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.

Recovery of bovine Babesia spp. after long-term cryostorage and comparison of bovine donor erythrocytes and serum.

Cultured Babesia bovis and Babesia bigemina were recovered from liquid nitrogen storage nearly 30 years after they were cryopreserved. Four cattle were compared as donors of erythrocytes and serum for microaerophilous stationary phase (MASP) cultures for recovery of B. bigemina. Erythrocytes and serum from only one (#913) of the four animals supported growth of B. bigemina. Two B. bigemina (frozen in 1986 and 1987) and two B. bovis (both frozen in 1986) cryostocks were recovered from liquid nitrogen storage and all four recovered and thrived in #913 erythrocytes and serum. In the third passage after recovery, B. bovis cultures were cryopreserved. Six months later they were successfully recovered using #913 erythrocytes and serum. This study shows that B. bovis and B. bigemina stored nearly 30 years in liquid nitrogen can be successfully recovered in the MASP system. This study also confirms previous observations that selection of a suitable bovine donor of erythrocytes and serum is critical to the success of the culture.

Loss of protein targeting to glycogen sensitizes human hepatocellular carcinoma cells towards glucose deprivation mediated oxidative stress and cell death.

Protein targeting to glycogen (PTG) is a ubiquitously expressed scaffolding protein that critically regulates glycogen levels in many tissues, including the liver, muscle and brain. However, its importance in transformed cells has yet to be explored in detail. Since recent studies have demonstrated an important role for glycogen metabolism in cancer cells, we decided to assess the effect of PTG levels on the ability of human hepatocellular carcinoma (HepG2) cells to respond to metabolic stress. Although PTG expression did not significantly affect the proliferation of HepG2 cells under normal culture conditions, we determined that PTG plays an important role during glucose deprivation. Overexpression of PTG protected cells from cell death in the absence of glucose, whereas knocking down PTG further promoted cytotoxicity, as measured by the release of lactate dehydrogenase (LDH) into the media. Additionally, we demonstrated that PTG attenuates glucose deprivation induced haeme oxygenase-1 (HO-1) expression, suggesting that PTG protects against glucose deprivation-induced oxidative stress. Indeed, treating cells with the antioxidant N-acetyl cysteine (NAC) rescued cells from cytotoxicity caused by glucose deprivation. Finally, we showed that loss of PTG resulted in enhanced autophagy. In control cells, glucose deprivation suppressed autophagy as determined by the increase in the levels of p62, an autophagy substrate. However, in knockdown cells, this suppression was relieved. Blockade of autophagy also attenuated cytotoxicity from glucose deprivation in PTG knockdown cells. Taken together, our findings identify a novel role for PTG in protecting hepatocellular carcinoma cells from metabolic stress, in part by regulating oxidative stress and autophagy.