NKX2-5 congenital heart disease mutations show diverse loss and gain of epigenomic, biochemical and chromatin search functions underpinning pathogenicity.

Congenital heart defects (CHD) occur in ∼1% of live births, with both inherited and acquired mutations and environmental factors known to contribute to causation. However, network perturbations and epigenetic changes in CHD remain poorly characterised. We report an integrated functional-epigenomics approach to understanding CHD, focusing on the cardiac homeodomain (HD) family transcription factor NKX2-5, mutations in which cause diverse congenital heart structural and conduction defects.

Functional analysis of JPH2-knockout cardiomyocytes identifies ECCD as a novel indicator in a human cardiac modelJPH2.

Background: Junctophilin-2 (JPH2) is a vital protein in cardiomyocytes, anchoring T-tubule and sarcoplasmic reticulum membranes to facilitate excitation-contraction coupling, a process essential for cardiac contractile function. Dysfunction of JPH2 is associated with cardiac disorders such as heart failure; however, prior studies using mouse models or primary human cardiomyocytes are limited by interspecies differences or poor cell viability, respectively. This study aimed to investigate JPH2's role in human cardiac function and disease using a novel stem cell-derived model, while introducing a new indicator to evaluate related cardiac impairments.

Combined Proteomic and Phosphoproteomic Characterization of the Molecular Regulators and Functional Modules During Pancreatic Progenitor Cell Development.

Differentiated multipotent pancreatic progenitors have major advantages for both modeling pancreas development and preventing or treating diabetes. Despite significant advancements in inducing the differentiation of human pluripotent stem cells into insulin-producing cells, the complete mechanism governing proliferation and differentiation remains poorly understood. This study used large-scale mass spectrometry to characterize molecular processes at various stages of human embryonic stem cell (hESC) differentiation toward pancreatic progenitors.

The application of quantitative telomerase activity measurement as an important indicator to monitor the cardiomyocyte differentiation process of human induced pluripotent stem cells under defined conditions.

The construction of an in vitro differentiation system for human induced pluripotent stem cells (hiPSCs) has made exciting progress, but it is still of great significance to clarify the differentiation process. The use of conventional genetic and protein-labeled microscopes to observe or detect different stages of hiPSC differentiation is not specific enough and is cumbersome and time-consuming. In this study, in addition to analyzing the expression of gene/protein-related markers, we used a previously reported simple and excellent quantitative method of cellular telomerase activity based on a quartz crystal microbalance (TREAQ) device to monitor the dynamic changes in cellular telomerase activity in hiPSCs during myocardial differentiation under chemically defined conditions.

A review of protocols for engineering human cardiac organoids.

The use of human cardiac organoids (hCOs) as 3D in vitro models for cardiovascular research has shown great promise. Human pluripotent stem cells (hPSCs) have proven to be a potent source for engineering hCOs. However, various protocols for generating hCOs from hPSCs result in significant differences in heart development, maturity, complexity, vascularization, and spatial structure, all of which can influence their functional and physiological properties.

Establishment of human embryonic stem cell WAe009-A-88 carrying a long QT syndrome mutation in KCNH2.

Long-QT syndrome type 2 (LQT2) is a life-threatening Mendelian disease caused by genetic variants in KCNH2. Herein, we generated a human embryonic stem cell line (WAe009-A-88) carrying a LQT2 related mutation in KCNH2, c.1720 A>G.

Novel CaMKII-δ Inhibitor Hesperadin Exerts Dual Functions to Ameliorate Cardiac Ischemia/Reperfusion Injury and Inhibit Tumor Growth.

Background: Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease, the leading cause of morbidity and mortality worldwide. At present, there is no effective therapy for reducing cardiac I/R injury. CaMKII (Ca/calmodulin-dependent kinase II) plays a pivotal role in the pathogenesis of severe heart conditions, including I/R injury.

Establishment of human embryonic stem cell WAe009-A-74 carrying a Long QT syndrome mutation in KCNH2.

