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In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.

ABSTRACT: Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.

SUBMITTER: Lee J

PROVIDER: S-EPMC7471119 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The extracellular matrix (ECM) of the embryonic heart guides assembly and maturation of cardiac cell types and, thus, may serve as a useful template, or blueprint, for fabrication of scaffolds for cardiac tissue engineering. Surprisingly, characterization of the ECM with cardiac development is scattered and fails to comprehensively reflect the spatiotemporal dynamics making it difficult to apply to tissue engineering efforts. The objective of this work was to define a blueprint of the spatiotemporal organization, localization, and relative amount of the four essential ECM proteins, collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN) in the left ventricle of the murine heart at embryonic stages E12.5, E14.5, and E16.5 and 2 days postnatal (P2). Second harmonic generation (SHG) imaging identified fibrillar collagens at E14.5, with an increasing density over time. Subsequently, immunohistochemistry (IHC) was used to compare the spatial distribution, organization, and relative amounts of each ECM protein. COLIV was found throughout the developing heart, progressing in amount and organization from E12.5 to P2. The amount of COLI was greatest at E12.5 particularly within the epicardium. For all stages, FN was present in the epicardium, with highest levels at E12.5 and present in the myocardium and the endocardium at relatively constant levels at all time points. ELN remained relatively constant in appearance and amount throughout the developmental stages except for a transient increase at E16.5. Expression of ECM mRNA was determined using quantitative polymerase chain reaction and allowed for comparison of amounts of ECM molecules at each time point. Generally, COLI and COLIII mRNA expression levels were comparatively high, while COLIV, laminin, and FN were expressed at intermediate levels throughout the time period studied. Interestingly, levels of ELN mRNA were relatively low at early time points (E12.5), but increased significantly by P2. Thus, we identified changes in the spatial and temporal localization of the primary ECM of the developing ventricle. This characterization can serve as a blueprint for fabrication techniques, which we illustrate by using multiphoton excitation photochemistry to create a synthetic scaffold based on COLIV organization at P2. Similarly, fabricated scaffolds generated using ECM components, could be utilized for ventricular repair.

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In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.

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