Dynamic Double Network Hydrogel for Generating Pancreatic Organoids from InducedPluripotent Stem Cells

PROJECT SUMMARY Human induced pluripotent stem cells (hiPSCs) can be differentiated to cells in all three germ layers (ectoderm, mesoderm, and endoderm), providing an invaluable cell source for basic research and translational applications. While recent years have witnessed breakthroughs in lineage-specific differentiation of hiPSC, the effect of matrix stiffness, viscoelasticity, and integrin ligand presentation during multi-stage exocrine pancreatic organoid (exoPO) development remain largely unexplored. Furthermore, current three-dimensional (3D) matrices for hiPSC culture and differentiation do not provide sufficient controls over matrix biophysical properties (e.g., viscoelasticity, stiffness) and biochemical motifs (e.g., cell-adhesive ligands). Additionally, no prior work has employed dynamic xeno-free hydrogels to study the effect of matrix mechanics and cell- adhesive ligand presentation on the development of hiPSC-derived exoPO. We hypothesize that exoPO differentiation can be drastically improved by presenting the cells with fine-tuned 3D matrix properties during the developmental stages. To achieve this goal, we will develop a viscoelastic dynamic double network (DDN) hydrogel platform with unprecedented tunability in matrix mechanical properties and biochemical motifs. Specifically, we will control matrix stiffness by forming an elastic hydrogel network with inverse Electron Demand Diels-Alder (iEDDA) click reaction. We will tune matrix stress-relaxation through a set of linear polymers complexed by reversible boronate-diol bonding. Uniquely, the elastic iEDDA click hydrogel network will be engineered to exhibit tunable hydrolytic degradation. On the other hand, the viscoelastic network will allow conjugation of cell adhesive ligands to permit viscoelasticity mediated engagement of integrins. With this viscoelastic DDN hydrogel platform, we will define the impact of matrix viscoelasticity, stiffness, and integrin ligand presentation on multipotent pancreatic progenitor cell differentiation and exoPO formation. In Aim 1, we will study the role of matrix viscoelasticity on pancreatic progenitor differentiation. In Aim 2, we will describe the requirements of matrix stiffness during pancreatic progenitor differentiation. In Aim 3, we will identify the role of cell adhesive ligands on pancreatic ductal/acinar cell specification. In the long-term, this project will produce a dynamic hydrogel platform to advance the use of chemically-defined matrices as xeno-free artificial stem cell niches for organoid development and tissue regeneration applications.