Publications by authors named "Joseph C Wu"

531 Publications

Deciphering pathogenicity of variants of uncertain significance with CRISPR-edited iPSCs.

Trends Genet 2021 Sep 8. Epub 2021 Sep 8.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Division of Cardiology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Genetic variants play an important role in conferring risk for cardiovascular diseases (CVDs). With the rapid development of next-generation sequencing (NGS), thousands of genetic variants associated with CVDs have been identified by genome-wide association studies (GWAS), but the function of more than 40% of genetic variants is still unknown. This gap of knowledge is a barrier to the clinical application of the genetic information. However, determining the pathogenicity of a variant of uncertain significance (VUS) is challenging due to the lack of suitable model systems and accessible technologies. By combining clustered regularly interspaced short palindromic repeats (CRISPR) and human induced pluripotent stem cells (iPSCs), unprecedented advances are now possible in determining the pathogenicity of VUS in CVDs. Here, we summarize recent progress and new strategies in deciphering pathogenic variants for CVDs using CRISPR-edited human iPSCs.
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http://dx.doi.org/10.1016/j.tig.2021.08.009DOI Listing
September 2021

Leaders in Cardiovascular Research: Joseph C. Wu.

Cardiovasc Res 2021 Aug;117(10):e126-e128

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.

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http://dx.doi.org/10.1093/cvr/cvab252DOI Listing
August 2021

Highlights from Stanford Drug Discovery Symposium 2021.

Cardiovasc Res 2021 08;117(10):e132-e134

Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA.

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http://dx.doi.org/10.1093/cvr/cvab250DOI Listing
August 2021

Generation of three induced pluripotent stem cell lines (SCVIi014-A, SCVIi015-A, and SCVIi016-A) from patients with LQT1 caused by heterozygous mutations in the KCNQ1 gene.

Stem Cell Res 2021 Aug 5;55:102492. Epub 2021 Aug 5.

Stanford Cardiovascular Institute, United States; Department of Medicine, Division of Cardiovascular Medicine, United States; Department of Radiology, Stanford University School of Medicine, United States. Electronic address:

Congenital long QT syndrome type 1 (LQT1) results from KCNQ1 mutations that cause loss of Kv7.1 channel function, leading to arrhythmias, syncope, and sudden cardiac death. Here, we generated three human-induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of LQT1 patients carrying pathogenic variants (c.569 G>A, c.585delG, and c.573_577delGCGCT) in KCNQ1. All lines show typical iPSC morphology, high expression of pluripotent markers, normal karyotype, and are able to differentiate into three germ layers in vitro. These lines are valuable resources for studying the pathological mechanisms of LQT1 caused by KCNQ1 mutations.
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http://dx.doi.org/10.1016/j.scr.2021.102492DOI Listing
August 2021

Generation of three induced pluripotent stem cell lines from hypertrophic cardiomyopathy patients carrying MYH7 mutations.

Stem Cell Res 2021 Aug 12;55:102455. Epub 2021 Jul 12.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

MYH7 heterozygous mutations are common genetic causes of hypertrophic cardiomyopathy (HCM). HCM is characterized by hypertrophy of the left ventricle and diastolic dysfunction. We generated three human induced pluripotent stem cell (iPSC) lines from three HCM patients each carrying a single heterozygous mutation in MYH7, c.2167C > T, c.4066G > A, and c.5135G > A, respectively. All lines expressed high levels of pluripotent markers, had normal karyotype, and possessed capability of differentiation into derivatives of the three germ layers, which can serve as valuable tools for modeling HCM in vitro and investigating the pathological mechanisms related to MYH7 mutations.
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http://dx.doi.org/10.1016/j.scr.2021.102455DOI Listing
August 2021

CRISPRi/a Screening with Human iPSCs.

Methods Mol Biol 2021 ;2320:261-281

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.

Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.
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http://dx.doi.org/10.1007/978-1-0716-1484-6_23DOI Listing
August 2021

Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular non-compaction.

Eur Heart J 2021 Jul 19. Epub 2021 Jul 19.

Department of Biology, Stanford University, Stanford, CA 94305, USA.

