Establishment of mouse endometrial assembloids with luminal epithelial-like structures in vitro

Embryo implantation requires coordinated interactions between the embryo and the maternal endometrium. Endometrial organoids (EOs) have emerged as promising in vitro models for studying this process. At present, however, mouse-derived EOs (mEOs) are predominantly composed of a single epithelial cell type and lack a functional luminal epithelium (LE), cellular diversity, and hormonally responsive architecture, limiting their capacity to recapitulate the dynamic receptive phase of the endometrium. In this study, a robust three-dimensional mouse endometrial assembloid model was established by co-culturing endometrial gland-like organoids (GLOs) with primary endometrial stromal cells (ESCs) under air-liquid interface (ALI) conditions. Optimization of the GLO culture was achieved by fine-tuning the concentrations of Wnt3a and R-Spondin1, while stromal viability and functionality were enhanced by supplementation with hydrocortisone, L-ascorbic acid, and ITS-X. Comparative analysis demonstrated that ALI conditions significantly enhanced epithelial proliferation, gland morphogenesis, and metabolic activity across both epithelial and stromal compartments. Notably, the resulting ALI-grown mouse endometrial assembloids (ALI-mEnAOs) developed a well-defined LE layer and recapitulated molecular and cellular features characteristic of both pre-receptive and receptive endometrial states in vivo. Compared to existing mEOs, analyses confirmed that ALI-mEnAOs closely mimic in vivo endometrial architecture, lineage composition, phase-specific gene expression signatures, and dynamic hormonal response. This model offers a physiologically relevant platform for mechanistic investigation of endometrial function and embryo implantation dynamics.