Human Stem Cell Fate Decisions Dictated by Decoupled Biophysical Cues

多能干细胞可以转化为构成我们体内组织和器官的所有类型的细胞。特定的细胞类型（干细胞命运）取决于它们经历的生化和生物物理线索。生物物理学线索取决于周围支架的机械特性，例如弹性模量和泊松比（泊松比是材料的横向收缩与拉伸时长度增加的比率）。 该项目旨在了解这些机械性能如何通过调整弹性模量和泊松比来确定干细胞的命运，以揭示生物物理和生物化学信号对干细胞命运的协同作用。这项研究将有利于生物工程应用和生物制造（例如，新型微载体），为生物技术和制药工业带来更好的药物筛选和疾病建模。该项目还将建立一个互动学习平台，以减少性别差距，增加少数民族学生对工程的参与。将努力通过参加拟议的研究和教育活动，鼓励非裔美国学生和女学生追求高级培训和职业。该项目将包括与数学，科学和工程组织，全国黑人工程师协会，少数民族网络质量教育以及国家高磁场实验室的SciGirls计划合作，以吸引年轻女孩和非洲裔美国学生接受先进的科学和工程培训和职业。本项目的目标是阐明三维聚氨酯（PU）支架的弹性模量和泊松比的生物物理线索之间的相互作用及其对iPSCs在谱系特异性定型期间分泌内源性ECM和Yes相关蛋白（雅普）定位的影响。该项目的中心假设是，具有可调弹性模量和泊松比的支架影响细胞组织和生物物理信号，以调节内源性ECM和雅普表达的概况，并影响iPSC的谱系定型。这一假设是基于初步结果，这些结果证明了使用具有凹入结构的3D支架增强多能干细胞（PSC）的神经和血管分化-具有向内指向的角度的结构，如果在一个方向上拉伸，则在所有三个方向上扩展，即，具有负泊松比的结构。研究计划有三个目标。第一个目标是制造和表征具有不同泊松比和弹性模量的支架阵列。具有网状结构的规则PU支架将被加热/软化，并且受控屈曲将用于产生在固定弹性模量下具有不同泊松比（0.3至-0.4）的拉胀支架谱。同样地，通过将具有不同模量的规则支架压缩到相同的屈曲程度来制造在固定泊松比下具有不同弹性模量（1-100 kPa）的拉胀支架。支架将根据有限元建模的预测制作，输入温度依赖性弹性模量和视频数据。第二个目的是检查泊松比和弹性模量对iPSC谱系定型的差异影响。未分化的iPSC或iPSC衍生的神经祖细胞（NPC）将被接种到不同的支架中并向神经谱系或血管谱系诱导。将针对神经元标志物或血管标志物表征细胞，期望模拟组织弹性的拉胀支架将促进iPSC的神经分化，并且弹性模量和泊松比对神经谱系定型具有不同的影响。 第三个目的是确定泊松比和弹性模量对雅普定位和内源性ECM分泌的影响，所述内源性ECM调节经典Wnt信号传导并有助于iPSC的谱系定型。将检查雅普定位，并揭示雅普对Wnt信号传导的影响，期望拉胀支架诱导细胞质雅普定位，核雅普定位激活Wnt信号传导，细胞将响应支架弹性分泌不同的ECM谱并影响Wnt信号传导，最后，雅普与Wnt信号的相互作用有助于iPSCs的谱系承诺。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值进行评估，被认为值得支持和更广泛的影响审查标准。

Pluripotent stem cells can turn into all types of cells that make up the tissues and organs in our body. The specific cell type (stem cell fate) depends on the biochemical and biophysical cues they experience. The biophysical cues depend on the surrounding scaffold's mechanical properties, such as the elastic modulus and the Poisson's ratio (which is the ratio of a material's lateral contraction to its increase in length upon stretching). This project seeks to understand how these mechanical properties determine stem cell fate by tuning both elastic modulus and Poisson's ratio to reveal the synergistic roles of biophysical and biochemical signaling on stem cell fate. This research will benefit bioengineering applications and biomanufacturing (e.g., novel microcarriers), leading to better drug screening and disease modeling for the biotechnology and pharmaceutical industries. The project will also establish an interactive learning platform to reduce gender disparity and increase the participation of minority students in engineering. Efforts will be made to stimulate African American and women students to pursue an advanced training and career by participating in the proposed research and educational activities. This project will include working with Women in Math, Science and Engineering organization, National Society of Black Engineers, Quality Education for Minorities Network, and the SciGirls program at National High Magnetic Field Laboratory to attract young girls and African American students for advanced science and engineering training and career. The goal of this project is to elucidate the interactions between biophysical cues of elastic modulus and Poisson's ratio of 3D polyurethane (PU) scaffolds and their influence on the secretion of endogenous ECMs and Yes-associated protein (YAP) localization by iPSCs during lineage-specific commitment. The project's central hypothesis is that the scaffolds with tunable elastic modulus and Poisson's ratio affect cell organization and transduce biophysical signals to modulate the profile of endogenous ECMs and YAP expression and influence lineage commitment of iPSCs. This hypothesis is based on preliminary results that demonstrated enhanced neural and vascular differentiation of pluripotent stem cells (PSCs) using 3D scaffolds with re-entrant structures - structures with angles pointing inward that expand in all three directions if stretched in one direction, i.e., structures with negative Poisson's ratios. The research plan is organized under three objectives. The FIRST Objective is to fabricate and characterize scaffold arrays with different Poisson's ratio and elastic modulus. Regular PU scaffolds with reticulate structure will be heated/softened and controlled buckling will be used to produce a spectrum of auxetic scaffolds with varying Poisson's ratio (0.3 to -0.4) at a fixed elastic modulus. Likewise, auxetic scaffolds with different elastic modulus (1-100 kPa) at a fixed Poisson's ratio will be fabricated by compressing the regular scaffold with different modulus to the same degree of buckling. The scaffolds will be fabricated according to the prediction of finite element modeling with the inputs of temperature-dependent elastic modulus and on video data. The SECOND Objective is to examine the differential effects of Poisson's ratio and elastic modulus on iPSC lineage commitment. Undifferentiated iPSCs or iPSC-derived neural progenitor cells (NPCs) will be seeded into different scaffolds and induced toward neural lineage or vascular lineage. The cells will be characterized for neuronal markers or vascular markers with expectations that auxetic scaffolds that mimic tissue elasticity will promote neural differentiation of iPSCs and that elastic modulus and Poisson's ratio have differential effects on neural lineage commitment. The THIRD Objective is to determine the influences of Poisson's ratio and elastic modulus on YAP localization and the secretion of endogenous ECMs, which modulate canonical Wnt signaling and contribute to the lineage commitment of iPSCs. YAP localization will be examined and the influence of YAP on Wnt signaling will be revealed with expectations that auxetic scaffolds induce cytoplasmic YAP localization, that nuclear YAP localization activates Wnt signaling, that cells will secrete different profiles of ECMs in response to the scaffold elasticity and influence Wnt signaling and finally, that interactions of YAP with Wnt signaling contribute to the lineage commitment of iPSCs.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.