The Specification and Functional Maturation of Sub-Cerebral Projection Neurons Derived from Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSC) are an invaluable resource for investigating the molecular mechanisms regulating cell fate specification during brain development. However, most directed differentiation methods exhibit significant cell fate heterogeneity and require several months to become functional. To address this challenge, we developed a green fluorescent protein (GFP) reporter system in hiPSC by targeting the genomic locus of Forebrain Enriched Zinc Finger 2 (<i>FEZF2</i>), which encodes a transcription factor essential for the fate specification of sub-cerebral projection neurons (SCPN) during forebrain development. Using this FEZF2-GFP reporter hiPSC line, we optimized a directed differentiation protocol to rapidly and efficiently generate pallial progenitors and glutamatergic neuronal subgroups after 3 weeks. Through fluorescence activated cell sorting for both GFP and CD200, isolated post-mitotic SCPN immediately displayed electrophysiological properties and formed glutamatergic synapses within 4 additional weeks of <i>in vitro</i> cell culture. Co-culture with hiPSC-derived spinal motor neurons further enhanced these electrophysiological characteristics, improved viability, and increased synapse formation in SCPN. This study presents a streamlined and effective strategy to generate, isolate, and characterize human motor neuron circuits, providing insights into the molecular determinants regulating synaptogenesis and functional maturation.