A novel protocol for the efficient generation of all three major hippocampal neuronal sub-populations from human pluripotent stem cells

The diverse computational functions of the human hippocampus rely on coordinated interactions among dentate gyrus (DG), CA3, and CA1 subfields, yet generating all three neuronal identities in vitro - particularly CA1 - has remained challenging. Here we establish a reproducible and modular differentiation protocol that directs human pluripotent stem cells (hPSCs) through dorsomedial telencephalic progenitors to yield DG, CA3, and CA1 neuronal subtypes together with hippocampal regionally specified astrocytes. Early tri-inhibition combined with Sonic hedgehog suppression produced dorsal forebrain progenitors (FOXG1+, PAX6+), while FGF2 treatment supported progenitor maintenance and induced TBR2+ intermediate progenitors. Controlled WNT activation using CHIR99021 drove progressive enrichment of PROX1 hippocampal progenitors across two independent donor lines. Terminal differentiation produced MAP2+/TAU+ neurons that expressed DG (PROX1), CA3 (GRIK4), and CA1 (WFS1, OCT6) markers, with maturing synaptic puncta. Defined progenitors generated long-lived (>400 days) hippocampal organoids exhibiting mixed neuronal-glial populations and spontaneous activity characterized by increased firing rates, high information entropy, and hub-like causal connectivity relative to monolayers, whereas astrocytes-supplemented monolayers displayed intermediate maturation. Population level electrophysiological analysis was also conducted to explore the dynamics of these different cultures. This platform enables systematic experimental control over neuron-astrocyte ratios, culture geometry, and developmental timing, providing a foundation for mechanistic studies of human hippocampal development, circuit function, and disease. Note on figure qualityThis is the preprint version of the manuscript. Figures are included adjacent to described results for the convenience of the reader but may be lower resolution due to file size restrictions on bioRxiv. High resolution figures are included as separate .tiff files for download.