CAREER: Engineering Stem Cell-Based Cardiac Organoids

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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.