BMP-9 dependent pathways required for the chondrogenic differentiation of pluripotent stem cells

Current cartilage repair therapies focus on the delivery of chondrocytes differentiated from mesenchymal stem cells, and thus understanding the factors that promote chondrogenesis may lead to improved therapies. Several bone morphogenetic proteins (BMPs) have been implicated in chondrogenic differentiation and/or chondrocyte function. Although the signaling pathways downstream of BMPs have been studied in other systems, their role in chondrogenesis is less well characterized. Here, we investigated the effects of BMP-9 in chondroprogenitor cells. Compared to BMP-2 and BMP-6, we showed that BMP-9 was significantly more potent in inducing chondrogenic differentiation in mouse C3H10T1/2 and ATDC5 cells. Moreover, we demonstrated that BMP-9 induces the phosphorylation of SMAD1/5 in a dose and time dependent manner. Confocal immunofluorescence microscopy further demonstrated an accumulation of phosphorylated SMAD1/5 in the nuclei of BMP-9 treated cells. Consistent with activation of the SMAD signaling pathway, we also observed an up-regulation of Id1 and PAI-I expression. Importantly, we demonstrated that the simultaneous knockdown of SMAD1 and SMAD5 was able to inhibit chondrogenesis. Additionally, we also observed activation of p38 by BMP-9, and pharmacological inhibition of this pathway blocked chondrogenesis. In contrast, inhibition of p44/42 ERK had no effect. Finally, we tested the ability of Noggin to block the actions of BMP-9. While Noggin potently inhibited the ability of BMP-2 to mediate differentiation, it had no significant effect on BMP-9. Our findings provide a clearer understanding of the cellular pathways utilized by BMP-9 for chondrogenesis that may help improve current therapies for regenerative cartilage repair.