Engineering hPSC-Derived Endometrial Organoids for Modeling Regeneration and Disease

Project summary The human endometrium is a uniquely regenerative tissue, undergoing cyclical remodeling without fibrosis ap- proximately 450 times in a woman's lifetime. This remarkable regenerative capacity is essential for reproductive health but is often disrupted in disorders such as endometriosis, Asherman's syndrome, uterine fibroids, and unexplained infertility. These conditions, which collectively affect millions of women and represent hundreds of billions of dollars in economic burden, are associated with impaired regulation of the stem cell niche and epithe- lial–stromal interactions. Despite its clinical importance, the mechanisms governing endometrial regeneration and stem cell niche formation remain poorly understood due to the lack of suitable human models. This exploratory R21 proposal seeks to address this critical biotechnological gap by developing an innovative human pluripotent stem cell (hPSC)-derived endometrial organoid platform that closely recapitulates the phys- iological interactions and developmental trajectories of the endometrium. Unlike biopsy-derived organoids, which lack developmental plasticity and are difficult to genetically manipulate, our hPSC-derived platform fol- lows the developmental trajectory of the Müllerian duct, allowing the spontaneous emergence of the endometrial epithelium and its associated stem cell niche. This provides an unprecedented platform to investigate endome- trial homeostasis, regeneration, and disease mechanisms. To establish this system, we will define the differentiation trajectory from Müllerian duct progenitors to endo- metrial organoids using single-cell RNA sequencing, computational lineage inference, and machine learning- driven tissue crosstalk analysis. By benchmarking against human reproductive single-cell atlases, we will refine key signaling inputs—including RA, BMP4, and Wnt—to optimize epithelial-stromal interactions and identify transitional states where stem-like populations emerge. We will then apply a kinome-wide CRISPRi screen to uncover genetic regulators of endometrial stem cell specification and maintenance, leveraging a validated sgRNA library to systematically inhibit kinase activity during niche formation. Pharmacological validation of top candi- dates in both hPSC-derived and patient-derived endometrial organoids will confirm functional relevance, while single-cell RNA sequencing of inhibitor-treated organoids will reveal how lineage trajectories are altered in re- sponse to kinase inhibition. By integrating cutting-edge stem cell biology, large-scale functional genomics, and machine learning-driven sig- naling analysis, this project will generate a scalable, physiologically relevant platform for dissecting human en- dometrial stem cell niche regulation. Our novel microphysiological platform will fuel future hypothesis-driven research and lay the groundwork for therapeutic strategies in reproductive medicine and regenerative biology. 1