Defined matrices for epithelial organoid culture and disease modelling

Advances in our understanding of the biology of stem cells and their niches have provided the necessary foundation to create in vitro complex self-organizing three-dimensional (3D) structures called organoids. While organoids represent a highly promising new class of biological model systems, the translational potential of these systems is currently limited by their dependence on animal-derived matrices, particularly Matrigel, an extracellular matrix (ECM) derived from mouse tumors. Apart from their immunogenicity and batch-to-batch variability, these matrices do not allow modulation of ECM composition and, accordingly, systematic adaptation of properties to specific cells and tissues. To address this issue, new biomaterial-based approaches have focused on the development of synthetic alternatives to Matrigel. This work focuses on the development of chemically defined matrices to elucidate the specific effects of ECM, with the goal of further increasing the translational relevance of epithelial organoids for regenerative medicine and disease modeling applications. Given the critical functionality of the intestine and the liver, and emergence of debilitating diseases due to damage to these organs, I have mainly focused on intestinal and liver organoids. First, I developed a new family of synthetic poly(ethylene glycol) (PEG)-based hydrogels, so-called low-defect thiol-Michael addition (LDTM) hydrogels, by modifying the conventional crosslinking strategy of existing PEG-co-peptide hydrogels formed by Michael addition of bis-cysteine peptides and vinyl sulfone-terminated PEG macromers. These mechanically tunable hydrogels are chemically defined and can be prepared with minimal batch-to-batch variability. They also allow the derivation of mouse and human intestinal and liver organoids, the latter without any animal components. Furthermore, I leveraged the modularity of synthetic matrices and derivation of liver ductal organoids in these matrices, to model some aspects of liver fibrosis, a global health problem affecting millions of people, and for which organ transplantation is the only medical solution. I demonstrated the role of tissue stiffness, a feature usually neglected in fibrosis models, on a regenerative mechanism associated with liver fibrosis called ductular reaction (DR). Human ductal liver organoids showed reduced proliferative capacity in LDTM gels with stiffness values similar to those of fibrotic liver tissue. In addition, transcriptomic analysis revealed impaired stemness potential, as well as concomitant induction of inflammatory responses. Overall, the newly developed hydrogels provide a reproducible, chemically defined environment that can be readily prepared with minimal batch-to-batch variability for multiple stem cell types and for organoid culture. Successful derivation of human intestinal and liver organoids in these matrices may open exciting prospects for clinical applications of organoids. In addition, the modularity of these matrices to mimic the ECM in pathological tissues, particularly the aberrant mechanical properties of fibrotic tissues, will enable the development of powerful disease models and predictive drug screening platforms and promote the development of new therapies