Joseph C. Wu, MD, PhD's Profile
Joseph  C. Wu, MD, PhD is part of Stanford Profiles, official site for faculty, postdocs, students and staff information (Expertise, Bio, Research, Publications, and more). The site facilitates research and collaboration in academic endeavors.
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This study will assess the safety and efficacy of intracoronary artery administered autologous bone marrow derived stem cells in subjects post ST segment elevation myocardial infarction (STEMI). This will be assessed by evaluating and comparing the autologous stem cell treatment group to the control group in terms of the occurrence of AE's, SAE's and Major Adverse Cardiac Events (MACE), by the change in myocardial perfusion (RTSS) measured quantitatively by gated single photon emission computed tomography myocardial perfusion imaging (gated SPECT MPI), and other secondary endpoints such as LVEF measured by cardiac MRI in addition to other endpoints.

Stanford is currently not accepting patients for this trial. For more information, please contact Tom Moss, MD, 818-903-4036.

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Abstract

The common aldehyde dehydrogenase 2 (ALDH2) alcohol flushing variant known as ALDH2*2 affects ∼8% of the world's population. Even in heterozygous carriers, this missense variant leads to a severe loss of ALDH2 enzymatic activity and has been linked to an increased risk of coronary artery disease (CAD). Endothelial cell (EC) dysfunction plays a determining role in all stages of CAD pathogenesis, including early-onset CAD. However, the contribution of ALDH2*2 to EC dysfunction and its relation to CAD are not fully understood. In a large genome-wide association study (GWAS) from Biobank Japan, ALDH2*2 was found to be one of the strongest single-nucleotide polymorphisms associated with CAD. Clinical assessment of endothelial function showed that human participants carrying ALDH2*2 exhibited impaired vasodilation after light alcohol drinking. Using human induced pluripotent stem cell-derived ECs (iPSC-ECs) and CRISPR-Cas9-corrected ALDH2*2 iPSC-ECs, we modeled ALDH2*2-induced EC dysfunction in vitro, demonstrating an increase in oxidative stress and inflammatory markers and a decrease in nitric oxide (NO) production and tube formation capacity, which was further exacerbated by ethanol exposure. We subsequently found that sodium-glucose cotransporter 2 inhibitors (SGLT2i) such as empagliflozin mitigated ALDH2*2-associated EC dysfunction. Studies in ALDH2*2 knock-in mice further demonstrated that empagliflozin attenuated ALDH2*2-mediated vascular dysfunction in vivo. Mechanistically, empagliflozin inhibited Na+/H+-exchanger 1 (NHE-1) and activated AKT kinase and endothelial NO synthase (eNOS) pathways to ameliorate ALDH2*2-induced EC dysfunction. Together, our results suggest that ALDH2*2 induces EC dysfunction and that SGLT2i may potentially be used as a preventative measure against CAD for ALDH2*2 carriers.

View details for DOI 10.1126/scitranslmed.abp9952

View details for PubMedID 36696485

Abstract

Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.

View details for DOI 10.1016/j.cell.2022.04.005

View details for PubMedID 35489334

Abstract

Human induced pluripotent stem cells (iPSCs) have emerged as an effective platform for regenerative therapy, disease modeling, and drug discovery. iPSCs allow for the production of limitless supply of patient-specific somatic cells that enable advancement in cardiovascular precision medicine. Over the past decade, researchers have developed protocols to differentiate iPSCs to multiple cardiovascular lineages, as well as to enhance the maturity and functionality of these cells. Despite significant advances, drug therapy and discovery for cardiovascular disease have lagged behind other fields such as oncology. We speculate that this paucity of drug discovery is due to a previous lack of efficient, reproducible, and translational model systems. Notably, existing drug discovery and testing platforms rely on animal studies and clinical trials, but investigations in animal models have inherent limitations due to interspecies differences. Moreover, clinical trials are inherently flawed by assuming that all individuals with a disease will respond identically to a therapy, ignoring the genetic and epigenomic variations that define our individuality. With ever-improving differentiation and phenotyping methods, patient-specific iPSC-derived cardiovascular cells allow unprecedented opportunities to discover new drug targets and screen compounds for cardiovascular disease. Imbued with the genetic information of an individual, iPSCs will vastly improve our ability to test drugs efficiently, as well as tailor and titrate drug therapy for each patient.

View details for DOI 10.1124/pr.116.013003

View details for PubMedID 31871214

Abstract

The diversity of cardiac lineages contributes to the heterogeneity of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). Here, we report the generation of a hiPSC TBX5Clover2 and NKX2-5TagRFP double reporter to delineate cardiaclineages and isolate lineage-specific subpopulations. Molecular analyses reveal that four different subpopulations can be isolated based on the differential expression of TBX5 and NKX2-5, TBX5+NKX2-5+, TBX5+NKX2-5-, TBX5-NKX2-5+, and TBX5-NKX2-5-, mimicking the first heart field, epicardial, second heart field, and endothelial lineages, respectively. Genetic and functional characterization indicates that each subpopulation differentiates into specific cardiac cells. We further identify CORIN as a cell-surface marker for isolating the TBX5+NKX2-5+ subpopulation and demonstrate the use of lineage-specific CMs for precise drug testing. We anticipate that this tool will facilitate theinvestigation of cardiac lineage specification and isolation of specific cardiac subpopulations for drug screening, tissue engineering, and disease modeling.

View details for PubMedID 30880024

Abstract

Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-β (PDGFRB) as a potential therapeutic target.

View details for DOI 10.1038/s41586-019-1406-x

View details for PubMedID 31316208

Abstract

Cancer cells and embryonic tissues share a number of cellular and molecular properties, suggesting that induced pluripotent stem cells (iPSCs) may be harnessed to elicit anti-tumor responses in cancer vaccines. RNA sequencing revealed that human and murine iPSCs express tumor-associated antigens, and we show here a proof of principle for using irradiated iPSCs in autologous anti-tumor vaccines. In a prophylactic setting, iPSC vaccines prevent tumor growth in syngeneic murine breast cancer, mesothelioma, and melanoma models. As an adjuvant, the iPSC vaccine inhibited melanoma recurrence at the resection site and reduced metastatic tumor load, which was associated with fewer Th17 cells and increased CD11b+GR1himyeloid cells. Adoptive transfer of T cells isolated from vaccine-treated tumor-bearing mice inhibited tumor growth in unvaccinated recipients, indicating that the iPSC vaccine promotes an antigen-specific anti-tumor T cell response. Our data suggest an easy, generalizable strategy for multiple types of cancer that could prove highly valuable in clinical immunotherapy.

View details for PubMedID 29456158

Abstract

Cardiac development requires coordinated and large-scale rearrangements of the epigenome. The roles and precise mechanisms through which specific epigenetic modifying enzymes control cardiac lineage specification, however, remain unclear. Here we show that the H3K4 methyltransferase SETD7 controls cardiac differentiation by reading H3K36 marks independently of its enzymatic activity. Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that SETD7 targets distinct sets of genes to drive their stage-specific expression during cardiomyocyte differentiation. SETD7 associates with different co-factors at these stages, including SWI/SNF chromatin-remodeling factors during mesodermal formation and the transcription factor NKX2.5 in cardiac progenitors to drive their differentiation. Further analyses revealed that SETD7 binds methylated H3K36 in the bodies of its target genes to facilitate RNA polymerase II (Pol II)-dependent transcription. Moreover, abnormal SETD7 expression impairs functional attributes of terminally differentiated cardiomyocytes. Together, these results reveal how SETD7 acts at sequential steps in cardiac lineage commitment, and they provide insights into crosstalk between dynamic epigenetic marks and chromatin-modifying enzymes.

View details for PubMedID 29499155

Abstract

Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.

