The human endometrium is a mucosal barrier that undergoes cycles of growth, differentiation, and breakdown in response to sex hormone fluctuations. Dynamic tissue responses to hormones are primarily driven by epithelial-stromal communication and its dysregulation is linked to myriad gynecological disorders. The lack of robust in vitro models for the long-term 3D co-culture of patient-derived endometrial epithelial and stromal cells hinders dissection of this crosstalk and thus impairs progress in disease treatment. Here, we describe a versatile synthetic extracellular matrix tailored to the endometrium that enables the in vitro modeling of human healthy and disease states across the menstrual cycle. We used a tissue-inspired approach to semi-empirically screen a parameter space that encompasses the biophysical and molecular features of the endometrial microenvironment. Leveraging cell-specific integrin expression profiles, we defined a modular polyethylene glycol (PEG)-based hydrogel that fosters hormone-driven expansion and differentiation of epithelial organoids co-cultured with stromal cells. Characteristic morphological and molecular responses of each cell type to hormone changes were observed when cells were co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Using transcriptomic and functional assays, we demonstrate the ability to recapitulate menstrual-cycle specific reproductive events and identified that inflammation-induced dysregulation of epithelial proliferation is mediated via the stromal compartment. Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand endometriotic diseases.

8Epithelial organoids are now an important tool in fields ranging from regenerative medicine to 9 drug discovery. Organoid culture requires Matrigel, a complex, tumor-derived, extracellular 10 matrix. An alternative completely synthetic matrix could improve culture reproducibility, clarify 11 mechanistic phenomena, and enable applications involving human implantation. Here, we 12 designed synthetic matrices with tunable biomolecular and biophysical properties that allowed us 13 to identify critical gel parameters in organoid formation. Inspired by known epithelial integrin 14 expression in the proliferative niche of the human intestine, we identified an a2b1 integrin-binding 15 peptide as a critical component of the synthetic matrix that supports human duodenal colon and 16 endometrial organoid propagation. We show that organoids emerge from single cells, retain their 17 proliferative capacity, are functionally responsive to basolateral stimulation and have correct 18 apicobasal polarity upon induction of differentiation. The local biophysical presentation of the 19 cues, rather than bulk mechanical properties, appears to be the dominant parameter governing 20 epithelial cell proliferation and organoid formation in the synthetic matrix. 21 . 22