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. hESCs were induced into pancreatic progenitor cells in a five-stage differentiation protocol. A high-performance liquid chromatography-mass spectrometry platform was used to undertake comprehensive proteome and phosphoproteome profiling of cells at different stages. A series of bioinformatic explorations, including coregulated modules, gene regulatory networks, and phosphosite enrichment analysis, were then conducted. A total of 27,077 unique phosphorylated sites and 8122 proteins were detected, including several cyclin-dependent kinases at the initial stage of cell differentiation. Furthermore, we discovered that ERK1, a member of the MAPK cascade, contributed to proliferation at an early stage. Finally, Western blotting confirmed that the phosphosites from SIRT1 and CHEK1 could inhibit the corresponding substrate abundance in the late stage. Thus, this study extends our understanding of the molecular mechanism during pancreatic cell development.

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. Finally, by integrating these results, we analyzed the relationship between telomerase activity and the expression of marker genes/proteins as well as the cell type at each study time point. This dynamic quantitative measurement of cellular telomerase activity should be a promising indicator for monitoring dynamic changes in a stage of hiPSC differentiation and inducing cell types. This study provided a quantitative, dynamic and simple monitoring index for the in vitro differentiation process of hiPSC-CMs, which was a certain reference value for the optimization and improvement of the induction system.

The pseudoenzyme ADPRHL1 affects cardiac function by regulating the ROCK pathway.

BACKGROUND: Pseudoenzymes, catalytically deficient variants of active enzymes, have a wide range of regulatory functions. ADP-ribosylhydrolase-like 1 (ADPRHL1), a pseudoenzyme belonging to a small group of ADP-ribosylhydrolase enzymes that lacks the amino acid residues necessary for catalytic activity, may have a significant role in heart development based on accumulating evidence. However, the specific function of ADPRHL1 in this process has not been elucidated. To investigate the role of ADPRHL1 in the heart, we generated the first in vitro human embryonic stem cell model with an ADPRHL1 knockout. METHOD: Using the CRISPR/Cas9 system, we generated ADPRHL1 knockout in the human embryonic stem cell (hESC) H9 line. The cells were differentiated into cardiomyocytes using a chemically defined and xeno-free method. We employed confocal laser microscopy to detect calcium transients and microelectrode array (MEA) to assess the electrophysiological activity of ADPRHL1 deficiency cardiomyocytes. Additionally, we investigated the cellular mechanism of ADPRHL1 by Bulk RNA sequencing and western blot. RESULTS: The results indicate that the absence of ADPRHL1 in cardiomyocytes led to adhered abnormally, as well as perturbations in calcium transients and electrophysiological activity. We also revealed that disruption of focal adhesion formation in these cardiomyocytes was due to an excessive upregulation of the ROCK-myosin II pathway. Notably, inhibition of ROCK and myosin II effectively restores focal adhesions in ADPRHL1-deficient cardiomyocytes and improved electrical conduction and calcium activity. CONCLUSIONS: Our findings demonstrate that ADPRHL1 plays a critical role in maintaining the proper function of cardiomyocytes by regulating the ROCK-myosin II pathway, suggesting that it may serve as a potential drug target for the treatment of ADPRHL1-related diseases.

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. This protocol review aims to highlight different strategies for generating hCOs using hPSCs while also critically discussing their challenges and limitations.

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. The WAe009-A-88 line maintained stem cell-like morphology, expressed high levels of pluripotent markers, had a normal karyotype, and could differentiate into all three germ layers in vivo. The cell line can serve as valuable tools for modeling LQT2 in vitro and investigating the pathological mechanisms related to KCNH2 mutations.

