CRISPRi/a screens in human iPSC-cardiomyocytes identify glycolytic activation as a druggable target for doxorubicin-induced cardiotoxicity.

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12).

Simultaneous Optical Imaging of Action Potentials and Calcium Transients in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiovascular diseases have emerged as one of the leading causes of human mortality, but the discovery of new drugs has been hindered by the absence of suitable in vitro platforms. In recent decades, continuously refined protocols for differentiating human induced pluripotent stem cells (hiPSCs) into hiPSC-derived cardiomyocytes (hiPSC-CMs) have significantly advanced disease modeling and drug screening; however, this has led to an increasing need to monitor the function of hiPSC-CMs. The precise regulation of action potentials (APs) and intracellular calcium (Ca) transients is critical for proper excitation-contraction coupling and cardiomyocyte function.

Cracking the code of the cardiovascular enigma: hPSC-derived endothelial cells unveil the secrets of endothelial dysfunction.

Endothelial dysfunction is a central contributor to the development of most cardiovascular diseases and is characterised by the reduced synthesis or bioavailability of the vasodilator nitric oxide together with other abnormalities such as inflammation, senescence, and oxidative stress. The use of patient-specific and genome-edited human pluripotent stem cell-derived endothelial cells (hPSC-ECs) has shed novel insights into the role of endothelial dysfunction in cardiovascular diseases with strong genetic components such as genetic cardiomyopathies and pulmonary arterial hypertension. However, their utility in studying complex multifactorial diseases such as atherosclerosis, metabolic syndrome and heart failure poses notable challenges.

The new era of cardiovascular research: revolutionizing cardiovascular research with 3D models in a dish.

Cardiovascular research has heavily relied on studies using patient samples and animal models. However, patient studies often miss the data from the crucial early stage of cardiovascular diseases, as obtaining primary tissues at this stage is impracticable. Transgenic animal models can offer some insights into disease mechanisms, although they usually do not fully recapitulate the phenotype of cardiovascular diseases and their progression.

Sphingolipid metabolism controls mammalian heart regeneration.

Utilization of lipids as energy substrates after birth causes cardiomyocyte (CM) cell-cycle arrest and loss of regenerative capacity in mammalian hearts. Beyond energy provision, proper management of lipid composition is crucial for cellular and organismal health, but its role in heart regeneration remains unclear. Here, we demonstrate widespread sphingolipid metabolism remodeling in neonatal hearts after injury and find that SphK1 and SphK2, isoenzymes producing the same sphingolipid metabolite sphingosine-1-phosphate (S1P), differently regulate cardiac regeneration.

Recent advances in regulating the proliferation or maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

In the last decade, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM)-based cell therapy has drawn broad attention as a potential therapy for treating injured hearts. However, mass production of hiPSC-CMs remains challenging, limiting their translational potential in regenerative medicine. Therefore, multiple strategies including cell cycle regulators, small molecules, co-culture systems, and epigenetic modifiers have been used to improve the proliferation of hiPSC-CMs.

TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death.

Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.

Background: pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive.

Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes.

Multiple strategies have been developed to efficiently differentiate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we describe a protocol for measuring three key functional parameters of hiPSC-CMs, including contractile function, calcium (Ca) handling, and action potential. For complete details on the use and execution of this protocol, please refer to Zhang et al.

DUSP26 induces aortic valve calcification by antagonizing MDM2-mediated ubiquitination of DPP4 in human valvular interstitial cells.

Aims: The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. Here, we evaluated the role and therapeutic value of dual-specificity phosphatase 26 (DUSP26) in CAVD.

Methods And Results: Microarray profiling of human calcific aortic valves and normal controls demonstrated that DUSP26 was significantly up-regulated in calcific aortic valves.

Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies.

Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy-based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation.

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death.

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS).

Effects of Cryopreservation on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Assessing Drug Safety Response Profiles.

Burgeoning applications of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in disease modeling, regenerative medicine, and drug screening have broadened the usage of hiPSC-CMs and entailed their long-term storage. Cryopreservation is the most common approach to store hiPSC-CMs. However, the effects of cryopreservation and recovery on hiPSC-CMs remain poorly understood.

Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy.

Mutations in , the gene that encodes lamin A and C, causes LMNA-related dilated cardiomyopathy (DCM) or cardiolaminopathy. LMNA is expressed in endothelial cells (ECs); however, little is known about the EC-specific phenotype of LMNA-related DCM. Here, we studied a family affected by DCM due to a frameshift variant in Human induced pluripotent stem cell (iPSC)-derived ECs were generated from patients with LMNA-related DCM and phenotypically characterized.

Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy.

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).

Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients.

Aims: Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. However, its cellular mechanisms are not fully understood, and presently there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD in HCM and as a platform for drug discovery.

A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles.

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 TBX5 and NKX2-5 double reporter to delineate cardiac lineages 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.

Human-Induced Pluripotent Stem Cell Model of Trastuzumab-Induced Cardiac Dysfunction in Patients With Breast Cancer.

Background: Molecular targeted chemotherapies have been shown to significantly improve the outcomes of patients who have cancer, but they often cause cardiovascular side effects that limit their use and impair patients' quality of life. Cardiac dysfunction induced by these therapies, especially trastuzumab, shows a distinct cardiotoxic clinical phenotype in comparison to the cardiotoxicity induced by conventional chemotherapies.

Methods: We used the human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) platform to determine the underlying cellular mechanisms in trastuzumab-induced cardiac dysfunction.

Single-Cell RNA Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development.

Nicotine, the main chemical constituent of tobacco, is highly detrimental to the developing fetus by increasing the risk of gestational complications and organ disorders. The effects of nicotine on human embryonic development and related mechanisms, however, remain poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) of human embryonic stem cell (hESC)-derived embryoid body (EB) in the presence or absence of nicotine.

Disruption of mesoderm formation during cardiac differentiation due to developmental exposure to 13-cis-retinoic acid.

13-cis-retinoic acid (isotretinoin, INN) is an oral pharmaceutical drug used for the treatment of skin acne, and is also a known teratogen. In this study, the molecular mechanisms underlying INN-induced developmental toxicity during early cardiac differentiation were investigated using both human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Pre-exposure of hiPSCs and hESCs to a sublethal concentration of INN did not influence cell proliferation and pluripotency.

Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells.

Background: The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity.

Applications of genetically engineered human pluripotent stem cell reporters in cardiac stem cell biology.

The advent of human pluripotent stem cells (hPSCs) has benefited many fields, from regenerative medicine to disease modeling, with an especially profound effect in cardiac research. Coupled with other novel technologies in genome engineering, hPSCs offer a great opportunity to delineate human cardiac lineages, investigate inherited cardiovascular diseases, and assess the safety and efficacy of cell-based therapies. In this review, we provide an overview of methods for generating genetically engineered hPSC reporters and a succinct synopsis of a variety of hPSC reporters, with a particular focus on their applications in cardiac stem cell biology.

Comparison of Non-human Primate versus Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Treatment of Myocardial Infarction.

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 × 10 NHP 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.