Engineered Viscoelastic Hydrogels for Optimized Generation and Transplantation of Human iPSC-Derived Salivary Gland Organoids to Treat Hyposalivation

PROJECT SUMMARY Human salivary gland organoid (SGO) are promising in vitro models for drug discovery, disease modeling, regenerative therapy. However, the vast majority of organoid derivation protocols rely on ill-defined Matrigel with tumorigenic origin and immunogenic risks, preventing their translational applications. The dependence of organoid assembly on self-organization capacity of pluripotent stem cells is another major limitation, leading to heterogeneous organoids due to stochastic self-assembly. Shifting SGO derivations from stochastic to deterministic remains a challenge due to lack of information on the regulatory factors dictating pluripotent stem cells fate during early embryonic development. Such cues – arise from complex cell-cell and cell-extracellular matrix (ECM) interactions – change spatially and temporally during developmental stages to guide stem/progenitor cells differentiation, self-organization, and tissue morphogenesis, making in vitro recapitulation using engineered synthetic matrices is difficult. Indeed, salivary gland embryonic development is a highly regulated multistage process, starting from pluripotent embryonic precursors, to germ-layer specification, then to lineage-restricted proximal and distal epithelial progenitors, which specializes to form epithelial compartments of ductal and acinar cells. Here, we propose development of human induced pluripotent stem cell (iPSC)-derived SGO with secretory function and well-defined architecture, and revealing the regulatory factors affecting salivary gland epithelial progenitor (SGEP) colony growth, differentiation, and branching morphogenesis. To achieve this goal, programmable differentiation of human iPSCs into SGEPs will be conducted. Afterwards, engineered dynamic hydrogels with fine-tuned physicochemical and biological properties will be utilized to probe the SGEPs colony response to matrix stiffness, stress-relaxation, degradability, and bioactivity (Aim 1). The information will be used in a feedback loop to optimize the hydrogel properties for generation of functional SGO, and thus avoiding the immunogenic risk of the commonly used Matrigel. Furthermore, photoactive hydrogel will be employed to enable on-demand modulation of matrix mechanics, to enable colony expansion at early stage and controlled symmetry breaking (onset of morphogenesis) at later stage, thereby generating reproducible SGO for translational applications (Aim 2). Finally, the secretory function of the SGO will be validated in a preclinical mouse model of hyposalivation (Aim 3). Overall, the proposed project marks as a conceptual advance in development of SGO using innovative viscoelastic matrices.