View details for DOI 10.1126/scitranslmed.aaf2584

View details for PubMedID 28202772

Abstract

Patient-specific pluripotent stem cells (PSCs) can be generated via nuclear reprogramming by transcription factors (i.e., induced pluripotent stem cells, iPSCs) or by somatic cell nuclear transfer (SCNT). However, abnormalities and preclinical application of differentiated cells generated by different reprogramming mechanisms have yet to be evaluated. Here we investigated the molecular and functional features, and drug response of cardiomyocytes (PSC-CMs) and endothelial cells (PSC-ECs) derived from genetically relevant sets of human iPSCs, SCNT-derived embryonic stem cells (nt-ESCs), as well as in vitro fertilization embryo-derived ESCs (IVF-ESCs). We found that differentiated cells derived from isogenic iPSCs and nt-ESCs showed comparable lineage gene expression, cellular heterogeneity, physiological properties, and metabolic functions. Genome-wide transcriptome and DNA methylome analysis indicated that iPSC derivatives (iPSC-CMs and iPSC-ECs) were more similar to isogenic nt-ESC counterparts than those derived from IVF-ESCs. Although iPSCs and nt-ESCs shared the same nuclear DNA and yet carried different sources of mitochondrial DNA, CMs derived from iPSC and nt-ESCs could both recapitulate doxorubicin-induced cardiotoxicity and exhibited insignificant differences on reactive oxygen species generation in response to stress condition. We conclude that molecular and functional characteristics of differentiated cells from human PSCs are primarily attributed to the genetic compositions rather than the reprogramming mechanisms (SCNT vs. iPSCs). Therefore, human iPSCs can replace nt-ESCs as alternatives for generating patient-specific differentiated cells for disease modeling and preclinical drug testing.

View details for PubMedID 29203658

Abstract

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 this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling 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.

View details for DOI 10.1038/ncb3411

View details for PubMedID 27642787

Abstract

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

View details for DOI 10.1016/j.cell.2016.04.002

View details for PubMedID 27114035

Abstract

Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but it causes a dose-related cardiotoxicity that can lead to heart failure in a subset of patients. 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 the predilection to DIC of individual patients at the cellular level. hiPSC-CMs derived from individuals with breast cancer who experienced DIC were consistently more sensitive to doxorubicin toxicity than hiPSC-CMs from patients who did not experience DIC, with decreased cell viability, impaired mitochondrial and metabolic function, impaired calcium handling, decreased antioxidant pathway activity, and increased reactive oxygen species production. Taken together, our data indicate that hiPSC-CMs are a suitable platform to identify and characterize the genetic basis and molecular mechanisms of DIC.

View details for DOI 10.1038/nm.4087

View details for PubMedID 27089514

Abstract

Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.

View details for DOI 10.1126/scitranslmed.3009027

View details for Web of Science ID 000343316800004

Abstract

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require 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 an optimized cardiac differentiation strategy, using 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 induction of differentiation, 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 chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.

View details for DOI 10.1038/nmeth.2999

View details for PubMedID 24930130

Abstract

Background Induced pluripotent stem cells and their differentiated cardiomyocytes (iCMs) have tremendous potential as patient-specific therapy for ischemic cardiomyopathy following myocardial infarctions, but difficulties in viable transplantation limit clinical translation. Exosomes secreted from iCMs (iCM-Ex) can be robustly collected in vitro and injected in lieu of live iCMs as a cell-free therapy for myocardial infarction. Methods and Results iCM-Ex were precipitated from iCM supernatant and characterized by protein marker expression, nanoparticle tracking analysis, and functionalized nanogold transmission electron microscopy. iCM-Ex were then used in in vitro and in vivo models of ischemic injuries. Cardiac function in vivo was evaluated by left ventricular ejection fraction and myocardial viability measurements by magnetic resonance imaging. Cardioprotective mechanisms were studied by JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) assay, immunohistochemistry, quantitative real-time polymerase chain reaction, transmission electron microscopy, and immunoblotting. iCM-Ex measured 140nm and expressed CD63 and CD9. iCM and iCM-Ex microRNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. Mice treated with iCM-Ex demonstrated significant cardiac improvement post-myocardial infarction, with significantly reduced apoptosis and fibrosis. In vitro iCM apoptosis was significantly reduced by hypoxia and exosome biogenesis inhibition and restored by treatment with iCM-Ex or rapamycin. Autophagosome production and autophagy flux was upregulated in iCM-Ex groups in vivo and in vitro. Conclusions iCM-Ex improve post-myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyoytes, enabling a cell-free, patient-specific therapy for ischemic cardiomyopathy.

View details for DOI 10.1161/JAHA.119.014345

View details for PubMedID 32131688

Abstract

BACKGROUND: Although cardiomyopathy has emerged as a leading cause of death in Duchenne muscular dystrophy (DMD), limited studies and therapies have emerged for dystrophic heart failure.OBJECTIVES: The purpose of this study was to model DMD cardiomyopathy using DMD patient-specific human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and to identify physiological changes and future drug therapies.METHODS: To explore and define therapies for DMD cardiomyopathy, the authors used DMD patient-specific hiPSC-derived cardiomyocytes to examine the physiological response to adrenergic agonists and beta-blocker treatment. The authors further examined these agents invivo using wild-type and mdx mouse models.RESULTS: At baseline and following adrenergic stimulation, DMD hiPSC-derived cardiomyocytes had a significant increase in arrhythmic calcium traces compared to isogenic controls. Furthermore, these arrhythmias were significantly decreased with propranolol treatment. Using telemetry monitoring, the authors observed that mdx mice, which lack dystrophin, had an arrhythmic death when stimulated with isoproterenol; the lethal arrhythmias were rescued, in part, by propranolol pre-treatment. Using single-cell and bulk RNA sequencing (RNA-seq), the authors compared DMD and control hiPSC-derived cardiomyocytes, mdx mice, and control mice (in the presence or absence of propranolol and isoproterenol) and defined pathways that were perturbed under baseline conditions and pathways that were normalized after propranolol treatment in the mdx model. The authors also undertook transcriptome analysis of human DMD left ventricle samples and found that DMD hiPSC-derived cardiomyocytes have dysregulated pathways similar to the human DMD heart. The authors further determined that relatively few patients with DMD see a cardiovascular specialist or receive beta-blocker therapy.CONCLUSIONS: The results highlight mechanisms and therapeutic interventions from human to animal and back to human in the dystrophic heart. These results may serve as a prelude for an adequately powered clinical study that examines the impact of beta-blocker therapy in patients with dystrophinopathies.

View details for DOI 10.1016/j.jacc.2019.12.066

View details for PubMedID 32164890

Abstract

Coronary artery disease (CAD) is the most common health problem worldwide and remains the leading cause of morbidity and mortality. Over the past decade, it has become clear that the inhabitants of our gut, the gut microbiota, play a vital role in human metabolism, immunity, and reactions to diseases, including CAD. Although correlations have been shown between CAD and the gut microbiota, demonstration of potential causal relationships is much more complex and challenging. In this review, we will discuss the potential direct and indirect causal roots between gut microbiota and CAD development via microbial metabolites and interaction with the immune system. Uncovering the causal relationship of gut microbiota and CAD development can lead to novel microbiome-based preventative and therapeutic interventions. However, an interdisciplinary approach is required to shed light on gut bacterial-mediated mechanisms (e.g., using advanced nanomedicine technologies and incorporation of demographic factors such as age, sex, and ethnicity) to enable efficacious and high-precision preventative and therapeutic strategies for CAD.

