K4 - Asymmetrical Curvature Alters Cardiomyocyte Differentiation in Developing Cardiac Organoids

Introduction: : Current 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. We hypothesize that alterations in the initial pattern shape and curvature of pluripotent stem cells (hPSCs) will lead to changes in cardiac organoid organization.

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). 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 analyzed by calcium flux imaging, contractility analysis, immunofluorescence (Fig 1c.), flow cytometry, etc.

Results, Conclusions, and Discussions:: Results and

Discussion:
Microconfinement of stem cell colonies on compliant substrates facilitated cardiac organoid generation and release. Reducing circle pattern size led to an increase in the percentage of cardiomyocytes, but less CD140b+ cells and reduced organoid contractility and force production. Removal of the center of the initial colony, via ring patterns, lead to a reduction in cardiomyocyte percentage and contractility. Patterning shapes with variations in curvature (e.g., teardrop, heart, cat’s eye) lead to a high percentage of TNNT2+ cardiomyocytes and greater contractility. Edge regions of high curvature were found to induce upregulation of early mesodermal and cardiac markers (TBX6, T, HAND1), 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. Shapes with multiple neighborhoods (e.g. bowtie, figure 8, dumbbell) established multiple nodes of T+/FOXA2+ cells at D2 to lead to multiple lobe sites of contractile cardiomyocytes with unique spatial organization, achieving the first functional multi-lobular organoids, with pattern asymmetry and size 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 for organoid types of mesodermal and endodermal lineages, enabling the generation of a variety of reproducible tissue organoids for disease modeling and improved drug screening.

Acknowledgements and/or References (Optional):: Results and

Discussion:
Microconfinement of stem cell colonies on compliant substrates facilitated cardiac organoid generation and release. Reducing circle pattern size led to an increase in the percentage of cardiomyocytes, but less CD140b+ cells and reduced organoid contractility and force production (Fig. 1d). Removal of the center of the initial colony, via ring patterns, lead to a reduction in EMT cell localization (Fig. 1e), cardiomyocyte percentage, and contractility. Patterning shapes with variations in curvature (e.g., teardrop, heart, cat’s eye) lead to a high percentage of TNNT2+ cardiomyocytes and greater contractility. Edge regions of high curvature were found to induce upregulation of early mesodermal and cardiac markers (TBX6, T, HAND1), leading to a cascade of differentiation the creates outer shells of myocardium. Spatial marker distribution is driven by pattern area and local cell density, influencing the cardiac subtype compositions and spatial distributions in the final organoids. Shapes with multiple neighborhoods (e.g. bowtie, figure 8, dumbbell) established multiple nodes of T+/FOXA2+ cells at D2 to lead to multiple lobe sites of contractile cardiomyocytes with unique spatial organization (Fig. 1f), achieving the first functional multi-lobular organoids, with pattern asymmetry and size 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.