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

Background Embryo implantation involves complex interactions between the embryo and the maternal endometrium. Recently, the endometrial organoids (EOs) provide a new approach for studying embryo implantation. However, the currently constructed mouse EOs (mEOs) were mainly made by single cell types, so they lacked the interaction between different cell types and the luminal epithelium (LE) structure, which leads to a lack of dynamic hormonal responses, making them difficult to mimic the receptive phase of the endometrium in vivo. Results We here successfully developed mouse endometrial assembloids with LE structures in vitro by co-culturing mouse endometrial gland-like organoids (GLOs) and endometrial stromal cells (ESCs) using the air-liquid interface (ALI) method. To do this end, we first optimized the culture system for mouse GLOs by fine-tuning the concentrations of Wnt3a and R-Spondin1 and then improved the culture of ESCs by incorporating hydrocortisone, L-ascorbic acid, and ITS-X. Comparison analysis showed that the ALI culture methods significantly enhance epithelial cell proliferation, gland development, and metabolic activity of GLOs and ESCs. Importantly, mouse endometrial assembloids in the ALI condition (ALI-mEnAOs) exhibited LE characteristics, which effectively simulate the mouse pre-receptive and receptive endometrium in vivo, respectively. Conclusion Compared to existing mouse endometrial organoids, we successfully developed ALI-mEnAOs that closely resemble the receptive phase of the mouse endometrium in vivo in LE structure, cellular phenotypes, gene expression profiles and dynamic hormonal responses. This study provides a reliable and valuable in vitro platform for investigating functions of the endometrium and embryo implantation.