Poster T14 - Harnessing geometry to guide the spatial organization of developing cardiac organoids

Introduction: Current 2D in vitro monocultures and animal models fall short in accurately modeling genetic disorders, leading to inadequate therapeutic development. Recent advances have now centered on 3D organoid models due to their improved cellular heterogeneity. However cardiac organoids–in particular–still lack crucial structural characteristics, cell types, and cellular organization which limits their utility. While recent approaches focus on modifying morphogens, none have used mechanical cues and/or microcontact printing (to create asymmetric organoids) as guides for the differentiation process.

Materials and

Methods: We use a microcontact printing technique to pattern islands of vitronectin on polyacrylamide hydrogels to create symmetric, geometrically defined colonies of pluripotent stem cells (Fig. 1a). Precise modulation of acrylamide and bis-acrylamide monomer ratios and concentrations can create polyacrylamide hydrogels with highly reproducible stiffness of 10 kPa. We then seed 135,000 H9 hPSCs/cm2 onto the patterns (Fig 1b-c). An optimized cardiac differentiation protocol was created to lead to the generation of cardiac organoids after 7-9 days of culture. During and after differentiation, organoids are collected and used for downstream analyses, i.e. calcium flux imaging, contractility analysis, immunofluorescence (Fig 1d.), and flow cytometry.

Results, Conclusions, and Discussions: Microconfinement of stem cell colonies on compliant substrates facilitates cardiac organoid generation and release, and substrate stiffness and geometric asymmetry influencing the spatial organization of organoid structure. Edge regions of high curvature were found to induce upregulation of early mesodermal markers, leading to a cascade of differentiation the creates outer shells of myocardium. Spatial distribution of these markers is further driven by pattern area and local cellular density, influencing the cardiac subtype compositions and spatial distributions in the final organoids. Pattern size directs cardiac subtype specification ratios, with smaller patterns leading to more cardiomyocytes and less CD140b+ cells. Mirrored geometries, such as figure eights, for the first time achieve functional, multi-lobular organoids with pattern asymmetry directing lobe size, merging, and cellular organization.

Conclusions: This platform enables both the high throughput generation cardiac organoids as well as fundamental studies into the role of biophysical cues and mechanobiology on cardiac development. This work will also provide insight into specific molecular mechanisms that regulate spatial patterning of cardiac cell types and guide the architecture within the heart. Ultimately, there is scope for application towards other organoid types of mesodermal and endodermal linages, enabling the generation of a variety of reproducible tissue organoids for disease modeling and improved drug screening.

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