Supplementary Materials1. differentiation in to the postnatal mouse cortex, are practical and set up long-distance projections, as demonstrated using iDISCO entire brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based approaches for PPP2R2C disease cell and modeling therapy in CNS disorders. Within the last few years, strategies have been created to convert hPSCs into early neural lineages. An especially efficient strategy may be the usage of two small-molecule inhibitors of SMAD signaling (LDN193189 and SB431542; known as LSB process) to result in differentiation of human being embryonic stem cells (hESCs) or human being induced pluripotent stem cells (hiPSCs) into PAX6+ central anxious program (CNS) neural precursors within 11 times of differentiation1. Neural subtype specification can be further modulated using additional small molecules targeting pathways such as WNT signaling. Timed Fluoroclebopride exposure to compounds activating WNT signaling under dual SMAD inhibition conditions induces the neural crest lineage, marked by SOX10 expression. In contrast, inhibition of WNT signaling blocks the formation of neural crest cells and enhances the induction of forebrain precursors, marked by FOXG1 expression2C4. While those manipulations efficiently specify defined neural precursor cell populations, further differentiation into functional neurons is a lengthy process that can extend over weeks if not months. To accelerate neuronal fate acquisition, we have used two additional small molecules: SU5402, a potent inhibitor of fibroblast growth factor (FGF) signaling5 and DAPT, a -secretase inhibitor blocking Notch signaling6. Combinatorial application of those two inhibitors with dual SMAD inhibition and WNT activation yields 75% post-mitotic neurons in 11 days of differentiation7, the same time period required for neural precursor cell induction under standard dual SMAD inhibition conditions1. However, co-expression of BRN3A and ISL1 in those rapidly induced neurons defined them as peripheral sensory rather than PAX6-derived CNS neurons7. Therefore, it has remained unclear whether strategies to accelerate neuronal fate acquisition during sensory fate specification can be adapted for CNS fates. PAX6-derived cortical neurons are of particular interest for studies of human development and for modeling human neurodevelopmental and neurodegenerative disorders. While reliable protocols exist to derive cortical neurons from hPSCs, those conditions require between 30 C 90 days of differentiation from hPSCs to yield both lower and upper layer cortical neurons8, 9 and even more protracted time periods to achieve full maturation. Here we aim to identify small-molecule based conditions that greatly accelerate human cortical neuron fate induction to facilitate the routine application of hPSC-derived neurons in applications for disease modeling and regenerative medicine. Development of an accelerated CNS neuron differentiation protocol Given the critical roles of WNT signaling in determining fate choice between the CNS and neural crest3, 10, we hypothesized that developing a combinatorial small-molecule approach that inhibits rather than activates WNT signaling may trigger rapid differentiation toward cortical neurons (Fig. 1a). To test this hypothesis, we replaced the GSK3 inhibitor CHIR99021 (C; WNT agonist) with the tankyrase inhibitor XAV939 (X; WNT antagonist), which acts to stabilize Axin11. All other inhibitors used previously for the derivation of sensory neurons (LSB, SU5402 and DAPT) remained unchanged for these initial studies aimed at rapidly inducing forebrain neuron fates (LSB+X/S/D protocol). Given our past experience in unexpectedly triggering a CNS to peripheral nervous system (PNS) fate switch during rapid neuronal induction7, we first assessed the impact of the LSB+X/S/D condition on early ectodermal lineage choice using three engineered Fluoroclebopride hESC reporter lines: (CNS lineage), line12 (cranial placode fate). Faithfulness of reporter Fluoroclebopride manifestation was validated after aimed differentiation in to the particular fates1, 2, 13 (Fig. 1b). In keeping with earlier function, both LSB and LSB+X circumstances offered rise to a near-uniform inhabitants ( 95%) of PAX6+ cells, with few cells expressing SOX10 or 61 (Fig. 1c). On the other hand, LSB+C or LSB+C/S/D (generally known as.