Sequestered cell-secreted extracellular matrix proteins improve murine folliculogenesis and oocyte maturation for fertility preservation

Synthetic matrices offer a high degree of control and tunability for mimicking extracellular matrix functions of native tissue, allowing the study of disease and development in vitro. In this study, we functionalized degradable poly(ethylene glycol) hydrogels with extracellular matrix (ECM)-sequestering peptides aiming to recapitulate the native ECM composition for culture and maturation of ovarian follicular organoids. We hypothesized that ECM-sequestering peptides would facilitate deposition and retention of cell-secreted ECM molecules, thereby recreating cell-matrix interactions in otherwise bioinert PEG hydrogels. Specifically, heparin-binding peptide from antithrombin III (HBP), heparan sulfate binding peptide derived from laminin (AG73), basement membrane binder peptide (BMB), and heparan sulfate binding region of placental growth factor 2 (RRR) tethered to a PEG hydrogel significantly improved follicle survival, growth and maturation compared to PEG-Cys, a mechanically similar but biologically inert control. Immunohistochemical analysis of the hydrogel surrounding cultured follicles confirmed sequestration and retention of laminin, collagen I, perlecan, and fibronectin in ECM-sequestering hydrogels but not in bioinert PEG-Cys hydrogels. The media from follicles cultured in PEG-AG73, PEG-BMB, and PEG-RRR also had significantly higher concentrations of factors known to regulate follicle development compared to PEG-Cys. PEG-AG73 and PEG-BMB were the most beneficial for promoting follicle maturation, likely because AG73 and BMB mimic basement membrane interactions which are crucial for follicle development. Here we have shown that functionalizing PEG with ECM-sequestering peptides allows cell-secreted ECM to be retained within the hydrogels, restoring critical cell-matrix interactions and promoting healthy organoid development in a fully synthetic culture system. STATEMENT OF SIGNIFICANCE: Here we present a novel approach for sequestering and retaining cell-secreted extracellular matrix in a fully synthetic material for organoid culture. We have engineered a biomimetic poly(ethylene glycol) hydrogel functionalized with extracellular matrix-binding peptides to recapitulate the ovarian microenvironment. Incorporation of these peptides allows ovarian follicles to recreate their native matrix with the sequestered ECM that subsequently binds growth factors, facilitating follicle maturation. The novel design resulted in improved outcomes of folliculogenesis, potentially developing a fertility preservation option for young women undergoing sterilizing treatments for cancer. The fully synthetic and modular nature of this biomimetic material holds promise for other tissue engineering applications as it allows encapsulated cells to rebuild their native microenvironments in vitro.