Optimized feeder-dependent culture enables efficient hypothalamic-pituitary organoid induction from human pluripotent stem cells

Human pluripotent stem cell (hPSC)-derived hypothalamus-pituitary organoids recapitulate key processes of pituitary development in vitro, where cellular interactions between hypothalamic and oral ectodermal lineages are essential for organoid formation. However, how maintenance culture conditions for hPSCs influence the processes of lineage specification remains unclear. Here, we demonstrate that feeder-dependent culture conditions critically determine lineage specification during organoid formation. hPSCs maintained on SNL feeders preferentially differentiated into oral ectodermal cells, whereas maintenance on MEF feeders favored hypothalamic lineages. Co-culturing SNL- and MEF-maintained hPSCs or combining their conditioned media effectively balanced these lineages, enhancing pituitary induction efficiency. Furthermore, a stabilized FGF2 mutant enabled a "weekend-free" protocol that reduced labor and cost while maintaining high differentiation efficiency. Our findings underscore the significance of maintenance conditions and hypothalamus-oral ectoderm interactions in pituitary development, establishing a robust platform for efficient and reproducible generation of hypothalamus-pituitary organoids.