Long-QT syndrome type 2 (LQT2) is a common malignant hereditary arrhythmia. Due to the lack of suitable animal and human models, the pathogenesis of LQT2 caused by human ether-a-go-go-related gene (hERG) deficiency is still unclear. Herein, we have generated a human embryonic stem cell line (WAe009-A-74) carrying a LQTS related mutation in KCNH2.

Targeting CaMKII-δ9 Ameliorates Cardiac Ischemia/Reperfusion Injury by Inhibiting Myocardial Inflammation.

Background: CaMKII (Ca/calmodulin-dependent kinase II) plays a central role in cardiac ischemia/reperfusion (I/R) injury-an important therapeutic target for ischemic heart disease. In the heart, CaMKII-δ is the predominant isoform and further alternatively spliced into 11 variants. In humans, CaMKII-δ9 and CaMKII-δ3, the major cardiac splice variants, inversely regulate cardiomyocyte viability with the former pro-death and the latter pro-survival.

Base Editing of Human Pluripotent Stem Cells for Modeling Long QT Syndrome.

Human pluripotent stem cells (hPSCs) have great potential for disease modeling, drug discovery, and regenerative medicine as they can differentiate into many different functional cell types via directed differentiation. However, the application of disease modeling is limited due to a time-consuming and labor-intensive process of introducing known pathogenic mutations into hPSCs. Base editing is a newly developed technology that enables the facile introduction of point mutations into specific loci within the genome of living cells without unwanted genome injured.

The Role of METTL3-Mediated N6-Methyladenosine (m6A) of JPH2 mRNA in Cyclophosphamide-Induced Cardiotoxicity.

Cyclophosphamide (CYP)-induced cardiotoxicity is a common side effect of cancer treatment. Although it has received significant attention, the related mechanisms of CYP-induced cardiotoxicity remain largely unknown. In this study, we used cell and animal models to investigate the effect of CYP on cardiomyocytes.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

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Generation of a COL4A5 heterozygous mutation human embryonic stem cell line (WAe009-A-58) using an episomal vector-based CRISPR/Cas9 system.

X-linked Alport syndrome (XLAS) is the second most common inherited kidney disease which pathogenic variants related to a mutation in the COL4A5 gene encoding the type IV collagen α5 chain. Here, we have generated a COL4A5 heterozygous mutant human embryonic stem cell (hESC) line (H9-COL4A5 by an episomal vector-based CRISPR/Cas9 system. The generated H9-COL4A5 maintained a normal stem cell morphology, stably expressed pluripotent markers, and could differentiate into all three germ layers in vivo.

Establishment of a KCNQ1 homozygous knockout human embryonic stem cell line by episomal vector-based CRISPR/Cas9 system.

As a member of the voltage-gated potassium ion channels, KCNQ1 plays an important role in heart physiological functions. Numerous mutations in KCNQ1 were identified as primary causes to hereditary long-QT syndrome. To further study the role of KCNQ1 in human cardiac functions, here we generated a homozygous KCNQ1 knockout human embryonic stem cell line (KCNQ1-KO) using episomal vector-based CRISPR/Cas9 system.

hERG-deficient human embryonic stem cell-derived cardiomyocytes for modelling QT prolongation.

Background: Long-QT syndrome type 2 (LQT2) is a common malignant hereditary arrhythmia. Due to the lack of suitable animal and human models, the pathogenesis of LQT2 caused by human ether-a-go-go-related gene (hERG) deficiency is still unclear. In this study, we generated an hERG-deficient human cardiomyocyte (CM) model that simulates 'human homozygous hERG mutations' to explore the underlying impact of hERG dysfunction and the genotype-phenotype relationship of hERG deficiency.

Establishment of human embryonic stem cell WAe009-A-48 carrying a long QT syndrome mutation in SCN5A.

The long QT syndrome type 3 (LQT3) is currently the 3rd most prevalent of the 15 known types of LQT syndrome. Cardiac events in LQT3 are less frequent than LQT1 and LQT2, but more likely to be fatal. LQT3 is caused by mutation in gene SCN5A, which codes for the Nav1.

Generation of a TBX5 homozygous knockout embryonic stem cell line (WAe009-A-45) by CRISPR/Cas9 genome editing.

