Animal welfare and experimental procedures were conducted with the approval of the Institutional Animal Care and Use Committee of the Model Animal Research Center in Nanjing Medical University or college. brain. We propose that our work could inform clinical applications of hPSC-based cell therapy. after transplantation (13, 14). These overgrowth cells contained a great amount of undifferentiated human-specific NESTIN+ cells and enlarged the host brain. To safely use iPSC-based transplantation therapy in clinical applications, many efforts have been made to prevent tumor-like overgrowth. Removing remnant immature NS/PCs or differentiate these cells into more mature cell types may help to avoid tumor-like overgrowth following transplantation. For instance, a physiological medium (BrainPhys basal + ZM223 serum-free supplements) with adjustments to the concentrations of inorganic salts, neuroactive amino acids, and dynamic substrates improved maturation and enhanced the proportion of synaptically active neurons (15), which reduced tumor-like overgrowth. Another efficient method is usually to find the important signaling pathway controlling the induction and differentiation of NS/PCs. Inhibition of Notch signaling with a -secretase inhibitor (GSI) was shown to be able to induce NS/PCs to develop into a more mature state with limited proliferation (14, 16). In addition, treatment of iPSC-derived dopaminergic progenitor cells with GSIs prior to transplantation may control the growth of a potentially proliferative cell populace (16). Mouse monoclonal to AURKA However, the GSIs caused detrimental effects in patients with Alzheimer’s disease, and the toxic ZM223 side effects are the main concern for clinical application of this tool compound (17). It is important to find a way to enhance the induction and differentiation of NS/PCs. Azidothymidine (3-azido-3-deoxythymidine; AZT), a telomerase inhibitor, could inhibit the telomerase reverse ZM223 transcriptase (TERT) and interrupted the cell proliferation (18). Our previous study showed that AZT disrupted the proliferation of adult neural stem cells in the subventricular zone and hippocampus in mice without causing cell damage or apoptosis (19, 20). However, the effects of AZT in hPSC-derived neurons have not yet been explored. In this study, we show that this telomerase inhibitor AZT suppressed the proliferation of hPSC-derived neural progenitors, promoted the differentiation of hPSC-derived cortical neurons, and enhanced the maturation of ZM223 hPSC-derived neurons. Furthermore, we also found that AZT-pretreated, hPSC-derived precursors inhibited the proliferation and promoted the differentiation of cortical neurons and and and and and and = 5; 20 m AZT, = 5; 100 m AZT, = 6. *, < 0.05; **, < 0.01; represents control. represents Hoechst. represent S.E. We also compared the effect of AZT with current known tool compounds for enhancing the differentiation of neurons. The effects of AZT and the GSI (Fig. 1, and as well as DNA-binding and mitotic cell cycleCassociated genes compared with the controls (Fig. S3and and and and and = 4; 100 m AZT, = 6. *, ZM223 < 0.05. represents control. represent S.E. Next, we asked whether AZT promoted the maturation of hPSC-derived neurons. After 6 days of AZT treatment (3 days before and after cell plating on day 26), the percentages of TUJ-1+ (a marker for neurons) and MAP2+ (a marker for mature neurons) cells were increased by AZT treatment at day 35 (Fig. 3, and and and = 4; 20 m AZT, = 4; 100 m AZT, = 5. *, < 0.05. and represents control. signifies Hoechst. represent S.E. Electrophysiological features correlated well using the maturation position of neurons. Therefore, we analyzed the electrophysiological actions of AZT-treated neurons. The whole-cell clamp was performed on 7C10-day time plated neurons. The outward and inward currents had been documented through the use of voltage-clamp measures from ?80 to 60 mV (Fig. and and 3and and and and and = 3; AZT, = 4. *, < 0.05. represents control. represent S.E. AZT pretreatment improved the differentiation of cortical neurons from hPSC-derived cortical progenitors in vivo The hPSC-based cell treatment also.