View details for DOI 10.1186/s40168-020-00821-0

View details for PubMedID 32169105

Abstract

Microfluidic technologies offer new platforms for biosensing in various clinical and point-of-care (POC) applications. Currently, at the clinical settings, the gold standard diagnostic platforms for multiplexed sensing are multi-step, time consuming, requiring expensive and bulky instruments with a constant need of electricity which makes them unsuitable for resource-limited or POC settings. These technologies are often limited by logistics, costly assays and regular maintenance. Although there have been several attempts to miniaturize these diagnostic platforms, they stand short of batch fabrication and they are dependent on complementary components such as syringe pumps. Here, we demonstrated the development and clinical testing of a disposable, multiplexed sensing device (ToMMx), which is a portable, high-throughput and user-friendly microfluidic platform. It was built with inexpensive plastic materials and operated manually without requiring electrical power and extensive training. We validated this platform in a small cohort of 50 clinical samples from patients with cardiovascular diseases and healthy controls. The platform is rapid and gives quantifiable results with high sensitivity, as low as 5.29pg/mL, from only a small sample volume (4muL). ToMMx platform was compared side-by-side with commercial ELISA kits where the total assay time is reduced 15-fold, from 5h to 20min. This technology platform is broadly applicable to various diseases with well-known biomarkers in diagnostics and monitoring, especially with potential future impact at the POC settings.

View details for DOI 10.1016/j.bios.2019.111930

View details for PubMedID 31929083

Abstract

Bioluminescence imaging enables the real-time detection and tracking of engrafted cells in vivo noninvasively and dynamically. By detecting and quantifying the photons released from the oxidation of luciferin catalyzed by luciferase enzymes, this approach has proven effective in tracking engrafted stem cell survival and retention, making it a powerful tool to monitor cell fate after transplantation without animal sacrifice. Here we describe a protocol that allows luciferase-labeled stem cells to be imaged and tracked in vivo by bioluminescent imaging via an IVIS spectrum imaging system.

View details for DOI 10.1007/978-1-0716-0364-2_1

View details for PubMedID 32112374

Abstract

The disease burden associated with air pollution continues to grow. The World Health Organization (WHO) estimates ≈7 million people worldwide die yearly from exposure to polluted air, half of which-3.3 million-are attributable to cardiovascular disease (CVD), greater than from major modifiable CVD risks including smoking, hypertension, hyperlipidemia, and diabetes mellitus. This serious and growing health threat is attributed to increasing urbanization of the world's populations with consequent exposure to polluted air. Especially vulnerable are the elderly, patients with pre-existing CVD, and children. The cumulative lifetime burden in children is particularly of concern because their rapidly developing cardiopulmonary systems are more susceptible to damage and they spend more time outdoors and therefore inhale more pollutants. World Health Organization estimates that 93% of the world's children aged <15 years-1.8 billion children-breathe air that puts their health and development at risk. Here, we present growing scientific evidence, including from our own group, that chronic exposure to air pollution early in life is directly linked to development of major CVD risks, including obesity, hypertension, and metabolic disorders. In this review, we surveyed the literature for current knowledge of how pollution exposure early in life adversely impacts cardiovascular phenotypes, and lay the foundation for early intervention and other strategies that can help prevent this damage. We also discuss the need for better guidelines and additional research to validate exposure metrics and interventions that will ultimately help healthcare providers reduce the growing burden of CVD from pollution.

View details for DOI 10.1161/JAHA.119.014944

View details for PubMedID 32174249

Abstract

During the past decade, developments in genome editing technology have fundamentally transformed biomedical research. In particular, the CRISPR/Cas9 system has been extensively applied because of its simplicity and ability to alter genomic sequences within living organisms, and an ever increasing number of CRISPR/Cas9-based molecular tools are being developed for a wide variety of applications. While genome editing tools have been used for many aspects of biological research, they also have enormous potential to be used for genome editing therapy to treat a broad range of diseases. For some hematopoietic diseases, clinical trials of therapeutic genome editing with CRISPR/Cas9 are already starting phase I. In the cardiovascular field, genome editing tools have been utilized to understand the mechanisms of diseases such as cardiomyopathy, arrythmia, and lipid metabolism, which now open the door to therapeutic genome editing. Currently, therapeutic genome editing in the cardiovascular field is centered on liver-targeting strategies to reduce cardiovascular risks. Targeting the heart is more challenging. In this review, we discuss the potential applications, recent advances, and current limitations of therapeutic genome editing in the cardiovascular field.

View details for DOI 10.1016/j.addr.2020.02.003

View details for PubMedID 32092381

Abstract

Pulmonary artery smooth muscle cells (PASMCs) and pericytes are NG2+ mural cells that provide structural support to pulmonary arteries and capillaries. In pulmonary arterial hypertension (PAH), both mural cell types contribute to PA muscularization but whether similar mechanisms are responsible for their behavior is unknown.RNA-Seq was used to compare the gene profile of pericytes and PASMCs from PAH and healthy lungs. NG2-Cre-ER mice were used to generate NG2-selective reporter mice (NG2tdT) for cell lineage identification and tamoxifen-inducible mice for NG2-selective SDF1 knockout (SDF1NG2-KO).Hierarchical clustering of RNA-seq data demonstrated that the genetic profile of PAH pericytes and PASMCs is highly similar. Cellular lineage staining studies on NG2tdT mice in chronic hypoxia showed that similar to PAH, tdT+ cells accumulate in muscularized microvessels and demonstrate significant upregulation of SDF1, a chemokine involved in chemotaxis and angiogenesis. Compared to controls, SDF1NG2-KO mice in chronic hypoxia had reduced muscularization and lower abundance of NG2+ cells around microvessels. SDF1 stimulation in healthy pericytes induced greater contractility and impaired their capacity to establish endothelial-pericyte communications. In contrast, SDF1 knockdown reduced PAH pericyte contractility and improved their capacity to associate with vascular tubes in co-culture.SDF1 is upregulated in NG2+ mural cells and is associated with PA muscularization. Targeting SDF1 could help prevent and/or reverse muscularization in PAH.

View details for DOI 10.1165/rcmb.2019-0401OC

View details for PubMedID 32084325

Abstract

MicroRNAs are small, noncoding RNAs that play a key role in gene expression. Accumulating evidence suggests that aberrant microRNA expression contributes to the heart failure (HF) phenotype; however, the underlying molecular mechanisms are not well understood. A better understanding of the mechanisms of action of microRNAs could potentially lead to targeted therapies that could halt the progression or even reverse HF.We found that microRNA-152 (miR-152) expression was upregulated in the failing human heart and experimental animal models of HF. Transgenic mice with cardiomyocyte-specific miR-152 overexpression developed systolic dysfunction (mean difference, -38.74% [95% CI, -45.73% to -31.74%]; P<0.001) and dilated cardiomyopathy. At the cellular level, miR-152 overexpression perturbed mitochondrial ultrastructure and dysregulated key genes involved in cardiomyocyte metabolism and inflammation. Mechanistically, we identified Glrx5 (glutaredoxin 5), a critical regulator of mitochondrial iron homeostasis and iron-sulfur cluster synthesis, as a direct miR-152 target. Finally, a proof-of-concept of the therapeutic efficacy of targeting miR-152 in vivo was obtained by utilizing a locked nucleic acid-based inhibitor of miR-152 (LNA 152) in a murine model of HF subjected to transverse aortic constriction. We demonstrated that animals treated with LNA-152 (n=10) showed preservation of systolic function when compared with locked nucleic acid-control treated animals (n=9; mean difference, 18.25% [95% CI, 25.10% to 11.39%]; P<0.001).The upregulation of miR-152 expression in the failing myocardium contributes to HF pathophysiology. Preclinical evidence suggests that miR-152 inhibition preserves cardiac function in a model of pressure overload-induced HF. These findings offer new insights into the pathophysiology of HF and point to miR-152-Glrx5 axis as a potential novel therapeutic target.