Holt-Oram syndrome (HOS), which is caused by genetic changes in the TBX5 gene, affects the hands and heart. HOS patients have heart defects, including atrial septal defects (ASD), ventricular septal defects (VSD) and heart conduction disease. Here, we generated a homozygous TBX5 knockout human embryonic stem cell (hESC) line (TBX5-KO) using a CRISPR/Cas9 system.

Ascorbic acid can promote the generation and expansion of neuroepithelial-like stem cells derived from hiPS/ES cells under chemically defined conditions through promoting collagen synthesis.

Introduction: Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably.

Generation of a homozygous COX6A2 knockout human embryonic stem cell line (WAe009-A-47) via an epiCRISPR/Cas9 system.

COX6A2 protein is a structural subunit of Complex IV (CIV/Cytochrome c oxidase/COX) in the mitochondrial respiratory chain. It is mainly expressed in the heart and skeletal muscle, also in some interneurons, regulating the assembly and catalytic activity of CIV. Its mutations can lead to COX deficiency, causing human myopathies, and maybe a potential cause of neurological abnormalities.

CRISPR/Cas9-edited triple-fusion reporter gene imaging of dynamics and function of transplanted human urinary-induced pluripotent stem cell-derived cardiomyocytes.

Purpose: To investigate the post-transplantation behaviour and therapeutic efficacy of human urinary-induced pluripotent stem cell-derived cardiomyocytes (hUiCMs) in infarcted heart.

Methods: We used clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology to integrate a triple-fusion (TF) reporter gene into the AAVS1 locus in human urine-derived hiPSCs (hUiPSCs) to generate TF-hUiPSCs that stably expressed monomeric red fluorescent protein for fluorescence imaging, firefly luciferase for bioluminescence imaging (BLI) and herpes simplex virus thymidine kinase for positron emission tomography (PET) imaging.

Results: Transplanted cardiomyocytes derived from TF-hUiPSCs (TF-hUiCMs) engrafted and proliferated in the infarcted heart as monitored by both BLI and PET imaging and significantly improved cardiac function.

RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation.

Ras associated with diabetes (RAD) is a membrane protein that acts as a calcium channel regulator by interacting with cardiac L-type Ca channels (LTCC). RAD defects can disrupt intracellular calcium dynamics and lead to cardiac hypertrophy. However, due to the lack of reliable human disease models, the pathological mechanism of RAD deficiency leading to cardiac hypertrophy is not well understood.

Base Editing Mediated Generation of Point Mutations Into Human Pluripotent Stem Cells for Modeling Disease.

Human pluripotent stem cells (hPSCs) are a powerful platform for disease modeling and drug discovery. However, the introduction of known pathogenic mutations into hPSCs is a time-consuming and labor-intensive process. Base editing is a newly developed technology that enables facile introduction of point mutations into specific loci within the genome of living cells.

Generation of a Junctophilin-2 homozygous knockout human embryonic stem cell line (WAe009-A-36) by an episomal vector-based CRISPR/Cas9 system.

Mutations in Junctophilin-2(JPH2) gene is the cause of hypertrophic cardiomyopathy (HCM) and leading inherited cause of left ventricular hypertrophy and myofilaments disarray. JPH2 protein, a member of the Junctophilin family, is mainly expressed in heart and plays an important role in E-C coupling. We have generated a homozygous JPH2 knockout (JPH2-KO) human embryonic stem cell (hESC) line using an episomal vector-based CRISPR/Cas9 system.

Cardiac Ischemic Preconditioning Promotes MG53 Secretion Through HO-Activated Protein Kinase C-δ Signaling.

Background: Ischemic heart disease is the leading cause of morbidity and mortality worldwide. Ischemic preconditioning (IPC) is the most powerful intrinsic protection against cardiac ischemia/reperfusion injury. Previous studies have shown that a multifunctional TRIM family protein, MG53 (mitsugumin 53; also called TRIM72), not only plays an essential role in IPC-mediated cardioprotection against ischemia/reperfusion injury but also ameliorates mechanical damage.