CAREER: Engineering Stem Cell-Based Cardiac Organoids

人类诱导多能干细胞（hipsc）是一种能够变成任何细胞类型的细胞，为研究人类特异性组织和器官的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，微/迷你组织（称为类器官）可以发展成具有目标器官的许多特征。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类器官技术面临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的空间组织模式，这些类器官通常具有随机分布的细胞。这个CAREER项目试图在几何约束下控制hiPSC的生长和分化，以产生具有空间不同组织结构的跳动心脏类器官。这种心脏类器官系统可以提供关于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。本项目旨在将教育使命与研究计划相结合，增加不同层次学生参与干细胞生物学与微系统工程交叉领域研究的机会。此外，该项目致力于纽约中部地区周边地区的社区推广，计划开发并捐赠一个名为“干细胞的超级力量”的展览给锡拉丘兹米尔顿·j·鲁宾斯坦科学技术博物馆（MOST）。研究者的长期研究目标是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，本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，为理解生物物理线索（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奠定基础。研究计划有三个目标。第一个目的是研究生物物理信号对心脏类器官结构形态和收缩功能调控的影响。为了制造类器官，hipsc将被植入聚乙二醇模式的基质上，扩展到接近个体模式的汇合处，然后分化成心脏谱系。为了研究物理约束如何影响心脏类器官的形成和功能，hiPSCs将被植入不同直径的圆形图案中以强调大小差异，以及与圆形具有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。类器官将根据其收缩功能（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）进行表征。这些特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-功能关系如何随着图案几何形状（大小和形状）的变化而改变。客观结果有望确定最佳物理约束，从而可能产生具有最高形态一致性的心脏类器官。第二个目的是研究生物物理线索对调节心脏类器官细胞组成的影响，特别是心肌细胞（CMs）、心脏成纤维细胞（CFs）、心内膜细胞（EDCs）、平滑肌细胞（SMCs）和心外膜细胞（EPCs）。心脏类器官将在不同的模式几何下分化，并使用不同的细胞因子（如VEGF、维甲酸（RA）和BMP4）进行额外的分化，以促进向多个心脏特异性谱系的共分化。对不同细胞类型的空间分布和百分位数的评估将有助于探索物理限制如何与生化因素相互作用，从而有效地共分化为具有受控多细胞组成的空间组织的心脏类器官。预期较小的几何图形将有利于基质细胞群的分化（如CFs、EDs、EPs），而较大的几何图形将有利于肌肉细胞群的分化（如CMs、SMCs）。第三个目的是研究心脏类器官空间组织与机械转导和心脏发育信号通路相关的分子机制。我们的研究旨在验证这样的假设：物理禁闭会增强RhoA/ROCK活性，从而调节YAP/TAZ的核质穿梭，而YAP/TAZ在模式周的细胞核保留会抑制中胚层诱导过程中的内源性WNT信号，最终导致hiPSC微图案集落的空间分化。综上所述，研究成果有望：1)建立具有力学几何输入的体外类心脏器官模型；2)提供生物物理线索对控制心脏细胞规格的影响的知识；3)提供与心脏组织形态发生相关的发育机制生物学的机制见解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.