View details for DOI 10.1161/CIRCHEARTFAILURE.119.006298

View details for PubMedID 32160771

Abstract

With extended stays aboard the International Space Station (ISS) becoming commonplace, there is a need to better understand the effects of microgravity on cardiac function. We utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of microgravity on cell-level cardiac function and gene expression. The hiPSC-CMs were cultured aboard the ISS for 5.5weeks and their gene expression, structure, and functions were compared with ground control hiPSC-CMs. Exposure to microgravity on the ISS caused alterations in hiPSC-CM calcium handling. RNA-sequencing analysis demonstrated that 2,635 genes were differentially expressed among flight, post-flight, and ground control samples, including genes involved in mitochondrial metabolism. This study represents the first use of hiPSC technology to model the effects of spaceflight on human cardiomyocyte structure and function.

View details for DOI 10.1016/j.stemcr.2019.10.006

View details for PubMedID 31708475

Abstract

OBJECTIVES: There is a significant unmet need for a blood test with adequate sensitivity to detect colorectal cancer (CRC) and adenomas. We describe a novel circulating tumor cell (CTC) platform to capture colorectal epithelial cells associated with CRC and adenomas.METHODS: Blood was collected from 667 Taiwanese adults from 2012 to 2018 before a colonoscopy. The study population included healthy control subjects, patients with adenomas, and those with stage I-IV CRC. CTCs were isolated from the blood using the CellMax platform. The isolated cells were enumerated, and an algorithm was used to determine the likelihood of detecting adenoma or CRC. Nominal and ordinal logistic regression demonstrated that CTC counts could identify adenomas and CRC, including CRC stage.RESULTS: The CellMax test demonstrated a significant association between CTC counts and worsening disease status (Cuzick's P value < 0.0001) with respect to the adenoma-carcinoma sequence. The test showed high specificity (86%) and sensitivity across all CRC stages (95%) and adenomatous lesions (79%). The area under the curve was 0.940 and 0.868 for the detection of CRC and adenomas, respectively.DISCUSSION: The blood-based CTC platform demonstrated high sensitivity in detecting adenomas and CRC, as well as reasonable specificity in an enriched symptomatic patient population.TRANSLATIONAL IMPACT: If these results are reproduced in an average risk population, this test has the potential to prevent CRC by improving patient compliance and detecting precancerous adenomas, eventually reducing CRC mortality.

View details for DOI 10.14309/ctg.0000000000000088

View details for PubMedID 31663904

Abstract

Traditional African medicine is a source of new molecules that might be useful in modern therapeutics. We tested ten limonoids, six quinones, one xanthone, one alkaloid, and one cycloartane, isolated from four Cameroonian medicinal plants, and one plant-associated endophytic fungus, against Trypanosoma cruzi, the etiological agent of Chagas disease (CD). Vero cells, or human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes (hiPSC-CM) were infected with T. cruzi trypomastigotes (discrete typing unit types I or II). Infection took place in the presence of drugs, or 24 hours before drug treatment. Forty-eight hours after infection, infection rates and parasite multiplication were evaluated by Giemsa stain. Cell metabolism was measured to determine functional integrity. In Vero cells, several individual molecules significantly affected T. cruzi infection and multiplication with no, or minor, effects on cell viability. Reduced infection rates and multiplication by the quinone vismione B was superior to the commonly used therapeutic benznidazole (BNZ). The vismione B concentration inhibiting 50% of T. cruzi infection (IC50) was 1.3 M. When drug was applied after infection, anti-Trypanosoma effects of vismione B [10 M) were significantly stronger than effects of BNZ (23 M). Furthermore, in hiPSC-CM cultures, infection and multiplication rates in the presence of vismione B (10 M) were significantly lower than in BNZ (11.5 M), without showing signs of cytotoxicity. Our data indicate that vismione B is more potent against T. cruzi infection and multiplication than BNZ, with stronger effects on established infection. Vismione B, therefore, might become a promising lead molecule for treatment development for CD.

View details for DOI 10.4269/ajtmh.19-0350

View details for PubMedID 31571568

Abstract

In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human arteries and found that these cells transform into unique fibroblast-like cells, termed 'fibromyocytes', rather than into a classical macrophage phenotype. SMC-specific knockout of TCF21-a causal CAD gene-markedly inhibited SMC phenotypic modulation in mice, leading to the presence of fewer fibromyocytes within lesions as well as within the protective fibrous cap of the lesions. Moreover, TCF21 expression was strongly associated with SMC phenotypic modulation in diseased human coronary arteries, and higher levels of TCF21 expression were associated with decreased CAD risk in human CAD-relevant tissues. These results establish a protective role for both TCF21 and SMC phenotypic modulation in this disease.

View details for DOI 10.1038/s41591-019-0512-5

View details for PubMedID 31359001

Abstract

Over a century ago, it was reported that immunization with embryonic/fetal tissue could lead to the rejection of transplanted tumors in animals. Subsequent studies demonstrated that vaccination of embryonic materials in animals induced cellular and humoral immunity against transplantable tumors and carcinogen-induced tumors. Therefore, it has been hypothesized that the shared antigens between tumors and embryonic/fetal tissues (oncofetal antigens) are the key to anti-tumor immune responses in these studies. However, early oncofetal antigen-based cancer vaccines usually utilize xenogeneic or allogeneic embryonic stem cells or tissues, making it difficult to tease apart the anti-tumor immunity elicited by the oncofetal antigens vs. graft-vs.-host responses. Recently, one oncofetal antigen-based cancer vaccine using autologous induced pluripotent stem cells (iPSCs) demonstrated marked prophylactic and therapeutic potential, suggesting critical roles of oncofetal antigens in inducing anti-tumor immunity. In this review, we present an overview of recent studies in the field of oncofetal antigen-based cancer vaccines, including single peptide-based cancer vaccines, embryonic stem cell (ESC)- and iPSC-based whole-cell vaccines, and provide insights on future directions.

View details for DOI 10.3389/fimmu.2019.01510

View details for PubMedID 31338094

View details for PubMedCentralID PMC6628907

Abstract

AIMS: The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation.METHODS AND RESULTS: Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude.CONCLUSION: This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

View details for DOI 10.1093/eurheartj/ehz308

View details for PubMedID 31114854

Abstract

Long-QT syndrome, a frequently fatal inherited arrhythmia syndrome caused by genetic variants (congenital) or drugs (acquired), affects 1 in 2000 people worldwide. Its sentinel event is often sudden cardiac death, which makes preclinical diagnosis by genetic testing potentially life-saving. Unfortunately, clinical experience with genetic testing has shown that it is difficult to correctly identify genetic variants as disease causing. These current deficiencies in accurately assigning pathogenicity led to the discovery of increasing numbers of rare variants classified as variant of uncertain significance. To overcome these challenges, new technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) genome editing can be combined with human induced pluripotent stem cell-derived cardiomyocytes to provide a new approach to decipher pathogenicity of variants of uncertain significance and to better predict arrhythmia risk. To that end, the overarching goal of our network is to establish the utility of induced pluripotent stem cell-based platforms to solve major clinical problems associated with long-QT syndrome by determining how to (1) differentiate pathogenic mutations from background genetic noise, (2) assess existing and novel variants associated with congenital and acquired long-QT syndrome, and (3) provide genotype- and phenotype- guided risk stratification and pharmacological management of long-QT syndrome. To achieve these goals and to further advance the use of induced pluripotent stem cells in disease modeling and drug discovery, our team of investigators for this Leducq Foundation Transatlantic Networks of Excellence proposal will work together to (1) improve differentiation efficiency, cellular maturation, and lineage specificity, (2) develop new assays for high throughput cellular phenotyping, and (3) train young investigators to clinically implement patient-specific genetic modeling.

