Development of polarity-reversed endometrial epithelial organoids

Polarity-reversed endometrial epithelial organoids exhibit histological and physiological characteristics resembling uterine epithelium in vivo, respond to hormones, and undergo secretory cell transformation. The ability to modify the polarity without impairing functionality, coupled with successful coculture with microbial and embryonic entities, paves the way for investigating complex interactions at the endometrial epithelial surface.

The uterine epithelium comprises a single layer of hormone-responsive polarized epithelial cells that line the lumen and form tubular glands. Endometrial epithelial organoids (EEO) can be generated from uterine epithelia and recapitulate cell composition and hormone responses in vitro. As such, the development of EEO represents a significant advance in facilitating mechanistic studies in vitro. However, a major limitation of the use of EEO cultured in basement membrane extract and other hydrogels is the inner location of the apical membrane (apical-in EEO), thereby hindering direct access to the apical surface of the epithelium to study interactions with the embryo or infectious agents such as viruses and bacteria. To address this challenge, we developed a suspension culture method to reverse the polarity of EEO. The result is an apical-out organoid that preserves a distinct apical-basolateral orientation and remains responsive to ovarian steroid hormones. Apical-out EEO were positive for the gland marker, FOXA2, and exhibited appropriate hormonal regulation of steroid hormone receptor expression. Notably, progesterone treatment resulted in secretory transformation in apical-out EEO, including a decrease in microvilli and cilia, and an increase in secretory granules. Likewise, reflective of in vivo conditions, ENPP3, a P4-regulated gene, was localized apically in steroid hormone-treated organoids. Coculture experiments with apical out EEO demonstrate the model's utility in studying uterine epithelium interactions with bacteria (E. coli) and blastocysts. The apical out EEO model lays the foundation for developing new in vitro functional assays, particularly regarding epithelial interactions with embryos during pregnancy or other luminal constituents in a pathological or diseased state.