CAREER: Engineering Stem Cell-Based Cardiac Organoids

人类诱导多能干细胞（hiPSC），具有成为任何细胞类型的能力的细胞，为研究人类特异性组织和器官发育提供了前所未有的机会。通过将hiPSC分化为器官特异性细胞类型，可以开发称为类器官的微/迷你组织，以具有感兴趣器官的许多特征。这些基于干细胞的类器官被假设以类似于胚胎中实际器官的方式发育。然而，当前3D类器官技术的挑战之一是缺乏空间物理控制来促进这些自组装类器官中的空间组织图案化，这些自组装类器官通常看起来具有随机分布的细胞。该CAREER项目旨在控制hiPSC在几何限制下的生长和分化，以产生具有空间上不同的组织结构的跳动的心脏类器官。这种心脏类器官系统可以提供有关物理线索如何调节干细胞类器官的结构，功能和细胞特性的见解。该项目旨在将教育使命与研究计划相结合，以增加不同层次的学生参与干细胞生物学和微系统工程界面跨学科研究的机会。此外，该项目致力于社区外展到中央纽约地区周围的地区，这是通过计划开发和捐赠展览，“干细胞的超级力量”，以锡拉丘兹米尔顿J.鲁宾斯坦科学技术博物馆&（MOST）。深入了解生物物理因素在人类心脏发育和疾病中的作用。为了实现这一目标，这个CAREER项目的目标是设计一个具有机械几何输入的心脏类器官系统，以建立理解生物物理线索，特别是物理限制，如何影响类器官形成，组织功能和空间细胞分化的基础。 研究计划有三个目标。 第一个目的是研究生物物理线索对调节心脏类器官的结构形态和收缩功能的影响。 为了产生类器官，hiPSC将被接种到PEG图案化的基质上，在各个图案上扩增至接近汇合，然后分化成心脏谱系。为了研究物理限制如何影响心脏类器官的形成和功能，将hiPSC接种到具有不同直径的圆形图案中以强调尺寸差异，以及具有与圆形相同几何面积的三角形、正方形和矩形图案中以强调形状差异。 将相对于它们的收缩功能（收缩运动、动作电位、钙转运和心率）和结构形态（健康、高度和宽度）来表征类器官。 这些表征将用于建立类器官结构与心脏功能之间的相关性，并研究结构-功能关系如何通过图案几何形状（大小和形状）的变化而改变。 预期客观结果能够确定可能产生具有最高形态一致性的心脏类器官的最佳物理限制。 第二个目的是研究生物物理线索对调节心脏类器官的细胞组成的影响，特别是心肌细胞（CM）、心脏成纤维细胞（CF）、内皮细胞（EDC）、平滑肌细胞（SMC）和心外膜细胞（EPC）。心脏类器官将在不同的模式几何结构下用具有不同细胞因子的额外分化方案（例如，VEGF、视黄酸（RA）和BMP 4）以促进共分化成多种心脏特异性谱系。 对不同细胞类型的空间分布和百分位数的评估将使得能够探索物理限制如何与生物化学因素相互作用，以有效地共分化成具有受控多细胞组成的空间组织的心脏类器官。预期小图案几何形状将有利于基质细胞群（例如CF、ED、EP）的分化，而较大图案几何形状将有利于肌细胞群（例如CM、SMC）的分化。第三个目的是研究心脏类器官空间组织的分子机制，并探讨机械力信号转导和心脏发育的信号通路。 设计研究以检验以下假设：物理限制将增强RhoA/ROCK活性，其调节雅普/TAZ的核质穿梭，并且雅普/TAZ在图案周边处的细胞中的核保留将抑制中胚层诱导期间的内源性WNT信号传导，这最终导致微图案化hiPSC集落上的空间分化。 综上所述，研究结果有望：1）建立具有力学-几何输入的体外心脏类器官模型; 2）提供关于生物物理线索对控制心脏细胞特化的影响的知识;和3）提供与心脏组织形态发生相关的发育机械生物学的机械见解。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的学术价值和更广泛的影响审查标准。

Human induced pluripotent stem cells (hiPSCs), cells that have the ability to become any cell type, have provided an unprecedented opportunity to study human-specific tissue and organ development. By differentiating hiPSCs into organ-specific cell types, micro/mini tissues, termed organoids, can be developed to have many of the characteristics of the organ of interest. These stem cell-based organoids have been hypothesized to develop in a manner similar to actual organs in an embryo. However, one of the challenges of current 3D organoid technology is lack of spatial physical controls to promote spatial tissue patterning in these self-assembled organoids, which often appear to have a random distribution of cells. This CAREER project seeks to control hiPSC growth and differentiation under geometrical confinement in order to generate beating cardiac organoids with spatially distinct tissue architecture. This cardiac organoid system could provide insights about how physical cues regulate the structural, functional and cellular properties of stem cell organoids. The project aims to synergize the educational mission with the research program to increase the opportunities for students at different levels to participate in interdisciplinary research at the interface of stem cell biology and microsystem engineering. Furthermore, the project is dedicated to community outreach to the area surrounding the Central New York Region, which is demonstrated by plans to develop and donate an exhibit, “The Superpower of Stem Cells,” to the Syracuse Milton J. Rubenstein Museum of Science & Technology (MOST).The investigator’s long-term research goal is to gain in-depth mechanistic understanding of the role of biophysical factors in human heart development and diseases. Towards this goal, the goal of this CAREER project is to engineer a cardiac organoid system with mechano-geometrical inputs to establish a basis for