br Conclusion br Acknowledgements br

Conclusion
Acknowledgements
Introduction Since the first successful reprogramming of somatic methysergide into induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006), countless efforts have been made to fully explore the potential applications of iPSCs in biomedical research and remarkable progress has been made. Human iPSCs hold great promise in personalized disease modeling and drug screening, and could provide unique advantages over animal models by circumventing the intrinsic dissimilarities between species (Avior et al., 2016; Ebert et al., 2012). iPSCs have also opened numerous new opportunities in regenerative medicine as a potentially unlimited cell source for generating patient-specific cells to repair damaged or dysfunctional tissues, such as cardiac repair post myocardial infarction (Lalit et al., 2014), reversal of diabetes (Rezania et al., 2014), and bone regeneration (Tang et al., 2014). For all the above mentioned iPSC applications, it is critical to efficiently generate the desired cell types iPSCs, and the first step of differentiation is to induce iPSCs into the corresponding germ layer (ectoderm, mesoderm or endoderm). Inhibition of glycogen synthesis kinase 3 (GSK3), generally by Chir99021, is one of the most employed strategies to activate Wnt pathway and hence induce differentiation of iPSCs into early mesoderm cells. For instance, differentiation of human iPSCs into cardiomyocytes (Lian et al., 2012) or endothelial progenitor cells (Bao et al., 2016) employs Chir99021 during early time period of differentiation. In addition to promoting mesoderm lineage, GSK3 inhibition has also been used to generate insulin-producing cells (Takeuchi et al., 2014) and motor neuron progenitor cells (Du et al., 2015). However, despite the extensive use of GSK3 inhibitors in iPSC differentiation, its effects on iPSC cellular activities and cell survival, besides activating Wnt pathway, have yet to be examined. Thiol-containing antioxidants, such as N-acetyl cysteine (NAC) and beta-mercaptoethanol (BME) are widely used in cell culture to scavenge reactive oxygen species (ROS), reduce cellular oxidative stress and hence promote cellular functions and cell survival (Kim et al., 2009; Takahashi et al., 2002). NAC was shown to reduce DNA damages and genomic aberration during reprogramming of somatic cells into iPSCs without altering transgene expression (Ji et al., 2014). 1mM NAC has been shown to improve cell viability and functionality during hematopoietic differentiation of iPSCs (Berniakovich et al., 2012).
Materials and methods
Results
Discussion GSK3 inhibition is one of the most frequently employed methods in iPSC differentiation (Lian et al., 2012; Takeuchi et al., 2014; Du et al., 2015). Inhibition of GSK3 leads to the stabilization and nuclear translocation of β-catenin, where it binds TCF/LEF transcriptional factors and activates the Wnt target genes (Nelson and Nusse, 2004). Activation of Wnt would subsequently facilitate the formation of early mesodermal cells (Lindsley et al., 2006). Additionally, GSK3 is also broadly involved in the regulation of numerous cellular activities including cell division, cell adhesion, cell motility and cell survival (Frame and Cohen, 2001). Indeed, many downstream substrates of GSK3 are proapoptotic (Frame and Cohen, 2001), thus the inhibition of GSK3 by Akt generally promotes cell survival (Fang et al., 2000). Despite the broad impacts GSK3 has on cellular behaviors, how the inhibition of GSK3 affects iPSCs besides activating Wnt has rarely been investigated. In this work, we report that the inhibition of GSK3 greatly sensitized iPSCs to apoptosis induced by thiol-based antioxidants including NAC, BME and MTG, whereas non-thiol based antioxidant trolox, had no such combined effect with GSK3 inhibitors. Additionally, none of the compounds alone caused significant apoptosis at the concentrations we tested. In fact, NAC has been generally used to promote cell survival. Specifically, 1mM NAC was shown to increase cell viability and reduce DNA damages during iPSC reprogramming (Ji et al., 2014) or differentiation (Berniakovich et al., 2012). In addition, the fast-acting nature of this combined effect is also surprising. Upon co-treatment of NAC and Chir99021, substantial caspase 3 activity was detected after just 3h of treatment, and extensive cell death could be observed after merely 6h. Such rapid apoptosis indicates that it was most likely initiated via direct modification of protein/enzyme activities, and independent of transcriptional changes.