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. 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. 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. 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. 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.

Background— The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease– and human patient–specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line. Methods and Results— With the use of a standard embryoid body–based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell–derived cardiomyocytes show typical features of ES cell–derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), α-myosin heavy chain (α-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric α-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca ²⁺ fluctuations with amplitudes of Ca ²⁺ transients comparable to ES cell cardiomyocytes. Simultaneous Ca ²⁺ release within clusters of iPS cell–derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the β-adrenergic and muscarinic signaling cascade in these cells. Conclusions— iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.

Background—: Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. Methods and Results—: Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2–associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. Conclusions—: Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.