Modeling human prenatal adrenocortical functional zonation dynamics from pluripotent stem cells.

The adrenal cortex produces essential steroid hormones through a concentric zonal architecture, established by the centripetal transdifferentiation of subcapsular progenitors within a capsule-derived niche. To capture this complexity, we establish a human pluripotent stem cell-derived adrenal organoid system that faithfully recapitulates this process. RSPO3/WNT signaling from the capsule specifies definitive zone (DZ) progenitors from the adrenal primordium, which then differentiate into a cortisol-producing transitional zone and an androgen-producing fetal zone under the influence of RSPO3 and ACTH. Loss of NR0B1 impairs DZ specification and triggers direct adrenal primordium-to-fetal zone conversion, mirroring the mechanism of X-linked adrenal hypoplasia congenita. When DZ cells are encapsulated with capsule cells separately derived from pluripotent stem cells, they reconstitute zonation in vivo, forming ACTH-responsive tissue that produces both cortisol and androgens. This organoid platform offers a powerful tool to dissect human adrenal development and establishes a foundation for regenerative therapies targeting adrenal diseases.