bones of the 2 groups of mice. After the obtained cells were identified with flow cytometry for the surface markers, β-galactosidase staining was employed to compare the senescence level of BMSCs, MTT and EdU incorporation assays were conducted to compare the proliferation and self-renewal abilities of between the 2 groups. Western blotting was employed to analyze the expression of CyclinD1 and P21 in BMSCs. Then ALP staining, Alizarin Red staining and RT-qPCR were used to evaluate the osteogenic differentiation ability of the cells. RNA sequencing was performed to compare the differential gene expression in BMP2-induced BMSCs. Lastly, the sequencing results were re-confirmed by using flow cytometry. Results Flow cytometry showed that the sorted and cultured mouse BMSCs met the criteria for MSCs. The results of β-galactosidase staining indicated that the senescence level of BMSCs in the old group was significantly higher than that in the young group (P < 0.05). MTT and EdU doping experiments revealed that the cell viability and proliferation ability of BMSCs were significantly lower in the old group than the young group (P < 0.05). Western blotting displayed that the expression level of cell cycle protein CyclinD1 was lower, whereas that of cell cycle inhibitory factor P21 was significantly higher in the BMSCs from the old group than the cells from the young group (P < 0.05). ALP/Alizarin Red staining and RT-qPCR demonstrated that the BMSCs from the young group had stronger osteogenic differentiation capacity after BMP2 treatment when compared the cells of the old group (P < 0.05). RNA sequencing results displayed that the changing profile of CD51 expression was in opposite trends in the young and old BMSCs after BMP2 treatment. Finally, flow cytometry revealed that the percentage of CD51+ cells within the CD45- cells was significantly higher in the young group than the old group. Conclusion The decrease in the percentage of CD51+ cells among CD45- cells in aged BMSCs is closely associated with their decreased responsiveness to BMP2-induced osteogenic differentiation.

Keywords

• bone derived mesenchymal stem cells
• bmp2
• osteogenic differentiation
• aging
• cluster of differentiation 51