TSC haploinsufficient astrocytes presented a high proliferat

TSC haploinsufficient astrocytes presented a high proliferation rate and an increased saturation density when compared with WT astrocytes. Similarly, the conditional disruption of TSC1 in astrocytes using a human GFAP-Cre system was shown to produce mice that presented with astrocytosis (Han and Sahin, 2011). Moreover, the increase in astrocyte proliferation may precede the neuronal abnormalities, resulting in hippocampal neuronal disorganization and seizures. Previous studies indicated that NESTIN-immunoreactive neural cells were less developed than mature astrocytes, raising the possibility that new neurons were generated from these NESTIN-positive progenitors, resulting in hippocampal neuronal disorganization. In addition, recent studies demonstrated that astrocytes could give rise to new neurons in the adult mammalian hippocampus (Seri et al., 2001). In our studies, we observed NESTIN and TUJ1 protein expression in differentiated astrocytes, while the differentiated neurons expressed TUJ1 and NESTIN (data not shown), but not GFAP. Thus, our results partially supported the hypothesis that hippocampal neuronal disorganization in TSC results from the aberrant differentiation of NESTIN-positive immature astrocytes into neurons. Further studies are warranted to confirm our results. In other studies, while both TSC1 and TSC2 heterozygous mice exhibited an increased number of astrocytes in vivo, there was no increase in the saturation density of TSC2 haploinsufficient astrocytes compared with that of WT astrocytes in vitro. In addition, TSC1 knockout astrocytes and TSC1 knockout mouse embryonic fibroblasts presented a higher saturation density associated with decreased levels of p27kip1 (Uhlmann et al., 2002). The authors suggested that the non-cell-autonomous growth advantage conferred by TSC heterozygosity may involve p27kip1 expression. However, no gross or microscopic evidence of morphological rho inhibitor abnormalities was detected in the TSC1 or TSC2 haploinsufficient mice, while our patient presented with tubers in the brain. However, we did not study p27kip1 expression in our patient\'s astrocytes. Further experiments on the relationship between TSC heterozygosity and brain abnormalities, tuberin/hamartin signaling, and p27kip1 expression are required. The current study has some limitations. First, we did not investigate further the link between mTOR hyperactivation and synaptic transmission and plasticity, which might influence neuronal excitability and trigger epileptogenesis. However, a recent study on neurons derived from TSC2-deleted pluripotent stem cells demonstrated that heterozygous and homozygous loss of TSC2 led to altered synaptogenesis and transmission and that pharmacological inhibition of mTORC1 could ameliorate synaptic dysfunction (Costa et al., 2016). Second, only the proband who was carrying TSC2 gene mutation was compared with the normal control subject in our study. Identifying the possible role of TSC mutations in complex genetic and neural network processes pertaining to cell survival, structure, and function will provide a better understanding of the pathogenesis of TSC-related neurological symptoms (Mayer et al., 2004; Talos et al., 2008). Although our results may partially confirm the pathophysiological mechanisms underlying the neurological manifestations in TSC, additional efforts must be undertaken to explore these mechanisms further. In conclusion, abnormal pNSCs, neurons, and astrocytes differentiated from TSC iPSCs partially recapitulated the development of neurological abnormalities as observed in patients with mutations in TSC2, suggesting that TSC1/2 haploinsufficiency might be sufficient to contribute to the neuropathology of TSC. Furthermore, these cells provide an unprecedented model for studying the abnormal neural development and the potential underlying mechanisms of TSC in vitro, screening novel drugs for personalized therapies, and potentially complementing/implementing mTOR-directed inhibitory approaches.