Human Stem Cell Fate Decisions Dictated by Decoupled Biophysical Cues

多能干细胞可以转化为构成我们身体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)取决于他们所经历的生化和生物物理线索。生物物理线索取决于周围支架的机械性能，如弹性模数和泊松比(材料横向收缩与拉伸时长度增加的比率)。该项目试图通过调节弹性模数和泊松比来揭示生物物理和生化信号在干细胞命运中的协同作用，以了解这些机械特性如何决定干细胞的命运。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)，为生物技术和制药行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，以缩小性别差距，增加少数族裔学生对工程学的参与。将努力通过参加拟议的研究和教育活动，鼓励非洲裔美国人和女性学生追求高级培训和职业生涯。该项目将包括与女性在数学、科学和工程组织、全国黑人工程师协会、少数族裔优质教育网络以及国家强磁场实验室的SciGirls计划合作，以吸引年轻女孩和非裔美国学生参加高级科学和工程培训和职业生涯。本项目的目的是阐明三维聚氨酯(PU)支架弹性模量和泊松比的生物物理线索之间的相互作用，以及它们对IPSCs在谱系特异性承诺过程中内源性ECM分泌和YAP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并传递生物物理信号，从而调节内源性ECM和YAP的表达，并影响IPSCs的谱系承诺。这一假说基于的初步结果表明，使用带有折返式结构的3D支架可以增强多潜能干细胞(PSCs)的神经和血管分化。折返式结构是指角度指向内部的结构，如果在一个方向上拉伸，这些结构可以在所有三个方向上扩展，即泊松比为负的结构。研究计划在三个目标下进行组织。第一个目标是制备和表征具有不同泊松比和弹性模数的支架阵列。将具有网状结构的常规PU支架加热/软化，并控制屈曲，以产生在固定弹性模数下具有不同泊松比(0.3~-0.4)的伸展支架谱。同样，在固定泊松比下，通过将不同弹性模数的规则支架压缩到相同的屈曲程度，可以得到不同弹性模数(1-100kpa)的伸展支架。支架将根据有限元模型的预测，输入随温度变化的弹性模量，并根据视频数据进行制作。第二个目标是检验泊松比和弹性模数对IPSC谱系承诺的不同影响。未分化的IPSC或IPSC来源的神经前体细胞将被种植到不同的支架上，并被诱导为神经系或血管系。这些细胞将被表征为神经元标记或血管标记，期望模仿组织弹性的伸展支架将促进IPSCs的神经分化，并且弹性模数和泊松比对神经谱系承诺有不同的影响。第三个目的是确定泊松比和弹性模量对YAP定位和内源性ECM分泌的影响，内源性ECM调节规范的Wnt信号并有助于IPSCs的谱系承诺。将研究YAP定位并揭示YAP对WNT信号的影响，期望伸展支架诱导细胞质YAP定位，核YAP定位激活WNT信号，细胞将分泌不同类型的ECM以响应支架弹性并影响WNT信号，最后，YAP与WNT信号的相互作用有助于IPSC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优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.