understanding how biophysical cues, particularly physical confinement, affect organoid formation, tissue functionality, and spatial cell differentiation. The Research Plan is organized under three objectives. The FIRST Objective is to investigate the influence of biophysical cues on regulating the structural morphology and contractile functions of cardiac organoids. To create the organoids, hiPSCs will be seeded onto PEG patterned substrata, expanded to near confluence on the individual patterns, and then differentiated into cardiac lineages. To investigate how physical confinement affects cardiac organoid formation and functions, hiPSCs will be seeded into circle-shaped patterns with different diameters to emphasize size differences, and triangle, square, and rectangular shaped patterns with the same geometrical areas as the circles to emphasize shape differences. The organoids will be characterized relative to their contractile function (contractile motion, action potential, calcium transport and heart rate) and structural morphology (wellness, height and width). These characterizations will be used to establish the correlation between organoid structure and cardiac functions, and to study how structure-function relationship will be shifted by the changes on pattern geometry (size and shape). Objective outcomes are expected to enable determination of the optimal physical confinement that might create cardiac organoids with the highest consistency in morphology. THE SECOND Objective is to investigate the influence of biophysical cues on regulating the cellular composition of cardiac organoids, particularly cardiomyocytes (CMs), cardiac fibroblasts (CFs), endocardial cells (EDCs), smooth muscle cells (SMCs), and epicardial cells (EPCs). Cardiac organoids will be differentiated under different pattern geometries with additional differentiation protocols with different cytokines (e.g., VEGF,, retinoic acid (RA) and BMP4)to promote co-differentiation into multiple cardiac-specific lineages. Assessment of the spatial distribution and percentile of the different cell types will enable exploration of how physical confinement interplays with biochemical factors for effective co-differentiation into spatial- organized cardiac organoids with controlled multicellular composition. Expectations are that small pattern geometry will favor the differentiation of stromal cell populations (e.g. CFs, EDs, EPs), while larger pattern geometry will favor the differentiation of muscle cell populations (e.g. CMs, SMCs). The THIRD Objective is to investigate the molecular mechanisms of spatial organization of cardiac organoid associated with the signaling pathways of mechanotransduction and cardiac development. Studies are designed to test the hypothesis that physical confinement will enhance RhoA/ROCK activity, which regulates the nucleocytoplamic shuttling of YAP/TAZ, and nuclear retention of YAP/TAZ in the cells at the pattern perimeter will inhibit endogenous WNT signaling during mesoderm induction, which eventually leads to the spatial differentiation on the micropatterned hiPSC colonies. In summary, research outcomes are expected to: 1) create of an in vitro cardiac organoid model with mechano-geometrical inputs; 2) provide knowledge on the effects of biophysical cues on controlling cardiac cell specification; and 3) provide mechanistic insights into developmental mechanobiology relevant to cardiac tissue morphogenesis.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.