Impact of Matrix Viscoelasticity on Induced Pluripotent Stem Cell Morphogenesis

Stanford University, Stanford CA

Investigators

Abstract

This project will study the use of engineered biomaterials to guide the life, death, and development of human induced pluripotent stem cells (hiPSCs), which will advance our understanding of hiPSC biology, identify design rules for development from the hiPSCs, reveal how hiPSCs are able to form organ-like structures (organoids) containing lumens, and provide insight into human development. hiPSCs are derived from normal adult cells and have the potential to turn into any type of cell in humans, similar to the potential of embryonic stem cells. Until now, the scientific community has mostly focused on using biochemical factors and Petri dish-based culture to study and drive the development of these stem cells. The engineered biomaterials-based work in this project represents a new approach to studying and using hiPSCs, and the successful completion of this work should lead to biologists, bioengineers, and clinicians adopting these engineered biomaterials as a matrix to grow cells for studies or applications involving hiPSCs. The results of this research will be disseminated through presentations at conferences and peer-reviewed journal publications. This project will support the training of graduate students, postdoctoral fellows, undergraduate students including those from groups underrepresented in science, high school students including those from high-need under-resourced schools, and high school teachers. Hands-on demonstrations related to the research targeted at a lay audience will be developed in parallel and delivered to audiences at various venues open to the broader public. The overall objective of this project is to determine how the biological and mechanical properties of hydrogels regulates the morphogenesis of human induced pluripotent stem cells (hiPSCs). Alginate hydrogels with independently tunable stiffness, viscoelasticity, and cell-adhesion ligand densities will be used for 3D culture of hiPSCs. Preliminary results have identified that hydrogels that are more viscous and contain high cell-adhesion ligand densities promote hiPSC growth, proliferation, and lumen formation in hiPSCs, with the hiPSCs maintaining their pluripotency over long times. The first objective of the project is to determine the effect of hydrogel properties on the morphogenesis and lumenogenesis of hiPSCs cultured within the hydrogels, and fully characterize the molecular state of the hiPSCs in the different conditions by use of phosphoprotein arrays, RNA-seq, and ATAC-seq. The second objective of the research is to elucidate the physics of lumen formation by hiPSCs in viscoelastic hydrogels using pharmacological inhibition and CRISPR/Cas9 knockouts combined with timelapse confocal and lattice light sheet microscopy. These studies will reveal how known mechanisms of force generation combine to drive lumen formation and expansion. Finally, hiPSCs undergo apoptosis, or programmed cell death, in more elastic hydrogels with no cell-adhesion ligands, but not in fast-relaxing viscoelastic hydrogels with no cell-adhesion ligands, which indicates that an adhesion-independent mechanism regulates apoptosis. The third objective of the project will be to determine the molecular mechanism mediating the effects of hydrogel viscoelasticity on apoptosis, focusing on the role of cell volume regulation and mechanosensitive ion channels, and utilizing inhibition studies and CRISPR/Cas9-mediated knockout cells. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.