View details for DOI 10.1161/CIRCRESAHA.119.315209

View details for PubMedID 31465267

Abstract

Dilated cardiomyopathy with ataxia syndrome (DCMA) is an understudied autosomal recessive disease caused by loss-of-function mutations in the poorly characterized gene DNAJC19. Clinically, DCMA is commonly associated with heart failure and early death in affected children through an unknown mechanism. DCMA has been linked to Barth syndrome, a rare but well-studied disorder caused by deficient maturation of cardiolipin (CL), a key mitochondrial membrane phospholipid.Peripheral blood mononuclear cells from 2 children with DCMA and severe cardiac dysfunction were reprogrammed into induced pluripotent stem cells (iPSCs). Patient and control iPSCs were differentiated into beating cardiomyocytes (iPSC-CMs) using a metabolic selection strategy. Mitochondrial structure and CL content before and after incubation with the mitochondrially targeted peptide SS-31 were quantified.Patient iPSCs carry the causative DNAJC19 mutation (rs137854888) found in the Hutterite population, and the iPSC-CMs demonstrated highly fragmented and abnormally shaped mitochondria associated with an imbalanced isoform ratio of the mitochondrial protein OPA1, an important regulator of mitochondrial fusion. These abnormalities were reversible by incubation with SS-31 for 24 hours. Differentiation of iPSCs into iPSC-CMs increased the number of CL species observed, but consistent, significant differences in CL content were not seen between patients and control.We describe a unique and novel cellular model that provides insight into the mitochondrial abnormalities present in DCMA and identifies SS-31 as a potential therapeutic for this devastating disease.

View details for DOI 10.1016/j.cjca.2019.09.021

View details for PubMedID 32046906

Abstract

The protein-level translational status and function of many alternative splicing events remain poorly understood. We use an RNA sequencing (RNA-seq)-guided proteomics method to identify protein alternative splicing isoforms in the human proteome by constructing tissue-specific protein databases that prioritize transcript splice junction pairs with high translational potential. Using the custom databases to reanalyze ∼80 million mass spectra in public proteomics datasets, we identify more than 1,500 noncanonical protein isoforms across 12 human tissues, including ∼400 sequences undocumented on TrEMBL and RefSeq databases. We apply the method to original quantitative mass spectrometry experiments and observe widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation. On a proteome scale, alternative isoform regions overlap frequently with disordered sequences and post-translational modification sites, suggesting that alternative splicing may regulate protein function through modulating intrinsically disordered regions. The described approach may help elucidate functional consequences of alternative splicing and expand the scope of proteomics investigations in various systems.

View details for DOI 10.1016/j.celrep.2019.11.026

View details for PubMedID 31825849

Abstract

Introduction: Although many current cancer therapies are effective, the mortality rate globally is unacceptably high. Cancer remains the second leading cause of death worldwide after heart disease and has caused nearly 10 million deaths in 2018. Additionally, current preventive therapies for cancer are underdeveloped, undermining the quality of life of high-risk individuals. Therefore, new treatment options for targeting cancer are urgently needed. In a recent study, researchers adopted an autologous iPSC-based vaccine to present a broad spectrum of tumor antigens to the immune system and succeeded in orchestrating a strong prophylactic immunity towards multiple types of cancer in mice. Areas Covered: In this review, we provide an overview of how cancer develops, the role of immune surveillance in cancer progression, the current status and challenges of cancer immunotherapy as well as the genetic overlap between pluripotent stem cells and cancer cells. Finally, we discuss the rationale for an autologous iPSC-based vaccine and its applications in murine cancer models. Expert Opinion: The autologous iPSC-based vaccine is a promising preventive and therapeutic strategy for fighting various types of cancers. Continuing efforts and clinical/translational follow-up studies may bring an autologous iPSC-based cancer vaccination approach from bench to bedside.

View details for DOI 10.1080/14712598.2019.1650909

View details for PubMedID 31364894

Abstract

The Safety Pharmacology Society (SPS) held a West Coast Regional Meeting in Foster City, CA on November 14, 2018 at the Gilead Sciences Inc. site. The meeting was attended by scientists from the pharmaceutical and biotechnology industry, contract research organizations (CROs) and academia. A variety of scientific topics were presented by speakers, covering a broad variety of topics in the fields of safety risk assessment; from pro-arrhythmia and contractility risk evaluation, to models of heart failure and seizure in-a-dish; and discovery sciences; from stem cells and precision medicine, to models of inherited cardiomyopathy and precision cut tissue slices. The present review summarizes the highlights of the presentations and provides an overview of the high level of innovation currently underlying many frontiers in safety pharmacology.

View details for PubMedID 31077805

Abstract

Given that cardiovascular safety concerns remain the leading cause of drug attrition at the preclinical drug development stage, the National Center for Toxicological Research of the US Food and Drug Administration hosted a workshop to discuss current gaps and challenges in translating preclinical cardiovascular safety data to humans. This white paper summarizes the topics presented by speakers from academia, industry, and government intended to address the theme of improving cardiotoxicity assessment in drug development. The main conclusion is that to reduce cardiovascular safety liabilities of new therapeutic agents, there is an urgent need to integrate human-relevant platforms/approaches into drug development. Potential regulatory applications of human-derived cardiomyocytes and future directions in employing human-relevant platforms to fill the gaps and overcome barriers and challenges in preclinical cardiovascular safety assessment were discussed. This paper is intended to serve as an initial step in a public-private collaborative development program for human-relevant cardiotoxicity tools, particularly for cardiotoxicities characterized by contractile dysfunction or structural injury.

View details for DOI 10.1161/CIRCRESAHA.119.315378

View details for PubMedID 31600125

Abstract

Matrix nanotopographical cues are known to regulate the structure and function of somatic cells derived from human pluripotent stem cell (hPSC) sources. High-throughput electrophysiological analysis of excitable cells derived from hPSCs is possible via multielectrode arrays (MEAs) but conventional MEA platforms use flat substrates and do not reproduce physiologically relevant tissue-specific architecture. To address this issue, we developed a high-throughput nanotopographically patterned multielectrode array (nanoMEA) by integrating conductive, ion-permeable, nanotopographic patterns with 48-well MEA plates, and investigated the effect of substrate-mediated cytoskeletal organization on hPSC-derived cardiomyocyte and neuronal function at scale. Using our nanoMEA platform, we found patterned hPSC-derived cardiac monolayers exhibit both enhanced structural organization and greater sensitivity to treatment with calcium blocking or conduction inhibiting compounds when subjected to high-throughput dose-response studies. Similarly, hPSC-derived neurons grown on nanoMEA substrates exhibit faster migration and neurite outgrowth speeds, greater colocalization of pre- and postsynaptic markers, and enhanced cell-cell communication only revealed through examination of data sets derived from multiple technical replicates. The presented data highlight the nanoMEA as a new tool to facilitate high-throughput, electrophysiological analysis of ordered cardiac and neuronal monolayers, which can have important implications for preclinical analysis of excitable cell function.

View details for DOI 10.1021/acs.nanolett.9b04152

View details for PubMedID 31845810

Abstract

Billions of US dollars are invested every year by the pharmaceutical industry in drug development, with the aim of introducing new drugs that are effective and have minimal side effects. Thirty percent of in-pipeline drugs are excluded in an early phase of preclinical and clinical screening owing to cardiovascular safety concerns, and several lead molecules that pass the early safety screening make it to market but are later withdrawn owing to severe cardiac side effects. Although the current drug safety screening methodologies can identify some cardiotoxic drug candidates, they cannot accurately represent the human heart in many aspects, including genomics, transcriptomics, and patient- or population-specific cardiotoxicity. Despite some limitations, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful and evolving technology that has been shown to recapitulate many attributes of human cardiomyocytes and their drug responses. In this review, we discuss the potential impact of the inclusion of the hiPSC-CM platform in premarket candidate drug screening.

