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    • To explore the potential mechanism underlying this interesti

    2018-11-08

    To explore the potential mechanism underlying this interesting phenomenon, we examined the role of mTOR in this process as mTOR is well known for its role in controlling cell growth and metabolism, and more importantly, it is known to crosstalk with GSK3 (Inoki et al., 2006). Indeed, we were able rescue the procollagen c proteinase with increasing concentrations of rapamycin, suggesting the critical role of mTOR. To our surprise, though the inhibition of mTOR was able to rescue the cells, we did not observe up-regulation of mTOR activity in cells treated with Chir99021 and NAC, alone or in combination. We also showed that Akt might have contributed to the apoptosis. NAC and Chir99021 lead to a lower Akt activity compared to the control sample and stimulation of insulin pathway with IGF-1 and insulin partially reduced cell death. However, we ruled out the possibility that Akt dysregulation was the main cause for the proapoptotic effect since NAC alone resulted in the greatest Akt inhibition but did not cause significant apoptosis. As GSK3 inhibition strongly promotes differentiation, this raises the possibility that the observed synergistic effect might be a general response to the initiation of differentiation. Bone morphogenetic protein (BMP)-4 and fibroblasts growth factors (FGFs) are known to be critical for embryonic development and also widely used for modulation of differentiation (Kimura et al., 2000; Teo et al., 2012; Zhang et al., 2008). In fact, BMP-4, when combined with retinoic acid, has been shown to synergistically cause apoptosis in embryonal carcinoma cells (Fujita et al., 1999). Therefore, we examined if BMP-4 or FGF-10 could induce apoptosis, like GSK3 inhibitors, when used in conjunction with 4mM NAC. The results showed that even at the dosage of 200ng/mL, neither of these two growth factors induced apoptosis in iPSCs, indicating that the phenomenon is not a general response to iPSC differentiation (Supplementary Fig. 4). Our work thus far demonstrates that this sensitizing phenomenon is potentially unique to human cells. All three tested iPSC cell lines exhibited significant apoptosis upon co-treatment with the GSK3 inhibitor and thiol based antioxidants regardless of their origin and derivation method. We also observed a similar trend in human breast cancer cell line: MDA-MB-231. Although the decrease in cell viability was smaller compared to that observed in iPSCs, it was statistically significant (Supplementary Fig. 2A), indicating that this synergistic effect might be present in more human cell types and manifests at different intensities. Notably, pharmacological inhibition of GSK3 is actually a potential therapeutic strategy currently under clinical trials for cancer intervention (McCubrey et al., 2014) and also a potential treatment for neuronal disorders (Eldar-Finkelman and Martinez, 2011). Given that NAC is an extensively used nutritional supplement, this raises an interesting question of whether NAC will synergize with or antagonize the intended therapeutic potency of GSK3 inhibition in those treatments. From a basic science perspective, this research provides a new avenue to understand the effects of GSK3 inhibition and commonly used antioxidants on the survival of cells, particularly of iPSCs. Second, new knowledge gained from this work may allow for the development of more efficient iPSCs differentiation protocols which rely on GSK3 inhibition, specifically by improving cell survival. Lastly, with further studies to investigate how this cell-sensitizing phenomenon varies between cell types, we can potentially take advantage of this effect in a broader spectrum of biomedical applications such as cancer treatment and neurodegenerative diseases therapy.
    Conclusions
    Acknowledgement The authors would like to thank Dr. Austin Cooney for kindly providing a iPSC cell line derived from human foreskin fibroblast using MKOS Sendai viral transfection and his valuable input in the discussion of this work. We also thank Amy Brock lab for providing the MDA-MB-231 cell line. We acknowledge the research funding from American Heart Association (15SDG25740035).