Senescence dependent BMP-2 effect on bone marrow mesenchymal stem cell differentiation

Abstract

Background: Mesenchymal stem cells (MSCs) are cells having potential to differentiate various type of cells including osteoblasts, adipocytes, or chondrocytes. Bone morphogenetic protein-2 (BMP-2) is considered as a powerful inducer to promote cascade of differentiation into osteoblasts. Recently, BMP-2 has been tried as a bone graft substitute when spinal fusion surgery is performed in old age patients. However, lack of knowledge about the differentiation of MSCs by BMP-2 and the effect of aging has been one of problems for extensive use of BMP-2. In this regard, we intended to compare the effect of recombinant human BMP-2 (rhBMP-2) on differentiation of MSCs into osteoblasts and adipocytes between aging MSCs and young (control) MSCs.

Materials and methods: Cellular aging (senescence) was induced by replicative passages with cultures under high level glucose. Senescence-associated ß-galactosidase (SA-ß-gal) staining, and senescence related markers (p16, p21, and p53) were used to confirm aging MSCs. Aging and young (control) MSCs were cultured in osteogenic and adipogenic differentiated medium with different concentration of BMP-2 (0, 25, 100, 250, and 500 ng/ml). We compared the phenotypes by Calcium assay, adipogenesis assay, and specific staining (Alkaline Phosphatase, Alizarin Red S, Oil red O), and gene expression patterns by real-time polymerase chain reaction (PCR), western blot assay, and microarray analyses between aging MSCs and control MSCs. Microarray analysis was conducted with 4 different cells (aging MSCs and control MSCs with or without the induction of BMP-2). GSEA (Gene Set Enrichment Analysis), leading-edge subset analysis, and MetaCore analysis were used as a microarray analysis.

Results: Aging cells by replicative passages under high level glucose (22 mM) were well characterized by senescence markers. Osteogenic differentiation by BMP-2 decreased in aging MSCs compared to control MSCs. Lower ALP activity and less Ca++ release was observed in aging MSCs (P < 0.001, and P = 0.005, respectively), and positive dose-response relationship was observed in both MSCs. The mRNA expression of Runx-2, bone sialoprotein (BSP) and osteocalcin (OCN) was lower in aging MSCs. However, adipogenic differentiation by BMP-2 was comparable between 2 cells. Adipogenesis assay showed no differences of adipogenic potential between 2 groups (P = 0.279). In addition, there was no superiority in the expression of PPARγ between 2 cells. These results were also supported in protein level by western blot assay. Microarray analysis showed more upregulated genes in rhBMP-2-treated aging MSCs than in rhBMP-2-treated control MSCs and non-treated aging MSCs. Aging and adipogenesis related gene sets were more upregulated and significantly enriched by the induction of rhBMP-2 in aging MSCs compared to control MSCs. In BMP related leading edge subsets, 6 clustered genes (CHRDL1, NOG, SMAD1, SMAD7, FST, and BAMBI) were upregulated in aging MSCs. In aging MSCs, NF-kB or p38 MAPK pathway could be important in response to BMP-2, whereas BMP/SMAD pathway in young MSCs, based on MetaCore analysis.

Conclusions: Aging MSCs showed less osteogenic differentiation and comparable adipogenic differentiation in response to BMP-2 compared to control MSCs. Relative superior adipogenic differentiation by BMP-2 in aging MSCs is supported by the result of microarray analysis. In addition, several genes might be candidates for senescence dependent BMP-2 effect on differentiation of MSCs. Different signaling pathway might play a role in different response to BMP-2 between aging and young MSCs. Further in vivo research along with the current study may contribute to effective use of BMP-2 in clinical fields by elucidating the signaling pathway whether MSCs differentiate into osteoblasts or adipocytes by BMP-2.