Aims: Non-compaction cardiomyopathy is a devastating genetic disease caused by insufficient consolidation of ventricular wall muscle that can result in inadequate cardiac performance. Despite being the third most common cardiomyopathy, the mechanisms underlying the disease, including the cell types involved, are poorly understood. We have previously shown that endothelial cell-specific deletion of the chromatin remodeller gene Ino80 results in defective coronary vessel development that leads to ventricular non-compaction in embryonic mouse hearts. We aimed to identify candidate angiocrines expressed by endocardial and ECs inwildtype and LVNC conditions in Tie2Cre;Ino80fl/fl transgenic embryonic mouse hearts, and test the effect of these candidates on cardiomyocyte proliferation and maturation.

Methods And Results: We used single-cell RNA-sequencing to characterize endothelial and endocardial defects in Ino80-deficient hearts. We observed a pathological endocardial cell population in the non-compacted hearts and identified multiple dysregulated angiocrine factors that dramatically affected cardiomyocyte behaviour. We identified Col15A1 as a coronary vessel-secreted angiocrine factor, downregulated by Ino80-deficiency, that functioned to promote cardiomyocyte proliferation. Furthermore, mutant endocardial and endothelial cells (ECs) up-regulated expression of secreted factors, such as Tgfbi, Igfbp3, Isg15, and Adm, which decreased cardiomyocyte proliferation and increased maturation.

Conclusions: These findings support a model where coronary ECs normally promote myocardial compaction through secreted factors, but that endocardial and ECs can secrete factors that contribute to non-compaction under pathological conditions.
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http://dx.doi.org/10.1093/eurheartj/ehab298DOI Listing
July 2021

Macrophages: Potential Therapeutic Target of Myocardial Injury in COVID-19.

Circ Res 2021 06 24;129(1):47-49. Epub 2021 Jun 24.

Stanford Cardiovascular Institute (M.N., J.C.W.), Stanford University School of Medicine, CA.

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http://dx.doi.org/10.1161/CIRCRESAHA.121.319446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221538PMC
June 2021

The role of metabolism in directed differentiation versus trans-differentiation of cardiomyocytes.

Semin Cell Dev Biol 2021 May 29. Epub 2021 May 29.

Stanford Cardiovascular Institute, Stanford University, School of Medicine, USA; Division of Cardiovascular Medicine, Department of Medicine, Stanford University, School of Medicine, USA; Department of Radiology, Stanford University, School of Medicine, USA. Electronic address:

The advent of induced pluripotent stem cells (iPSCs) and identification of transcription factors for cardiac reprogramming have raised hope to cure heart disease, the leading cause of death in the world. Our knowledge in heart development and molecular barriers of cardiac reprogramming is advancing, but many hurdles are yet to be overcome for clinical translation. Importantly, we lack a full understanding of molecular mechanisms governing cell fate conversion toward cardiomyocytes. In this review, we will discuss the role of metabolism in directed differentiation versus trans-differentiation of cardiomyocytes. Cardiomyocytes exhibit a unique metabolic feature distinct from PSCs and cardiac fibroblasts, and there are multiple overlapping molecular mechanisms underlying metabolic reprogramming during cardiomyogenesis. We will discuss key metabolic changes occurring during cardiomyocytes differentiation from PSCs and cardiac fibroblasts, and the potential role of metabolic reprogramming in the enhancement strategies for cardiomyogenesis. Only when such details are discovered will more effective strategies to enhance the de novo production of cardiomyocytes be possible.
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http://dx.doi.org/10.1016/j.semcdb.2021.05.018DOI Listing
May 2021

Generation of three heterozygous KCNH2 mutation-carrying human induced pluripotent stem cell lines for modeling LQT2 syndrome.

Stem Cell Res 2021 07 20;54:102402. Epub 2021 May 20.

