The pituitary gland regulates a wide range of physiological functions, including growth, metabolism, reproduction, and stress responses. During embryonic development, it forms through interactions between the hypothalamus and a neighboring tissue known as the oral ectoderm. Scientists have recreated aspects of this process using hypothalamic-pituitary organoids derived from human induced pluripotent stem cells, creating valuable models for investigating human development and disease. However, inconsistent differentiation outcomes have limited their broader utility.

To address this challenge, researchers examined how stem cell maintenance conditions influence subsequent organoid formation. Although stem cells cultured under different conditions retained their pluripotent characteristics, their ability to generate pituitary tissues varied substantially. While cells maintained using feeder-dependent culture methods consistently produced organoids containing both hypothalamic and oral ectodermal populations, several feeder-free approaches were less effective at generating pituitary progenitor cells under the study's differentiation protocol.

The team then compared two commonly used feeder cell types and found striking differences in developmental potential. Stem cells maintained on SNL feeder cells tended to form oral ectodermal tissues, while those maintained on mouse embryonic fibroblast feeders preferentially generated hypothalamic lineages. Because pituitary development relies on coordinated communication between these two tissues, favoring one tissue over the other reduced the efficiency of pituitary induction.

Seeking to restore this balance, the researchers combined stem cells maintained under the two feeder conditions before organoid formation. This approach produced a more balanced representation of hypothalamic and oral ectodermal tissues and significantly improved pituitary progenitor cell generation. Interestingly, the lineage preferences established during maintenance culture were not permanent. As organoids developed, cells from both populations contributed broadly to organoid formation, suggesting that interactions during differentiation can reshape developmental trajectories.

To make the process more practical, the team developed a culture system that used a mixture of the two feeder cell types during stem cell maintenance. This simplified strategy achieved similar improvements without the need to culture separate stem cell populations. Further experiments showed that feeder cells exert their effects not only through direct contact but also through soluble factors released into the culture environment. Stem cells maintained in feeder-conditioned media remained highly competent for pituitary differentiation, highlighting the importance of these secreted signals.

The researchers also streamlined the lengthy culture process by introducing a "weekend-free" protocol. Replacing conventional fibroblast growth factor with a more stable variant reduced the frequency of media changes while maintaining stem cell quality and organoid performance. The resulting organoids successfully generated hormone-producing pituitary cells and retained functional hormone secretion.

Together, these findings identify stem cell maintenance conditions as a critical determinant of pituitary organoid formation. By promoting balanced development of hypothalamic and oral ectodermal tissues while reducing culture workload, this work establishes a more robust and practical system for studying human pituitary biology, modeling endocrine diseases, and supporting regenerative medicine research.

• doi: 10.1016/j.stemcr.2026.103145