View details for PubMedID 28992430

Abstract

As the second term of our commitment to Journal begins, we, the editors, would like to reflect on a few topics that have relevance today. These include prognostication and paradigm shifts; Serial testing: How to handle data? Is the change in perfusion predictive of outcome and which one? Ischemia-guided therapy: fractional flow reserve vs perfusion vs myocardial blood flow; positron emission tomography (PET) imaging using Rubidium-82 vs N-13 ammonia vs F-18 Flurpiridaz; How to differentiate microvascular disease from 3-vessel disease by PET? The imaging scene outside the United States, what are the differences and similarities? Radiation exposure; Special issues with the new cameras? Is attenuation correction needed? Are there normal databases and are these specific to each camera system? And finally, hybrid imaging with single-photon emission tomography or PET combined with computed tomography angiography or coronary calcium score. We hope these topics are of interest to our readers.

View details for PubMedID 29110288

Abstract

During development, the formation of a mature, well-functioning heart requires transformation of the ventricular wall from a loose trabecular network into a dense compact myocardium at mid-gestation. Failure to compact is associated in humans with congenital diseases such as left ventricular non-compaction (LVNC). The mechanisms regulating myocardial compaction are however still poorly understood. Here, we show that deletion of the Ino80 chromatin remodeler in vascular endothelial cells prevents ventricular compaction in the developing mouse heart. This correlates with defective coronary vascularization, and specific deletion of Ino80 in the two major coronary progenitor tissues-sinus venosus and endocardium-causes intermediate phenotypes. In vitro, endothelial cells promote myocardial expansion independently of blood flow in an Ino80-dependent manner. Ino80 deletion increases the expression of E2F-activated genes and endothelial cell S-phase occupancy. Thus, Ino80 is essential for coronary angiogenesis and allows coronary vessels to support proper compaction of the heart wall.

View details for PubMedID 29371594

Abstract

Cardiotoxic effects from cancer therapy are a major cause of morbidity during cancer treatment. Unexpected toxicity can occur during treatment and/or after completion of therapy, into the time of cancer survivorship. While older drugs such as anthracyclines have well-known cardiotoxic effects, newer drugs such as tyrosine kinase inhibitors, proteasome inhibitors, and immunotherapies also can cause diverse cardiovascular and metabolic complications. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly being used as instruments for disease modelling, drug discovery, and mechanistic toxicity studies. Promising results with hiPSC-CM chemotherapy studies are raising hopes for improving cancer therapies through personalized medicine and safer drug development. Here, we review the cardiotoxicity profiles of common chemotherapeutic agents as well as efforts to model them in vitro using hiPSC-CMs.

View details for PubMedID 29377985

Abstract

Non-human primates (NHPs) can serve as a human-like model to study cell therapy using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, whether the efficacy of NHP and human iPSC-CMs is mechanistically similar remains unknown. To examine this, RNU rats received intramyocardial injection of 1 × 107NHP or human iPSC-CMs or the same number of respective fibroblasts or PBS control (n = 9-14/group) at 4 days after 60-min coronary artery occlusion-reperfusion. Cardiac function and left ventricular remodeling were similarly improved in both iPSC-CM-treated groups. To mimic the ischemic environment in the infarcted heart, both cultured NHP and human iPSC-CMs underwent 24-hr hypoxia in vitro. Both cells and media were collected, and similarities in transcriptomic as well as metabolomic profiles were noted between both groups. In conclusion, both NHP and human iPSC-CMs confer similar cardioprotection in a rodent myocardial infarction model through relatively similar mechanisms via promotion of cell survival, angiogenesis, and inhibition of hypertrophy and fibrosis.

View details for PubMedID 29398480

View details for PubMedCentralID PMC5830958

Abstract

Myocardial infarction is the leading cause of morbidity and mortality worldwide. Recent advances in cardiac regenerative therapy have allowed for novel modalities in replenishing the damaged myocardium. However, poor long-term engraftment and survival of transplanted cells have largely precluded effective cell replacement. As an alternative to direct cell replacement, the release of paracrine protective factors may be a more plausible effector for cardioprotection which may partially be mediated through secretion of microvesicles, or exosomes, that contribute to cell-cell communication. In this chapter, we describe the isolation of exosomes from induced pluripotent stem cells-derived cardiomyocytes for subsequent microRNA profiling for a better understanding of the biological cargo contained within exosomes.

View details for PubMedID 29435928

Abstract

The adult mammalian heart possesses only limited capacity for innate regeneration and the response to severe injury is dominated by the formation of scar tissue. Current therapy to replace damaged cardiac tissue is limited to cardiac transplantation and thus many patients suffer progressive decay in the heart's pumping capacity to the point of heart failure. Nanostructured systems have the potential to revolutionize both preventive and therapeutic approaches for treating cardiovascular disease. Here, we outline recent advancements in nanotechnology that could be exploited to overcome the major obstacles in the prevention of and therapy for heart disease. We also discuss emerging trends in nanotechnology affecting the cardiovascular field that may offer new hope for patients suffering massive heart attacks.

View details for DOI 10.1038/nnano.2017.167

View details for PubMedID 28875984

View details for PubMedCentralID PMC5717755

Abstract

Risk stratification in heart failure (HF) relies on several established clinical risk scores, however, myocardial deformation, right heart metrics, and exercise performance have not usually been considered. This study sought to assess the incremental value of advanced echocardiographic and cardiopulmonary exercise testing (CPX) parameters to validated risk scores in HF.The Meta-Analysis Global Group in Chronic Heart Failure (MAGGIC) and Metabolic Exercise Test Data Combined with Cardiac and Kidney Indexes (MECKI) scores were applied to 208 ambulatory patients with dilated cardiomyopathy (DCM) who completed echocardiography in conjunction with CPX as part of the Stanford Exercise Testing registry. Patients were followed for the composite end point of death, heart transplant, left ventricular device implantation, and hospitalization for acute HF. Mean age, left ventricular ejection fraction (LVEF), and left ventricular global longitudinal strain (LVGLS) were 47 ± 13 years, 33 ± 13%, and -10.6 ± 4.4%, respectively, while right ventricular free-wall longitudinal strain was -18.8 ± 5.5%. Partial correlation mapping identified strong correlations between LVEF, LVGLS, and LV systolic strain rate, with a moderate correlation between these metrics and peak VO2. Over a median follow up of 5.3 years, the composite end point occurred in 60 patients. Cox proportional hazards identified MAGGIC score [hazard ratio (HR) (2.04 [1.39-3.01], P < 0.01], peak VO2 HR (0.52 [0.28-0.97], P = 0.04), and right atrial volume indexed (RAVI) HR (1.31 [1.07-1.61], P < 0.01) as independent correlates of outcome. RAVI remained an independent correlate when combined with the MECKI score (2.21 [1.59-3.07]), P < 0.01, RAVI, 1.33 [1.06-1.67], P = 0.01).Our study demonstrates that RAVI is complementary to well-validated HF risk scores and highlights the importance of exercise performance in DCM.

View details for DOI 10.1093/ehjci/jex187

View details for PubMedID 28977353

Abstract

The creation of physiologically-relevant human cardiac tissue with defined cell structure and function is essential for a wide variety of therapeutic, diagnostic, and drug screening applications. Here we report a new scalable method using Faraday waves to enable rapid aggregation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into predefined 3D constructs. At packing densities that approximate native myocardium (10(8)-10(9) cells/ml), these hiPSC-CM-derived 3D tissues demonstrate significantly improved cell viability, metabolic activity, and intercellular connection when compared to constructs with random cell distribution. Moreover, the patterned hiPSC-CMs within the constructs exhibit significantly greater levels of contractile stress, beat frequency, and contraction-relaxation rates, suggesting their improved maturation. Our results demonstrate a novel application of Faraday waves to create stem cell-derived 3D cardiac tissue that resembles the cellular architecture of a native heart tissue for diverse basic research and clinical applications.

