FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-beta signaling

Abstract

Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days' culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days' culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-beta signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.

Fig. 1

Experimental protocol and quantitation of osteogenic and chondrogenic markers. hMSCs were maintained in the medium with or without FGF-2 for 15 day, and osteogenic or chondrogenic differentiation of hMSCs was induced for 21 days (A). Then, the protein levels of ALP (B), the mRNA expression levels of osteocalcin (C), type II collagen (D) and type X collagen (E) were measured

Fig. 2

Genes up-regulated (>2 fold) and down-regulated (<1/2 fold) by FGF-2 in hMSCs. hMSCs were maintained in the medium with or without FGF-2 for 15 days. Then, total RNA were extracted from the hMSCs and microarray analysis were performed. The x-axis showed the fold-change of FGF-2 against Control. The y-axis showed the raw expression levels of hMSCs cultured in the medium with FGF-2

Fig. 3

Pathway analysis of genes up-regulated and down-regulated by FGF-2 in hMSCs. Pathway analysis of up-regulated and down-regulated genes by FGF-2 (Fig. 2) was performed by Ingenuity Pathway Analysis. The y-axis showed the ratio of genes mapped in Fig. 2 against all of genes belongs to each canonical pathway

Fig. 4

Genes up-regulated and down-regulated by FGF-2 in IGF-I signaling pathway. Genes up-regulated and down-regulated by FGF-2 (Fig. 2) were mapped with the IGF-I signaling pathway by Ingenuity Pathway Analysis. The red color showed up-regulated genes and green color showed down-regulated genes

Fig. 5

Genes up-regulated and down-regulated by FGF-2 in TGF-β signaling pathway. Genes up-regulated and down-regulated by FGF-2 (Fig. 2) were mapped with the TGF-β signaling pathway by Ingenuity Pathway Analysis. The red color showed up-regulated genes and green color showed down-regulated genes

Fig. 6

Our hypothesis that FGF-2 increases the osteogenic and chondrogenic differentiation potentials of hMSCs by inactivation of IGF-I and TGF-β signaling