Developmentally Inspired Bioprinting of Nascent Multicellular Human Heart Tissue Through in Situ Differentiation and Morphogenesis of iPSCs

ABSTRACT Current approaches to heart tissue bioprinting typically rely on using human induced pluripotent stem cell (iPSC)‐derived cardiomyocytes that are pre‐differentiated in 2D culture. This differs fundamentally from embryonic heart development, where mesodermal progenitors differentiate into cardiomyocytes within 3D, matrix‐rich, and shape‐morphing microenvironments. Here, we introduce a developmentally inspired approach that enables in situ mesodermal and cardiac differentiation of iPSCs within bioprinted, shape‐morphing pluripotent tissues. Using embedded bioprinting, Matrigel bioinks with high‐density iPSC suspensions were deposited into granular support hydrogels to generate pluripotent tissue constructs with defined architectures. These constructs exhibited shape‐morphing behavior, tunable by modulating the support bath viscoelasticity. Support bath mechanics also regulated iPSC fate, with softer formulations reducing spontaneous differentiation. Building on this, mesodermal and cardiac differentiation were directly driven within the morphing constructs via temporal WNT pathway modulation, resulting in multicellular cardiac tissues in which cardiomyocytes and fibroblasts co‐emerge from a common progenitor pool. These nascent heart tissues exhibited a developmental phenotype, with immunofluorescence and gene expression profiling revealing cardiac progenitors alongside maturing cardiomyocytes. Together, these findings highlight the potential for an alternative developmental biofabrication paradigm focused on printing pluripotent organ rudiments that recapitulate early aspects of embryonic development via programmed in situ lineage specification and shape‐morphing.