Dynamic double network hydrogel for generating pancreatic organoids from induced pluripotent stem cells

项目总结 人诱导多能干细胞(Hipscs)可在三个生殖层分化为细胞。 (外胚层、中胚层和内胚层)，为基础研究和翻译提供了宝贵的细胞来源 申请。虽然近年来在HiPSC的谱系特异性分化方面取得了突破，但 多期胰腺外分泌过程中基质刚性、粘弹性和整合素配体呈递的影响 类有机物(ExoPO)的发育在很大程度上仍未被探索。此外，当前的三维(3D) 用于hPSC培养和分化的基质不能提供足够的基质生物物理控制 性质(例如，粘弹性、刚性)和生化基序(例如，细胞粘附性配体)。另外，没有 以前的工作已经使用动态无异种水凝胶来研究基质力学和细胞-凝胶的影响。 黏附配体介绍HiPSC来源的exoPO的发展。我们假设exoPO 通过在过程中向细胞呈现微调的3D基质属性，可以显著地改善分化 发展阶段。为了实现这一目标，我们将开发一种粘弹性动态双网络(DDN)。 水凝胶平台在基质机械性能和生化基元方面具有前所未有的可调性。 具体地说，我们将通过形成具有逆电子的弹性水凝胶网络来控制基质的硬度 Demand Diels-Alder(IEDDA)点击反应。我们将通过一组线性的 通过可逆硼酸二醇键复合的聚合物。独一无二的是，弹性iEDDA点击水凝胶网络 将被设计成表现出可调的水解性降解。另一方面，粘弹性网络将 允许细胞黏附配体的结合，以允许粘弹性介导的整合素结合。有了这个 粘弹性DDN水凝胶平台，我们将定义基质粘弹性、刚性和整合素的影响 多能胰祖细胞分化和外周血细胞形成的配体呈递。在目标1中，我们 将研究基质粘弹性在胰腺祖细胞分化中的作用。在目标2中，我们将描述 胰腺祖细胞分化过程中对基质硬度的要求。在目标3中，我们将确定 胰腺导管/腺泡细胞规范上的细胞黏附配体。从长远来看，这个项目将产生一个 动态水凝胶平台促进化学定义的基质作为无异种人工干细胞的使用 用于有机物开发和组织再生应用的壁龛。

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.