Reconstructing the human endometrium in vitro using co-cultures of endometrial epithelial organoids and stromal cells

Human endometrial epithelial organoids are valuable in vitro models that recapitulate key epithelial features of the endometrium. However, they lack the stromal compartment, preventing the physiological epithelial–stromal crosstalk involved in regulating endometrial homeostasis and function. This study investigated whether co-culturing human endometrial epithelial organoids with stromal cells improves their molecular resemblance to native endometrial tissue. Endometrial biopsies were obtained from healthy donors ( n = 6). Human endometrial epithelial organoids and autologous stromal cells were isolated and cultured for 7 days under monoculture or co-culture conditions using a Transwell system. A human endometrial stromal stem cell line (ICE7) was also evaluated. Five experimental conditions were established: organoid monoculture, primary stromal cell monoculture, ICE7 monoculture, organoids co-cultured with primary stromal cells, and organoids co-cultured with ICE7 cells. Cell identity and viability were assessed by immunocytochemistry and LIVE/DEAD assays. Transcriptomic profiling was performed by RNA sequencing on all cell populations and native tissue, whereas secretomes were characterized by tandem mass spectrometry of conditioned-medium samples. Co-cultured organoids exhibited a transcriptomic profile that more closely resembled native endometrial tissue than did organoid monocultures. Principal component analysis showed a shift in the co-cultured organoid profile toward the native tissue, accompanied by higher transcriptomic correlation coefficients ( r ≈ 0.79 versus r ≈ 0.70) and fewer differentially expressed genes relative to native tissue samples. Co-cultured organoids showed significant upregulation of genes associated with endometrial function and cellular proliferation, including CDK1 , TOP2A , BUB1B , PRC1 , and SOX17 ( p < 0.05). No differentially expressed genes were detected between organoids co-cultured with primary stromal cells and those co-cultured with ICE7 cells. Secretome analysis identified proteins uniquely associated with co-culture conditions, including SOX17, HDAC4, BAG1, and YY1, suggesting active epithelial–stromal communication. Co-culture with stromal cells enhances the molecular fidelity of human endometrial epithelial organoids by promoting transcriptomic and secretome profiles that more closely resemble those of native endometrium. These findings highlight the importance of epithelial–stromal communication in endometrial modeling and support the use of co-culture systems for studies of endometrial physiology, regeneration, disease mechanisms, and therapeutic development.