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 (Ca2+/calmodulin-dependent kinase II) plays a pivotal role in the pathogenesis of severe heart conditions, including I/R injury. Pharmacological inhibition of CaMKII is an important strategy in the protection against myocardial damage and cardiac diseases. To date, there is no drug targeting CaMKII for the clinical therapy of heart disease. Furthermore, at present, there is no selective inhibitor of CaMKII-δ, the major CaMKII isoform in the heart. METHODS: A small-molecule kinase inhibitor library and a high-throughput screening system for the kinase activity assay of CaMKII-δ9 (the most abundant CaMKII-δ splice variant in human heart) were used to screen for CaMKII-δ inhibitors. Using cultured neonatal rat ventricular myocytes, human embryonic stem cell-derived cardiomyocytes, and in vivo mouse models, in conjunction with myocardial injury induced by I/R (or hypoxia/reoxygenation) and CaMKII-δ9 overexpression, we sought to investigate the protection of hesperadin against cardiomyocyte death and cardiac diseases. BALB/c nude mice with xenografted tumors of human cancer cells were used to evaluate the in vivo antitumor effect of hesperadin. RESULTS: Based on the small-molecule kinase inhibitor library and screening system, we found that hesperadin, an Aurora B kinase inhibitor with antitumor activity in vitro, directly bound to CaMKII-δ and specifically blocked its activation in an ATP-competitive manner. Hesperadin functionally ameliorated both I/R- and overexpressed CaMKII-δ9-induced cardiomyocyte death, myocardial damage, and heart failure in both rodents and human embryonic stem cell-derived cardiomyocytes. In addition, in an in vivo BALB/c nude mouse model with xenografted tumors of human cancer cells, hesperadin delayed tumor growth without inducing cardiomyocyte death or cardiac injury. CONCLUSIONS: Here, we identified hesperadin as a specific small-molecule inhibitor of CaMKII-δ with dual functions of cardioprotective and antitumor effects. These findings not only suggest that hesperadin is a promising leading compound for clinical therapy of cardiac I/R injury and heart failure, but also provide a strategy for the joint therapy of cancer and cardiovascular disease caused by anticancer treatment.

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. The WAe009-A-74 line maintained stem cell like morphology, pluripotency, normal karyotype and could differentiate into all three germ layers in vivo.

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

BACKGROUND: CaMKII (Ca2+/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. However, it is unknown whether specific inhibition of the detrimental CaMKII-δ9 prevents cardiac I/R injury and, if so, what is the underlying mechanism. Here, we aim to investigate the cardioprotective effect of specific CaMKII-δ9 inhibition against myocardial I/R damage and determine the underlying mechanisms. METHODS: The role and mechanism of CaMKII-δ9 in cardiac I/R injury were investigated in mice in vivo, neonatal rat ventricular myocytes, and human embryonic stem cell-derived cardiomyocytes. RESULTS: We demonstrate that CaMKII-δ9 inhibition with knockdown or knockout of its feature exon, exon 16, protects the heart against I/R-elicited injury and subsequent heart failure. I/R-induced cardiac inflammation was also ameliorated by CaMKII-δ9 inhibition, and compared with the previously well-studied CaMKII-δ2, CaMKII-δ9 overexpression caused more profound cardiac inflammation. Mechanistically, in addition to IKKß (inhibitor of NF-κB [nuclear factor-κB] kinase subunit ß), CaMKII-δ9, but not δ2, directly interacted with IκBα (NF-κB inhibitor α) with its feature exon 13-16-17 combination and increased IκBα phosphorylation and consequently elicited more pronounced activation of NF-κB signaling and inflammatory response. Furthermore, the essential role of CaMKII-δ9 in myocardial inflammation and damage was confirmed in human cardiomyocytes. CONCLUSIONS: We not only identified CaMKII-δ9-IKK/IκB-NF-κB signaling as a new regulator of human cardiomyocyte inflammation but also demonstrated that specifically targeting CaMKII-δ9, the most abundant CaMKII-δ splice variant in human heart, markedly suppresses I/R-induced cardiac NF-κB activation, inflammation, and injury and subsequently ameliorates myocardial remodeling and heart failure, providing a novel therapeutic strategy for various ischemic heart diseases.

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. We describe an optimized stepwise protocol to introduce disease-specific mutations of long QT syndrome (LQTs) into hPSCs. We highlight technical issues, especially those associated with introducing a point mutation to obtain isogenic hPSCs without inserting any resistance cassette and reproducible cardiomyocyte differentiation. Based on the protocol, we succeeded in getting hPSCs carrying LQTs pathogenic mutation with excellent efficiency (31.7% of heterozygous clones, 9.1% of homozygous clones) in less than 20 days. In addition, we also provide protocols to analyze electrophysiological of hPSC-derived cardiomyocytes using multi-electrode arrays. This protocol is also applicable to introduce other disease-specific mutations into hPSCs.

