Impact of Matrix Viscoelasticity on Induced Pluripotent Stem Cell Morphogenesis

该项目将研究使用工程生物材料来指导人类诱导多能干细胞（hiPSC）的生命，死亡和发育，这将促进我们对hiPSC生物学的理解，确定hiPSC发育的设计规则，揭示hiPSC如何能够形成含有管腔的器官样结构（类器官），并提供对人类发育的见解。 hiPSC来源于正常的成体细胞，具有转化为人类任何类型细胞的潜力，类似于胚胎干细胞的潜力。 到目前为止，科学界主要集中在使用生化因子和基于培养皿的培养来研究和驱动这些干细胞的发育。 该项目中基于工程生物材料的工作代表了一种研究和使用hiPSC的新方法，这项工作的成功完成将导致生物学家，生物工程师和临床医生采用这些工程生物材料作为基质来生长细胞，用于涉及hiPSC的研究或应用。 这项研究的结果将通过在会议上的演讲和同行评审的期刊出版物传播。 该项目将支持培训研究生、博士后研究员、本科生（包括那些来自科学领域代表性不足的群体的学生）、高中生（包括那些来自资源不足的高需求学校的学生）和高中教师。 与针对外行观众的研究相关的实践演示将同时开发，并在向更广泛公众开放的各种场所向观众提供。 该项目的总体目标是确定水凝胶的生物学和机械特性如何调节人诱导多能干细胞（hiPSC）的形态发生。 具有独立可调刚度、粘弹性和细胞粘附配体密度的藻酸盐水凝胶将用于hiPSC的3D培养。 初步结果已经确定，更粘稠且含有高细胞粘附配体密度的水凝胶促进hiPSC中的hiPSC生长、增殖和管腔形成，其中hiPSC长时间保持其多能性。 该项目的第一个目标是确定水凝胶特性对在水凝胶内培养的hiPSC的形态发生和管腔形成的影响，并通过使用磷蛋白阵列、RNA-seq和ATAC-seq来充分表征hiPSC在不同条件下的分子状态。 该研究的第二个目的是使用药理学抑制和CRISPR/Cas9敲除结合时移共聚焦和晶格光片显微镜来阐明hiPSC在粘弹性水凝胶中形成管腔的物理学。这些研究将揭示已知的力产生机制如何结合联合收割机来驱动管腔形成和扩张。 最后，hiPSC在没有细胞粘附配体的更有弹性的水凝胶中经历凋亡或程序性细胞死亡，但在没有细胞粘附配体的快速松弛粘弹性水凝胶中不经历凋亡或程序性细胞死亡，这表明粘附非依赖性机制调节凋亡。 该项目的第三个目标是确定介导水凝胶粘弹性对细胞凋亡影响的分子机制，重点是细胞体积调节和机械敏感离子通道的作用，并利用抑制研究和CRISPR/Cas9-该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的智力价值和更广泛的影响审查评估的支持的搜索.

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.