Developmentally inspired 4D bioprinting of human heart tissue via shape-morphogenesis and in-situ lineage differentiation

Bioprinted heart tissues derived from human induced pluripotent stem cells (iPSCs) hold great potential as regenerative implants that can strengthen or replace the failing heart. However, current bioprinting approaches primarily aim to recreate the heart tissue’s end-stage anatomical form, often neglecting the dynamic morphogenetic processes that drive its natural development during embryogenesis. For instance, the heart initially forms as a linear tube that undergoes a series of complex shape transformations, such as looping and chamber formation, that are critical for defining tissue architecture and function. Despite this, existing bioprinting approaches typically utilise static bioinks with minimal capacity for morphogenetic shape changes. As a result, bioprinted heart tissues are structurally and functionally immature compared to their adult counterparts. This immaturity limits their effectiveness as therapeutic implants for heart repair. The overall objective of this thesis was therefore to bioprint human heart tissues that undergo developmentally inspired shape-morphing and to investigate how these morphogenetic behaviours influence cardiac differentiation and maturation compared to static controls. The central hypothesis was that integrating 4D shape-morphing into the bioprinting workflow would accelerate cell and tissue maturation trajectories. To achieve this, a 4D bioprinting platform was first developed using collagen and hyaluronic acid bioinks that underwent programmable shape-morphing in granular support hydrogels. Shape-morphing was driven by cell-generated contractile forces and was tunable via parameters such as cell density, cell type, bioink composition, and support hydrogel viscoelasticity. The geometry of the printed constructs also influenced the extent and nature of morphogenesis, with shape changes arising from mechanical instabilities under endogenous stress. Notably, shape-morphing was found to sculpt cell and extracellular matrix(ECM) alignment along the principal tissue axis through a stress-avoidance mechanism. Next, it was explored how 4D shape-morphing could impact the structural and functional maturity of human heart tissues derived from iPSCs. Bioprinted heart tissues containing a co-culture of iPSC-cardiomyocytes (iPSC-CMs) and cardiac fibroblasts (7:3 ratio) exhibited enhanced shape-morphing compared to controls composed solely of iPSC-CMs. Notably, fibroblast-mediated shape-morphing enhanced the structural organisation and alignment of iPSC-CMs within the bioprinted heart tissues, resulting in improved contractile properties compared to static controls. Transcriptomic analysis also revealed upregulation of cardiac differentiation markers, including genes encoding sodium ion channels (SCN5A), gap junctions (GJA1, GJA5) and cardiac muscle development (GATA4), confirming enhanced functional maturation. Building on these results, a developmentally inspired in-situ differentiation strategy was then implemented where bioprinted iPSCs were differentiated into cardiomyocytes within shape-morphing tissue constructs. This approach enabled simultaneous shape-morphing and lineage specification, as occurs during embryonic heart development. Bioprinted constructs composed of undifferentiated iPSCs were found to maintain pluripotency during early morphogenesis and could then be successfully directed toward mesodermal and cardiac fates using temporal WNT pathway modulation. The resulting constructs exhibited co-emergence of cardiomyocytes and fibroblast-like cells from a common progenitor population, closely mimicking native developmental processes, and gene expression analysis confirmed upregulation of early cardiac markers, including TNNI1, NKX2.5, MYH7, and POSTN. Notably, in-situ differentiated constructs exhibited greater structural organisation and cell-cell connectivity compared to those printed with pre-differentiated iPSC-CMs. In conclusion, this thesis presents a novel strategy for enhancing the structural and functional properties of bioprinted heart tissues by integrating cell-mediated shape-morphing and in-situ differentiation. This developmentally 4D bioprinting framework offers a powerful platform for engineering organ rudiments that undergo programmable morphogenesis to sculpt their final shape, composition, and function.

Funder

Irish Research Council
European Research Council

Publisher

University of Galway

Rights

CC BY-NC-ND

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Collections

University of Galway Theses (PhD Theses)