View details for DOI 10.1016/j.biomaterials.2017.03.037

View details for PubMedID 28376365

Abstract

Engineering 3D human cardiac tissues is of great importance for therapeutic and pharmaceutical applications. As cardiac tissue substitutes, extracellular matrix-derived hydrogels have been widely explored. However, they exhibit premature degradation and their stiffness is often orders of magnitude lower than that of native cardiac tissue. There are no reports on establishing interconnected cardiomyocytes in 3D hydrogels at physiologically-relevant cell density and matrix stiffness. Here we bioengineer human cardiac microtissues by encapsulating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in chemically-crosslinked gelatin hydrogels (1.25 × 10(8)/mL) with tunable stiffness and degradation. In comparison to the cells in high stiffness (16 kPa)/slow degrading hydrogels, hiPSC-CMs in low stiffness (2 kPa)/fast degrading and intermediate stiffness (9 kPa)/intermediate degrading hydrogels exhibit increased intercellular network formation, α-actinin and connexin-43 expression, and contraction velocity. Only the 9 kPa microtissues exhibit organized sarcomeric structure and significantly increased contractile stress. This demonstrates that muscle-mimicking stiffness together with robust cellular interconnection contributes to enhancement in sarcomeric organization and contractile function of the engineered cardiac tissue. This study highlights the importance of intercellular connectivity, physiologically-relevant cell density, and matrix stiffness to best support 3D cardiac tissue engineering.

View details for DOI 10.1016/j.biomaterials.2017.03.039

View details for PubMedID 28384492

Abstract

Purpose: 6"-(18)F-fluoromaltotriose is a novel positron emission tomography (PET) tracer that can potentially be used to image and localize most bacterial infections, much like 2-deoxy-2-(18)F-fluoro-D-glucose ((18)F-FDG) has been used to image and localize many cancers. However, unlike (18)F-FDG, 6"-(18)F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in most Gram-positive and Gram-negative strains of bacteria. Materials and Methods: 6"-(18)F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains incultures in vitro and in living mice. Results: 6"-(18)F-fluoromaltotriose was taken up in both Gram-positive and Gram-negative bacterial strains. 6"-[(18)F]-fluoromaltotriose was also able to detect Pseudomonas aeruginosa in a clinically relevant mouse model of wound infection. The utility of 6"-(18)F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. Conclusion: 6"-(18)F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients suffering from infectious diseases of bacterial origin.

View details for DOI 10.2967/jnumed.117.191452

View details for PubMedID 28490473

Abstract

Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.

View details for DOI 10.1038/srep44840

View details for Web of Science ID 000396983300001

View details for PubMedID 28327614

View details for PubMedCentralID PMC5361100

Abstract

Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.

View details for DOI 10.1016/j.stem.2016.11.018

View details for PubMedID 28089908

Abstract

Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome.The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development.Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

View details for DOI 10.1161/CIRCRESAHA.116.309948

View details for PubMedID 28246128

Abstract

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

View details for DOI 10.1073/pnas.1615375114

View details for PubMedID 28223521

Abstract

Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, enormous progress has been made in stem cell biology and regenerative medicine. Human iPSCs have been widely used for disease modelling, drug discovery and cell therapy development. Novel pathological mechanisms have been elucidated, new drugs originating from iPSC screens are in the pipeline and the first clinical trial using human iPSC-derived products has been initiated. In particular, the combination of human iPSC technology with recent developments in gene editing and 3D organoids makes iPSC-based platforms even more powerful in each area of their application, including precision medicine. In this Review, we discuss the progress in applications of iPSC technology that are particularly relevant to drug discovery and regenerative medicine, and consider the remaining challenges and the emerging opportunities in the field.

View details for DOI 10.1038/nrd.2016.245

View details for Web of Science ID 000393446800018

View details for PubMedID 27980341

Abstract

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present the evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by utilizing a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced re-seeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1x10(3) higher doses to form new teratomas. We demonstrate that radiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. This article is protected by copyright. All rights reserved.

View details for PubMedID 28600830

Abstract

The role of autoimmunity in cardiovascular (CV) diseases has been increasingly recognized. Autoimmunity is most commonly examined by the levels of circulating autoantibodies in clinical practices. Measurement of autoantibodies remains, however, challenging because of the deficiency of reproducible, sensitive, and standardized assays. The lack of multiplexed assays also limits the potential to identify a CV-specific autoantibody profile. To overcome these challenges, we developed a nanotechnology-based plasmonic gold chip for autoantibody profiling. This approach allowed simultaneous detection of 10 CV autoantibodies targeting the structural myocardial proteins, the neurohormonal regulatory proteins, the vascular proteins, and the proteins associated with apoptosis and coagulation. Autoantibodies were measured in four groups of participants across the continuum of hypertensive heart diseases. We observed higher levels of all 10 CV autoantibodies in hypertensive subjects (n= 77) compared with healthy participants (n= 30), and the autoantibodies investigated were related to each other, forming a highly linked network. In addition, we established that autoantibodies to troponin I, annexin-A5, and beta 1-adrenegic receptor best discriminated hypertensive subjects with adverse left ventricular (LV) remodeling or dysfunction (n= 49) from hypertensive subjects with normal LV structure and function (n= 28). By further linking these three significant CV autoantibodies to the innate and growth factors, we revealed a positive but weak association between autoantibodies to troponin I and proinflammatory cytokine IL-18. Overall, we demonstrated that this platform can be used to evaluate autoantibody profiles in hypertensive subjects at risk for heart failure.

View details for PubMedID 28630342

Abstract

Recent advances have improved our ability to generate cardiomyocytes from human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). However, our understanding of the transcriptional regulatory networks underlying early stages (ie, from mesoderm to cardiac mesoderm) of cardiomyocyte differentiation remains limited.To characterize transcriptome and chromatin accessibility during early cardiomyocyte differentiation from hiPSCs and hESCs.We profiled the temporal changes in transcriptome and chromatin accessibility at genome-wide levels during cardiomyocyte differentiation derived from 2 hiPSC lines and 2 hESC lines at 4 stages: pluripotent stem cells, mesoderm, cardiac mesoderm, and differentiated cardiomyocytes. Overall, RNA sequencing analysis revealed that transcriptomes during early cardiomyocyte differentiation were highly concordant between hiPSCs and hESCs, and clustering of 4 cell lines within each time point demonstrated that changes in genome-wide chromatin accessibility were similar across hiPSC and hESC cell lines. Weighted gene co-expression network analysis (WGCNA) identified several modules that were strongly correlated with different stages of cardiomyocyte differentiation. Several novel genes were identified with high weighted connectivity within modules and exhibited coexpression patterns with other genes, including noncoding RNA LINC01124 and uncharacterized RNA AK127400 in the module related to the mesoderm stage; E-box-binding homeobox 1 (ZEB1) in the module correlated with postcardiac mesoderm. We further demonstrated that ZEB1 is required for early cardiomyocyte differentiation. In addition, based on integrative analysis of both WGCNA and transcription factor motif enrichment analysis, we determined numerous transcription factors likely to play important roles at different stages during cardiomyocyte differentiation, such as T and eomesodermin (EOMES; mesoderm), lymphoid enhancer-binding factor 1 (LEF1) and mesoderm posterior BHLH transcription factor 1 (MESP1; from mesoderm to cardiac mesoderm), meis homeobox 1 (MEIS1) and GATA-binding protein 4 (GATA4) (postcardiac mesoderm), JUN and FOS families, and MEIS2 (cardiomyocyte).Both hiPSCs and hESCs share similar transcriptional regulatory mechanisms underlying early cardiac differentiation, and our results have revealed transcriptional regulatory networks and new factors (eg, ZEB1) controlling early stages of cardiomyocyte differentiation.

