![]() ![]() As methods to generate organoids representing other tissues typically use encapsulation of hPSCs in Matrigel 1, we here combine this strategy with directed cardiac differentiation by WNT pathway modulation. We describe a robust method to reproduce the first steps of human cardiogenesis in vitro. ![]() The outermost layer of the heart, the epicardium, is derived from a progenitor cell population called the ‘proepicardium’ and gives rise to cardiac fibroblasts and smooth muscle cells 14. Later, the heart tube undergoes looping, which positions the future heart chambers. Cells of the heart-forming regions extend across the midline and fuse into the heart tube, which consists of two layers, the myocardium and the endocardium. After precardiac mesoderm specification, progenies form the bilateral heart-forming regions on each side of the embryonic midline in close proximity and inductive exchange with the developing foregut endoderm 13. It originates from the splanchnic mesoderm, which emerges from the primitive streak during gastrulation. The heart is the first functional organ formed in the embryo. Our aim in the present study was to recapitulate early heart developmental patterns using hPSCs. However, these studies did not demonstrate the spatiotemporal patterning of early cardiogenesis, including the interplay with foregut endoderm. derived ‘precardiac organoids’ forming two heart fields, which showed high similarities to their in vivo counterparts at the corresponding developmental stage 12. generated ‘cardiac microchambers’ containing myofibroblasts at the perimeter and cardiomyocytes in the center by geometric confinement of hPSCs in micropatterns 11. Studies focusing on the morphological aspects of heart development are rare. These organoids reproduce some aspects of adult heart tissue, including stromal cells, an endothelial network and an epicardial layer, but do not reflect the processes of early heart development. 9 generated organoids by embedding hPSC-derived cardiomyocytes in a collagen I–Matrigel mixture. However, this work has focused on engineering pre-differentiated or primary cardiac cell types to mimic adult-like heart tissue 6, 7, 8, 9, 10. Recent studies of cardiac microtissues or organoids have improved on prior heart muscle engineering approaches 4, 5. Although organoid models have been described for a broad range of tissues 1, 2, 3, progress in the cardiovascular field has been limited. Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced PSCs (hiPSCs), have the capability to self-organize into three-dimensional (3D) structures called organoids resembling embryo-like tissue patterns in vitro. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. ![]() Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. ![]()
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