Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows the elucidation of cardiomyocyte macromolecular and metabolic requirements whilst providing a minimally complex system for the study of maturation and subtype specification.

Summary Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca2+) imaging indicated dysregulation of Ca2+ cycling and elevation in intracellular Ca2+ ([Ca2+]i) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca2+ homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

Doxorubicin (Adriamycin) is an anthracycline chemotherapy agent effective in treating a wide range of malignancies with a well–established dose–response cardiotoxic side effect that can lead to heart failure. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient–specific human induced pluripotent stem cell–derived cardiomyocytes (hiPSC–CMs) can recapitulate individual patients’ predilection to DIC at the single cell level. hiPSC–CMs derived from breast cancer patients who suffered clinical DIC are consistently more sensitive to doxorubicin toxicity, demonstrating decreased cell viability, mitochondrial and metabolic function, calcium handling, and antioxidant pathway activity, along with increased reactive oxygen species (ROS) production compared to hiPSC–CMs from patients who did not experience DIC. Together, our data indicate that hiPSC–CMs are a suitable platform for identifying and verifying the genetic basis and molecular mechanisms of DIC.

Fibroblast growth factor 2 (FGF-2) is a mitogen that is exported from cells by an endoplasmic reticulum/Golgiindependent secretory pathway. Recent findings have shown that FGF-2 export occurs by direct translocation from the cytoplasm across the plasma membrane into the extracellular space. Here, we report that FGF-2 contains a binding site for phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], the principal phosphoinositide species associated with plasma membranes. Intriguingly, in the context of a lipid bilayer, the interaction between FGF-2 and PI(4,5)P 2 is shown to depend on a lipid background that resembles plasma membranes. We show that the interaction with PI(4,5)P 2 is critically important for FGF-2 secretion as experimental conditions reducing cellular levels of PI(4,5)P 2 resulted in a substantial drop in FGF-2 export efficiency. Likewise, we have identified FGF-2 variant forms deficient for binding to PI(4,5)P 2 that were found to be severely impaired with regard to export efficiency. These data show that a transient interaction with PI(4,5)P 2 associated with the inner leaflet of plasma membranes represents the initial step of the unconventional secretory pathway of FGF-2.

Rational Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocte (ESC-CM) transplantation, thereby potentially preventing dilative remodelling and progression to heart failure. Objective Assessment of transport stability, long term survival, structural organisation, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction (MI) model. Methods and Results We constructed EHMs from ESC-CMs and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). After ischemia/reperfusion (I/R) injury, EHMs were implanted onto immunocompromised rat hearts at 1 month to simulate chronic ischemia. Bioluminescence imaging (BLI) showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving up to 25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs −6.7±1.4% vs control −10.9±1.5%, n>12, P=0.05), we observed no difference between EHMs containing viable or non-viable human cardiomyocytes in this chronic xenotransplantation model (n>12, P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. Conclusions EHM transplantation led to high engraftment rates, long term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic MI model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

Background Brugada Syndrome is a disorder associated with characteristic ECG precordial ST elevation and predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to lack of adequate human cellular models of the disorder. Methods and Results We recruited two patients with Type 1 Brugada Syndrome (BrS) carrying two different SCN5A variants and two healthy controls. We generated induced pluripotent stem cells (iPSCs) from their skin fibroblasts by using integration-free Sendai virus. We utilized directed differentiation to create purified populations of iPSC-derived cardiomyocytes (iPSC-CMs). BrS iPSC-CMs showed reductions in inward Na+ current density and reduced maximal upstroke velocity of action potential compared to healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs showed increased burden of triggered activity, abnormal Ca2+ transients, and beating interval variation. Correction of the causative variant by genome editing was performed and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared to controls. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression. Conclusions Patient-specific iPSC-CMs are able to recapitulate single cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate cellular disease mechanism and identify novel therapeutic targets.

The exact nature of the immune response elicited by autologous induced pluripotent stem cell (iPSC) progeny is still not well understood. Here we show in murine models that autologous iPSC-derived endothelial cells (iECs) elicit an immune response that resembles the one against a comparable somatic cell, the aortic endothelial cell (AEC). These cells exhibit long-term survival in vivo and prompt a tolerogenic contexture of intra-graft characterized by elevated IL-10 expression. In contrast, undifferentiated iPSCs elicit a very different immune response with high lymphocytic infiltration and elevated IFN-γ, granzyme-B, and perforin intra-graft. Furthermore, the clonal structure of infiltrating T cells from iEC grafts is statistically indistinguishable from that of AECs, but is different from that of undifferentiated iPSC grafts. Taken together, our results indicate that the differentiation of iPSCs results in a loss of immunogenicity and leads to the induction of tolerance, despite expected antigen expression differences between iPSC-derived versus original somatic cells.

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and was associated with perturbed transforming growth factor beta (TGFβ) signaling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGFβ signaling. TBX20 regulates the expression of TGFβ signaling modifiers including a known genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGFβ signaling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.