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. Our data demonstrated that CYP-induced a prolonged cardiac QT interval and electromechanical coupling time courses accompanied by JPH2 downregulation. Moreover, N6-methyladenosine (m6A) methylation sequencing and RNA sequencing suggested that CYP induced cardiotoxicity by dysregulating calcium signaling. Importantly, our results demonstrated that CYP induced an increase in the m6A level of JPH2 mRNA by upregulating methyltransferases METTL3, leading to the reduction of JPH2 expression levels, as well as increased field potential duration and action potential duration in cardiomyocytes. Our results revealed a novel mechanism for m6A methylation-dependent regulation of JPH2, which provides new strategies for the treatment and prevention of CYP-induced cardiotoxicity.

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

[Figure: see text].

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. This cell line offers an in vitro efficient platform to explore pathogenic mechanisms in XLAS and provides a cell-based disease model for drug testing.

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. This generated cell line presented typical stem cells colony morphology, maintained highly pluripotency and normal karyotype, also was able to differentiate into all three germ layers in vivo.

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. METHODS: The KCNH2 was knocked out in the human embryonic stem cell (hESC) H9 line using the CRISPR/Cas9 system. Using a chemically defined differentiation protocol, we obtained and verified hERG-deficient CMs. Subsequently, high-throughput microelectrode array (MEA) assays and drug interventions were performed to characterise the electrophysiological signatures of hERG-deficient cell lines. RESULTS: Our results showed that KCNH2 knockout did not affect the pluripotency or differentiation efficiency of H9 cells. Using high-throughput MEA assays, we found that the electric field potential duration and action potential duration of hERG-deficient CMs were significantly longer than those of normal CMs. The hERG-deficient lines also exhibited irregular rhythm and some early afterdepolarisations. Moreover, we used the hERG-deficient human CM model to evaluate the potency of agents (nifedipine and magnesium chloride) that may ameliorate the phenotype. CONCLUSIONS: We established an hERG-deficient human CM model that exhibited QT prolongation, irregular rhythm and sensitivity to other ion channel blockers. This model serves as an important tool that can aid in understanding the fundamental impact of hERG dysfunction, elucidate the genotype-phenotype relationship of hERG deficiency and facilitate drug development.

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. The TBX5-KO maintained stem cell like morphology, pluripotency markers, normal karyotype, and could differentiate into all three germ layers in vivo. This cell line can provide an in vitro platform for studying the pathogenic mechanisms and biological function of TBX5 in the heart development.

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. METHODS: We induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors. RESULTS: HESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genes PAX6, NESTIN, SOX1, and SOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express ß-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway. CONCLUSIONS: This new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons.

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. Here, we used the CRISPR/Cas9 editing system to establish a homozygous COX6A2 knockout (COX6A2-KO) human embryonic stem cell (hESC) line. This COX6A2-KO hESC has normal morphology, pluripotency, and karyotype, which can differentiate into three germ layers in vivo.

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 Nav1.5 Na+ channel. Herein, we have generated a human embryonic stem cell line (WAe009-A-48) carrying a LQTS related mutation in SCN5A (WAe009-A-48). The WAe009-A-48 line maintained stem cell like morphology, pluripotency, normal karyotype and could differentiate into all three germ layers in vivo.

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. Under ischaemic conditions, TF-hUiCMs enhanced cardiomyocyte (CM) glucose metabolism and promoted angiogenic activity. CONCLUSION: This study established a CRISPR/Cas9-mediated multimodality reporter gene imaging system that can determine the dynamics and function of TF-hUiCMs in myocardial infarction, which is helpful for investigating the application of stem cell therapy.

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 Ca2 + 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. In this study, we created a RRAD -/- H9 cell line using CRISPR/Cas9 technology. RAD disruption did not affect the ability and efficiency of cardiomyocytes differentiation. However, RAD deficient hESC-CMs recapitulate hypertrophic phenotype in vitro. Further studies have shown that elevated intracellular calcium level and abnormal calcium regulation are the core mechanisms by which RAD deficiency leads to cardiac hypertrophy. More importantly, management of calcium dysregulation has been found to be an effective way to prevent the development of cardiac hypertrophy in vitro.