View details for PubMedID 28663367

Abstract

Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome.Human ESCs and iPSCs were differentiated into cardiomyocytes using small molecule inhibitors. Fluorescence-activated cell sorting (FACS) analysis confirmed ∼85% and ∼83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable to the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective.We demonstrate for the first time that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future. This article is protected by copyright. All rights reserved.

View details for PubMedID 28710827

Abstract

Engineering of myocardial tissue constructs is a promising approach for treatment of coronary heart disease. To engineer myocardial tissues that better mimic the highly ordered physiological arrangement and function of native cardiomyocytes, we generated electrospun microfibrous polycaprolactone scaffolds with either randomly oriented (14 μm fiber diameter) or parallel-aligned (7 μm fiber diameter) microfiber arrangement and co-seeded the scaffolds with human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and endothelial cells (iECs) for up to 12 days after iCM seeding. Here we demonstrated that aligned microfibrous scaffolds induced iCM alignment along the direction of the aligned microfibers after 2 days of iCM seeding, as well as promoted greater iCM maturation by increasing the sarcomeric length and gene expression of myosin heavy chain adult isoform (MYH7), in comparison to randomly oriented scaffolds. Furthermore, the benefit of scaffold anisotropy was evident in the significantly higher maximum contraction velocity of iCMs on the aligned scaffolds, compared to randomly oriented scaffolds, at 12 days of culture. Co-seeding of iCMs with iECs led to reduced contractility, compared to when iCMs were seeded alone. These findings demonstrate a dominant role of scaffold anisotropy in engineering cardiovascular tissues that maintain iCM organization and contractile function.

View details for PubMedID 28715029

View details for PubMedCentralID PMC5567776

Abstract

The need for novel approaches to cardiovascular drug development served as the impetus to convene an open meeting of experts from the pharmaceutical industry and academia to assess the challenges and develop solutions for drug discovery in cardiovascular disease.The Novel Cardiovascular Therapeutics Summit first reviewed recent examples of ongoing or recently completed programs translating basic science observations to targeted drug development, highlighting successes (protein convertase sutilisin/kexin type 9 [PCSK9] and neprilysin inhibition) and targets still under evaluation (cholesteryl ester transfer protein [CETP] inhibition), with the hope of gleaning key lessons to successful drug development in the current era. Participants then reviewed the use of innovative approaches being explored to facilitate rapid and more cost-efficient evaluations of drug candidates in a short timeframe.We summarize observations gleaned from this summit and offer insight into future cardiovascular drug development.The rapid development in genetic and high-throughput drug evaluation technologies, coupled with new approaches to rapidly evaluate potential cardiovascular therapies with in vitro techniques, offer opportunities to identify new drug targets for cardiovascular disease, study new therapies with better efficiency and higher throughput in the preclinical setting, and more rapidly bring the most promising therapies to human testing. However, there must be a critical interface between industry and academia to guide the future of cardiovascular drug development. The shared interest among academic institutions and pharmaceutical companies in developing promising therapies to address unmet clinical needs for patients with cardiovascular disease underlies and guides innovation and discovery platforms that are significantly altering the landscape of cardiovascular drug development.

View details for PubMedID 28735360

Abstract

Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and inDrosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

View details for PubMedID 28749338

View details for PubMedCentralID PMC5584994

Abstract

There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

View details for PubMedID 28834758

Abstract

Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart.We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression.We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31+CD144+), cardiac progenitor cells (Sca-1+), fibroblasts (DDR2+), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression.Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases.

View details for PubMedID 29030344

View details for PubMedCentralID PMC5773062

Abstract

The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling.Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (hiPSCs, IMR-90 line) were differentiated to human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin(+) flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 x 10(6) hPSC-CMs were mixed with 0.4 x 10(6) human fibroblasts (IMR-90 line) (3:1 ratio) and Type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane (PDMS) reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable to rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, β-adrenergic receptors, and t-tubule protein caveolin-3.Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. This article is protected by copyright. All rights reserved.

View details for PubMedID 29086457

Abstract

Recent data suggest that circulating biomarkers may predict outcome in patients undergoing transcatheter aortic valve replacement (TAVR). We examined the association between inflammatory, myocardial, and renal biomarkers and their role in ventricular recovery and outcome after TAVR.A total of 112 subjects undergoing TAVR were included in the prospective registry. Plasma levels of B-type natriuretic peptide, hs-TnI (high-sensitivity troponin I), CRP (C-reactive protein), GDF-15 (growth differentiation factor 15), GAL-3 (galectin-3), and Cys-C (cystatin-C) were assessed before TAVR and in 100 sex-matched healthy controls. Among echocardiographic parameters, we measured global longitudinal strain, indexed left ventricular mass, and indexed left atrial volume. The TAVR group included 59% male, with an average age of 84 years, and 1-year mortality of 18%. Among biomarkers, we found GDF-15 and CRP to be strongly associated with all-cause mortality (P<0.001). Inclusion of GDF-15 and CRP to the Society of Thoracic Surgeons score significantly improved C index (0.65-0.79; P<0.05) and provided a category-free net reclassification improvement of 106% at 2 years (P=0.01). Among survivors, functional recovery in global longitudinal strain (>15% improvement) and indexed left ventricular mass (>20% decrease) at 1 year occurred in 48% and 22%, respectively. On multivariate logistic regression, lower baseline GDF-15 was associated with improved global longitudinal strain at 1 year (hazard ratio=0.29; P<0.001). Furthermore, improvement in global longitudinal strain at 1 month correlated with lower overall mortality (hazard ratio=0.45; P=0.03).Elevated GDF-15 correlates with lack of reverse remodeling and increased mortality after TAVR and improves risk prediction of mortality when added to the Society of Thoracic Surgeons score.

View details for PubMedID 29222133

Abstract

Brugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, 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 a lack of adequate human cellular models of the disorder.The objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).This study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs.BrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca(2+)) 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 Ca(2+) transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression.Patient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca(2+) handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.

View details for DOI 10.1016/j.jacc.2016.07.779

View details for PubMedID 27810048

Abstract

Although progress continues to be made in the field of stem cell regenerative medicine for the treatment of cardiovascular disease, significant barriers to clinical implementation still exist.To summarize the current barriers to the clinical implementation of stem cell therapy in patients with cardiovascular disease and to discuss potential strategies to overcome them.Information for this review was obtained through a search of PubMed and the Cochrane database for English-language studies published between January 1, 2000, and July 25, 2016. Ten randomized clinical trials and 8 systematic reviews were included.One of the major clinical barriers facing the routine implementation of stem cell therapy in patients with cardiovascular disease is the limited and inconsistent benefit observed thus far. Reasons for this finding are unclear but may be owing to poor cell retention and survival, as suggested by numerous preclinical studies and a small number of human studies incorporating imaging to determine cell fate. Additional studies in humans using imaging to determine cell fate are needed to understand how these factors contribute to the limited efficacy of stem cell therapy. Treatment strategies to address poor cell retention and survival are under investigation and include the following: coadministration of immunosuppressive and prosurvival agents, delivery of cardioprotective factors packaged in exosomes rather than the cells themselves, and use of tissue-engineering strategies to provide structural support for cells. If larger grafts are achieved using these strategies, it will be imperative to carefully monitor for the potential risks of tumorigenicity, immunogenicity, and arrhythmogenicity.Despite important achievements to date, stem cell therapy is not yet ready for routine clinical implementation. Significant research is still needed to address the clinical barriers outlined herein before the next wave of large clinical trials is under way.

View details for DOI 10.1001/jamacardio.2016.2750

View details for PubMedID 27579998

Abstract

The use of tissue engineering approaches in combination with exogenously produced cardiomyocytes offers the potential to restore contractile function after myocardial injury. However, current techniques assessing changes in global cardiac performance after such treatments are plagued by relatively low detection ability. Since the treatment is locally performed, this 