ABSTRACT: The human brain formation involves complicated processing, which is regulated by a gene regulatory network influenced by different signaling pathways. The cross-regulatory interactions between elements of different pathways affect the process of cell fate assignment during neural and astroglial tissue patterning. In this study, the interactions between Wnt and Notch pathways, the two major pathways that influence neural and astroglial differentiation of human induced pluripotent stem cells (hiPSCs) individually, were investigated. In particular, the synergistic effects of Wnt-Notch pathway on the neural patterning processes along the anterior-posterior or dorsal-ventral axis of hiPSC-derived cortical spheroids were explored. The human cortical spheroids derived from hiPSCs were treated with Wnt activator CHIR99021 (CHIR), Wnt inhibitor IWP4, and Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester [DAPT]) individually, or in combinations (CHIR + DAPT, IWP4 + DAPT). The results suggest that CHIR + DAPT can promote Notch signaling, similar or higher than CHIR alone, whereas IWP4 + DAPT reduces Notch activity compared to IWP4 alone. Also, CHIR + DAPT promoted hindbrain marker HOXB4 expression more consistently than CHIR alone, while IWP4 + DAPT promoted Olig2 expression, indicating the synergistic effects distinctly different from that of the individual small molecule. In addition, IWP4 simultaneously promoted dorsal and ventral identity. The patterned neural spheroids can be switched for astroglial differentiation using bone morphogenetic protein 4. This study should advance the derivations of neurons, astroglial cells, and brain region-specific organoids from hiPSCs for disease modeling, drug screening, as well as for hiPSC-based therapies. Impact Statement Wnt signaling plays a central role in neural patterning of human pluripotent stem cells. It can interact with Notch signaling in defining dorsal-ventral and rostral-caudal (or anterior-posterior) axis of brain organoids. This study investigates novel Wnt and Notch interactions (i.e., Wntch) in neural patterning of dorsal forebrain spheroids or organoids derived from human induced pluripotent stem cells. The synergistic effects of Wnt activator or inhibitor with Notch inhibitor were observed. This study should advance the derivations of neurons, astroglial cells, and brain region-specific organoids from human stem cells for disease modeling and drug screening, as well as for stem cell-based therapies. The results can be used to establish better in vitro culture methods for efficiently mimicking in vivo structure of central nervous system.
