Endocrine Abstracts

JOINT2959

Background: The pituitary gland is the hub of our endocrine system as it is responsible for the production of hormones that drive and regulate important physiological processes including body growth, lactation and stress. Organoids are complex 3D structures that mimic the biological functions of the target organ and allow the study of physiopathological mechanisms. The development of in vitro models using human induced pluripotent stem cells (hiPSC)-derived pituitary organoids has helped gain knowledge on pituitary biology and disorders^1,2. However, up to date the generation of hormone-secreting organoids remains challenging because of the paucity of cells that differentiate into hormone-secreting cells.ObjectiveIn this study, we aimed to generate pituitary organoids with an improved ratio of hormone-secreting cells.

Methods: AG08h and KOLF2.1J hiPS cell lines were both derived from healthy donors, and 10742L from a patient with Leber hereditary optic neuropathy. All three hiPS cell lines underwent to genomic characterization prior to their differentiation towards pituitary organoids. hiPSCs were differentiated into pituitary organoids as previously described 3. Cells were maintained in culture up to 108 days and the expression of pluripotency markers and pituitary differentiation markers as well as those of ACTH, GH and PRL was measured via real time- quantitative PCR (RT-qPCR). Protein levels of ACTH, GH and PRL were measured by immunofluorescence (IF) and ELISA.

Results: We observed the presence of an epithelial layer as early as day 10 in all hiPS cell lines. In line with this, as of day 30 mRNA levels of EpCAM increased. Whereas the expression of pluripotency markers (SOX2 and NANOG) strongly decreased along with differentiation, mRNA levels of POU1F1, PITX1 as early pituitary differentiation markers increased. In addition, we found increased levels of HESX1 between day 30–48 suggesting proliferation of pituitary progenitors, followed by increased TBX19 and PROP1 between day 48-60 pointing at further differentiation towards both corticotroph and somato-lactotroph cell lineages. Consistently, from day 60 onwards, we found an increase of ACTH, GH and PRL mRNA levels.

Conclusion: Here, we induced pituitary differentiation using three different hiPS cell lines. Of note, all three hiPS cell lines showed variable basal levels of ACTH secretion in culture media, and 10724L showed also secretion of prolactin, thus representing an interesting model to study both pituitary development and diseases.

References: 1. doi:10.3389/fendo.2023.1233714.

2. doi:10.7554/eLife.90875

3. doi:10.1016/j.stemcr.2023.05.002.