Organoid co-culture model of the cycling human endometrium in a fully-defined synthetic extracellular matrix reveals epithelial-stromal crosstalk

Abstract

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.

Integrating endometrial proteomic and single cell transcriptomic pipelines reveals distinct menstrual cycle and endometriosis-associated molecular profiles

“…Tyro3 undergoes enhanced shedding, producing prodigious amounts of soluble receptor, in rheumatoid arthritis (Vullings et al, 2020). These mechanisms could be further parsed in 3D co-cultures, building on advances in 3D tissue engineering models that incorporate the relevant cell types in synthetic ECMs (Below et al, 2022; Gnecco et al, 2021).…”

Bioengineered Hydrogels Recapitulate Fibroblast Heterogeneity in Cancer

“…In conclusion, using in vivo transcriptomic data to guide the selection of hydrogel parameters, [59] we established in vitro cultures of different CAF subsets in HNSCC using a bioengineered hydrogel. These in vitro myCAF and IL-iCAF cultures revealed useful insights, such as the effect of microtubule-targeting standard-of-care drugs on different CAF subsets.…”

An integrated single-cell reference atlas of the human endometrium

“…exhibited small fold changes between cases and controls), requiring further validation in a larger set of samples with detailed metadata and menstrual phase annotation. To dissect the molecular pathways and validate the involvement of WNT and insulin pathways in progesterone-mediated cellular responses could now be tested using 3D in vitro models of the endometrium encompassing both stromal and epithelial cells 82 . The HECA is a key stepping stone towards the generation of a future pan-endometrial atlas encompassing endometrial cellular heterogeneity across the lifespan and in diseases.…”