Stanford Cardiovascular Institute, United States; Depart of Medicine, Division of Cardiovascular Medicine, United States; Department of Radiology, Stanford University School of Medicine, United States. Electronic address:

Congenital long QT syndrome type 2 (LQT2) results from KCNH2 mutations that cause loss of Kv11.1 channel function which can lead to arrhythmias, syncope, and sudden death. Here, we generated three human-induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of two LQT2 patients carrying pathogenic variants (c.1714G > A and c.2960del) and one LQT2 patient carrying a variant of uncertain significance (c.1870A > T) in KCNH2. All lines show typical iPSC morphology, high expression of pluripotent markers, normal karyotype, and differentiate into three germ layers in vitro. These lines are valuable resources for studying the pathological mechanisms of LQTS caused by caused by KCNH2 mutations.
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http://dx.doi.org/10.1016/j.scr.2021.102402DOI Listing
July 2021

Human induced pluripotent stem cell-derived atrial cardiomyocytes carrying an SCN5A mutation identify nitric oxide signaling as a mediator of atrial fibrillation.

Stem Cell Reports 2021 Jun 20;16(6):1542-1554. Epub 2021 May 20.

Division of Cardiology, Department of Medicine, Chicago, IL, USA; Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA; Jesse Brown Veterans Administration Medical Center, Chicago, IL, USA. Electronic address:

Mutations in SCN5A, encoding the cardiac sodium channel, are linked with familial atrial fibrillation (AF) but the underlying pathophysiologic mechanisms and implications for therapy remain unclear. To characterize the pathogenesis of AF-linked SCN5A mutations, we generated patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) from two kindreds carrying SCN5A mutations (E428K and N470K) and isogenic controls using CRISPR-Cas9 gene editing. We showed that mutant AF iPSC-aCMs exhibited spontaneous arrhythmogenic activity with beat-to-beat irregularity, prolonged action potential duration, and triggered-like beats. Single-cell recording revealed enhanced late sodium currents (I) in AF iPSC-aCMs that were absent in a heterologous expression model. Gene expression profiling of AF iPSC-aCMs showed differential expression of the nitric oxide (NO)-mediated signaling pathway underlying enhanced I. We showed that patient-specific AF iPSC-aCMs exhibited striking in vitro electrophysiological phenotype of AF-linked SCN5A mutations, and transcriptomic analyses supported that the NO signaling pathway modulated the I and triggered AF.
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http://dx.doi.org/10.1016/j.stemcr.2021.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190590PMC
June 2021

Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes.

Stem Cell Reports 2021 Sep 20;16(9):2169-2181. Epub 2021 May 20.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CCSR Room 4215, 269 Campus Drive, Stanford, CA 94305-5175, USA; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA. Electronic address:

Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention.
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http://dx.doi.org/10.1016/j.stemcr.2021.04.018DOI Listing
September 2021

Antitumor effects of iPSC-based cancer vaccine in pancreatic cancer.

Stem Cell Reports 2021 Jun 6;16(6):1468-1477. Epub 2021 May 6.

Stanford Cardiovascular Institute, Stanford University, Stanford, CA 94305, USA; Department of Medicine, Division of Cardiovascular Medicine, Stanford University, 265 Campus Drive, Stanford, CA 94305, USA. Electronic address:

Induced pluripotent stem cells (iPSCs) and cancer cells share cellular similarities and transcriptomic profiles. Here, we show that an iPSC-based cancer vaccine, comprised of autologous iPSCs and CpG, stimulated cytotoxic antitumor CD8 T cell effector and memory responses, induced cancer-specific humoral immune responses, reduced immunosuppressive CD4 T regulatory cells, and prevented tumor formation in 75% of pancreatic ductal adenocarcinoma (PDAC) mice. We demonstrate that shared gene expression profiles of "iPSC-cancer signature genes" and others are overexpressed in mouse and human iPSC lines, PDAC cells, and multiple human solid tumor types compared with normal tissues. These results support further studies of iPSC vaccination in PDAC in preclinical and clinical models and in other cancer types that have low mutational burdens.
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http://dx.doi.org/10.1016/j.stemcr.2021.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190592PMC
June 2021

Reconstructing the heart using iPSCs: Engineering strategies and applications.

