Here we addressed these problems by focusing on control of

Here we addressed these problems by focusing on control of the regional identity of PSC-derived neural progenitors and neurons. The developing neural tube is subdivided into distinct regions along the anteroposterior (A-P) and dorsoventral (D-V) axes, and each region produces a specific subtype of neurons (Kiecker and Lumsden, 2012). Namely, neuronal subtype specification in the neural tube is determined in a region-specific manner. If the regional identity of PSC-derived neurons could be regulated at will based on the same protocol, then any desired subtypes could be induced with the same efficiency and under the same culture conditions. A protocol permitting such regulation would enable the reproduction of disease phenotypes in any given buy 69 8 region and also the comparison of phenotypes between different neuronal subtypes.
Results
Discussion Our findings are consistent with previous reports demonstrating that the graded signaling activities of Wnt/RA and Shh establish the A-P and D-V axes in vivo. Also noteworthy is that our study shows that this model for early neural patterning, which is proposed mostly from work on Xenopus, chicks, and mice, can be applied to human development. Because in vivo experiments of the human embryo are ethically challenging and technically difficult, our culture system now provides novel opportunities to uncover the mechanism of neural patterning in humans. A remaining issue regarding our present culture system is the ability to recapitulate the environment after regional specification. In some neuronal subtypes, additional signals are needed for further specification, maturation, and maintenance. When cells migrate from their birthplace or if they project to distant targets, they often receive such signals from cells with different regional identity. Such additional signals cannot be recapitulated in our present culture system. In the case of midbrain dopaminergic neurons, for example, transforming growth factor β (TGF-β) expressed in their projection targets is necessary for their maintenance (Poulsen et al., 1994). Dopaminergic neurons in our culture did not express late-stage markers such as PITX3 and DAT, probably because of the lack of such additional signals (data not shown). In addition to treatment with signaling modulators, co-culturing with cells of the destination of migration or cells of the projection targets would be a promising approach to overcome this issue. Specific brain regions are preferentially damaged in most neurological diseases, whereas regions other than the lesioned area remain relatively unaffected. For this reason, the differentiation of patient-derived iPSCs into specific neuronal subtypes representing the lesion site is a valuable approach for modeling human neurological diseases (Imaizumi and Okano, 2014; Okano and Yamanaka, 2014). The current investigation reproduced p-tau accumulation as an AD phenotype by endowing neurons with forebrain characteristics. On the other hand, a previous report using the same iPSC clones did not detect such a phenotype, probably because of the induction of inappropriate subtypes (Yagi et al., 2011). These results highlight the importance of correct neuronal subtype specification in reproducing neurological disease phenotypes with PSCs. Some investigators have reported the directed differentiation of PSCs into a limited number of neuronal subtypes, including midbrain dopaminergic neurons and spinal cord motor neurons (Kriks et al., 2011; Li et al., 2005). Given that each differentiation protocol is overly optimized for a particular subtype, the protocol in question cannot be readily applied to additional neuronal subtypes. Nevertheless, our culture system theoretically enables the differentiation of PSCs into all neuronal subtypes, making it possible to efficiently generate subtypes whose induction from PSCs has been difficult. Also, as emphasized above, our culture system is based on the same protocol to obtain neuronal subtypes with equivalent efficiency and under the same general culture conditions (with the exception of the modulation of different signaling cascades). Therefore, our method allows comparative analysis of a variety of neuronal subtypes, whereas earlier investigations on disease modeling have focused only on selectively affected subtypes. Moreover, other investigators have reported that the modulation of Wnt signaling can direct the regional identity of PSC-derived neurons in a manner similar to that observed in our culture system. Nevertheless, a parallel analysis of disease phenotypes between neurons with different regional identities was not performed in these studies (Kirkeby et al., 2012; Moya et al., 2014). To fully understand the mechanism of disease pathology and progression, one must investigate not only the manner in which specific neuronal subtypes are affected but also how other subtypes remain unaffected. In the case of genetic diseases, neural subtype specificity of disease phenotypes is not necessarily accounted for by the expression pattern of the responsible genes. For example, some mutations in TARDBP, which is globally expressed across the entire brain, cause motor neuron-selective degeneration (Kabashi et al., 2008, 2010; Sephton et al., 2010). Similarly, although PSEN1 and PSEN2 show widespread expression in the brain, their mutations predominantly affect forebrain neurons (Rogaev et al., 1995; Sherrington et al., 1995). However, the mechanism of neuronal subtype specificity of these disease phenotypes is unclear. Our culture system may permit investigations toward filling in the gaps in this knowledge base.