BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis

Abstract

The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. An interesting phenomenon was that BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism.

Autogenous regulation of a network of bone morphogenetic proteins (BMPs) mediates the osteogenic differentiation in murine marrow stromal cells

“…The current studies also do not support a positive regulatory role for BMP-4 in osteogenous since: a) as MSC differentiation progresses there is less BMP-4 expressed; b) both Noggin and BMP-2 antagonism lead to increased BMP-4 expression; c) both BMP-2 or BMP-7 induction of osteogenic differentiation leads to down regulation of endogenous BMP-4 expression. Finally, previous data from our laboratory had also shown that C3H10T½ cells in their undifferentiated state expressed high endogenous levels of BMP-4 whereas in a similar fashion as seen in this study, the exogenous addition of BMP-2 and BMP-7 downregualted its expression during skeletal cell differentiation [9].…”

“…Noggin is naturally expressed during developmental processes and is known to act as non-competitive inhibitor of BMPs 2, 4 and 7 with a low (Kd 1.0×10 −10 ) disassociation constant [20]. Interestingly we were unable to detect BMP-7 in this system, despite being present in control fracture callus tissue specimens at levels equivalent to BMP-2 and 3 in other mesenchymal cell populations [5,9]. Since BMP-7 expression is not detectable, its role in the differentiation of these cultures is considered negligible.…”

“…These data suggest that repair and developmental processes are regulated through the coordinated actions of multiple BMPs instead of a single master BMP. Early experiments using recombinant human BMP-2, BMP-4 or BMP-7, demonstrated that these proteins have the ability to individually stimulate osteoblastic or chondrogenic phenotypic expression in various mesenchymal precursor cell lines [4,[8][9][10]. However, unlike the in vitro investigations, in vivo studies suggest that these factors function in a coordinated manner [7,11].…”

“…Numerous studies have now demonstrated that the stromal cells have significant proliferative capacity and cells within this population contribute to the endothelial reticulum and possess the ability to differentiate into multiple cell types including osteoblasts, chondrocytes, and adipocytes. Previous studies have demonstrated that exogenous addition of multiple individual BMPs can stimulate osteogenic differentiation in mesenchymal cells [4,8,9]. However, these effects have not been fully characterized at a molecular level.…”

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

“…Mid-and late-stage osteogenic markers including OPN, bone sialoprotein (BSP), and OCN are temporal successors to RUNX2 and OSX expression after rhBMP-2 stimulation [63]. The complex temporal expression profiles of OPN, BSP, and OCN have been elucidated [15,16,23,25,26,30,53,59,66]; therefore, a coordinated, temporal RNAi treatment cycle may enhance the duration and intensity of gene silencing and prevent HA deposition in vitro. Consequently, the evolution of RNAi therapeutics must match RNAi targets to their expression profiles.…”