To investigate apoptotic and necrotic

To investigate apoptotic and necrotic cell death, we have used a phosphatidylserine-binding protein (Annexin-V-FITC) to detect apoptosis-dependent redistribution of phospholipid. Cells were also counter-stained with PI to distinguish apoptosis from necrosis. We found few apoptotic and necrotic HOBt manufacturer in shcontrol BMSCs, an increase in response to oxidant, and a substantial block of this effect by Vitamin C. However, with decreased transporter levels, shSVCT2 BMSCs showed significantly higher apoptotic and necrotic cells. Moreover, supplementation with vitamin C to shSVCT2 BMSCs did not block oxidative stress-induced apoptosis and necrosis. Recently, Song et al (2014) reported that autophagy is critical for the BMSCs\' survival under oxidative conditions. Furthermore, other investigators also reported that low level or acute exposure induces autophagic flux, whereas chronic or high dose oxidative treatment blocks autophagy and enhances apoptosis in a number of cell types (Song et al, 2014; Boya et al, 2005; Zhou et al, 2012). We speculate that knockdown or down-regulation of SVCT2 transporter disturbs cell anti-oxidant properties because of less availability of vitamin C which leads to weaker cell survival mechanism. Vitamin C is known for its antioxidant dependent autophagy and cell survival role in various cell types (Bridges, 1987; Martin et al, 2002; Gruss-Fischer and Fabian, 2002; Tannetta et al, 2008; Hung et al., 2010; Singletary and Milner, 2008; Tomasetti et al, 2012). Ours is the first study to demonstrate the critical role of SVCT2 transporter in antioxidant effect of vitamin C on autophagy and cell survival in BMSCs. In our previous studies, we showed that expression of SVCT2 decreases with aging (Fulzele et al, 2013) and in diabetic bone and bone marrow (Sangani et al, 2013). Therefore, decreased SVCT2 expression appears to overload the accumulation of ROS, which leads to a weakened cell survival mechanism, increased apoptotic and necrotic cell death, and a reduction in the differentiation of BMSCs into osteoblasts (osteogenesis). These events could be one of the reasons for bone loss, weaker bone structure, and increased fracture risk in aging, postmenopausal and diabetic medical conditions. The molecular mechanism of oxidative stress, supplementation of ascorbic acid, and SVCT2 transporter activity with regard to levels of autophagy and apoptosis need to be understood in detail.
Disclosure statement
Acknowledgments This publication is based upon work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development Program (VA Merit Award 1I01CX000930-01, WDH) and the National Institutes of Health (NIA-AG036675-01, WDH,CS,MH). The contents of this publication do not represent the views of the Department of Veterans Affairs, or the United States Government.
Introduction Motoneurons make contact with the skeletal muscle to form the neuromuscular junction (NMJ), which is required for signal transmission. Disorders of the NMJ such as myasthenia gravis lead to varying degrees of muscle weakness (Gomez et al., 2010). Muscle wasting and weakness in motoneuron diseases arise predominantly from the loss of motoneurons. Hence, research has focused on investigating the mechanisms of motoneuron cell death (de Carvalho et al., 2014). In addition, there is growing evidence indicating that alterations of NMJs and/or the skeletal muscle itself affect the pathogenic process in amyotrophic lateral sclerosis (ALS) (Miller et al., 2006; Murray et al., 2010; Wong and Martin, 2010). In vitro models could contribute to the analysis of the pathophysiology of motoneuron diseases and might help to screen for therapeutic options. Human induced pluripotent stem cell (hiPSC) technology has opened new avenues for elucidating underlying pathomechanisms and even putative treatment options. Cell culture systems generated from patient specific somatic tissue harbour the genetic defects giving the opportunity to analyse the functional impairment in vitro. Furthermore, the ability to repair underlying gene mutations in patient specific cells with modern gene editing systems provides the possibility of autologous cell transplantations. Recently, different methods have been published describing the generation of myoblasts from hiPSCs by introducing lentiviral systems forcing myogenic differentiation (Darabi et al., 2012; Goudenege et al., 2012; Tanaka et al., 2013; Abujarour et al., 2014). In addition, a sphere based culture system to generate hiPSC derived myoblast has recently been described but in these myogenic cultures a high degree of neuronal contamination was observed (Hosoyama et al., 2014). However, protocols giving rise to significant numbers of skeletal muscle cells without genetic engineering are therefore more physiological methods and are still in need to be developed. Promising candidate markers for the positive or negative selection of myogenic progenitors to enhance cell populations with myogenic potential are membrane bound surface proteins of the large family of CD factors, involved in a variety of cellular functions. One interesting candidate marker to select for myogenic precursors is CD34, expressed in quiescent satellite cells and proliferating myogenic precursors (Beauchamp et al., 2000; Jankowski et al., 2002; Zammit et al., 2006). Additionally, factors involved in promoting the myogenic potential of stem cells are produced and secreted by muscle stem or progenitor cells that in turn enhance in vitro differentiation of stem cells into the myogenic lineage (Stern-Straeter et al., 2014).