J Mol Cell Cardiol 2021 08 22;157:56-65. Epub 2021 Apr 22.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94025, USA; Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94025, USA. Electronic address:

Induced pluripotent stem cells (iPSCs) have emerged as a key component of cardiac tissue engineering, enabling studies of cardiovascular disease mechanisms, drug responses, and developmental processes in human 3D tissue models assembled from isogenic cells. Since the very first engineered heart tissues were introduced more than two decades ago, a wide array of iPSC-derived cardiac spheroids, organoids, and heart-on-a-chip models have been developed incorporating the latest available technologies and materials. In this review, we will first outline the fundamental biological building blocks required to form a functional unit of cardiac muscle, including iPSC-derived cells differentiated by soluble factors (e.g., small molecules), extracellular matrix scaffolds, and exogenous biophysical maturation cues. We will then summarize the different fabrication approaches and strategies employed to reconstruct the heart in vitro at varying scales and geometries. Finally, we will discuss how these platforms, with continued improvements in scalability and tissue maturity, can contribute to both basic cardiovascular research and clinical applications in the future.
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http://dx.doi.org/10.1016/j.yjmcc.2021.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8378256PMC
August 2021

A Call to Action for New Global Approaches to Cardiovascular Disease Drug Solutions.

Circulation 2021 Jul 20;144(2):159-169. Epub 2021 Apr 20.

Universite´ de Lorraine, INSERM CIC 1493, INI CRCT, CHRU, Nancy, France (F.Z.).

While we continue to wrestle with the immense challenge of implementing equitable access to established evidence-based treatments, substantial gaps remain in our pharmacotherapy armament for common forms of cardiovascular disease including coronary and peripheral arterial disease, heart failure, hypertension, and arrhythmia. We need to continue to invest in the development of new approaches for the discovery, rigorous assessment, and implementation of new therapies. Currently, the time and cost to progress from lead compound/product identification to the clinic, and the success rate in getting there reduces the incentive for industry to invest, despite the enormous burden of disease and potential size of market. There are tremendous opportunities with improved phenotyping of patients currently batched together in syndromic "buckets." Use of advanced imaging and molecular markers may allow stratification of patients in a manner more aligned to biological mechanisms that can, in turn, be targeted by specific approaches developed using high-throughput molecular technologies. Unbiased "omic" approaches enhance the possibility of discovering completely new mechanisms in such groups. Furthermore, advances in drug discovery platforms, and models to study efficacy and toxicity more relevant to the human disease, are valuable. Re-imagining the relationships among discovery, translation, evaluation, and implementation will help reverse the trend away from investment in the cardiovascular space, establishing innovative platforms and approaches across the full spectrum of therapeutic development.
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http://dx.doi.org/10.1161/CIR.0000000000000981DOI Listing
July 2021

A call to action for new global approaches to cardiovascular disease drug solutions.

Eur Heart J 2021 04;42(15):1464-1475

Université de Lorraine, INSERM CIC 1493, INI CRCT, CHRU Nancy, France.

Whilst we continue to wrestle with the immense challenge of implementing equitable access to established evidence-based treatments, substantial gaps remain in our pharmacotherapy armament for common forms of cardiovascular disease including coronary and peripheral arterial disease, heart failure, hypertension, and arrhythmia. We need to continue to invest in the development of new approaches for the discovery, rigorous assessment, and implementation of new therapies. Currently, the time and cost to progress from lead compound/product identification to the clinic, and the success rate in getting there reduces the incentive for industry to invest, despite the enormous burden of disease and potential size of market. There are tremendous opportunities with improved phenotyping of patients currently batched together in syndromic 'buckets'. Use of advanced imaging and molecular markers may allow stratification of patients in a manner more aligned to biological mechanisms that can, in turn, be targeted by specific approaches developed using high-throughput molecular technologies. Unbiased 'omic' approaches enhance the possibility of discovering completely new mechanisms in such groups. Furthermore, advances in drug discovery platforms, and models to study efficacy and toxicity more relevant to the human disease, are valuable. Re-imagining the relationships among discovery, translation, evaluation, and implementation will help reverse the trend away from investment in the cardiovascular space, establishing innovative platforms and approaches across the full spectrum of therapeutic development.
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http://dx.doi.org/10.1093/eurheartj/ehab068DOI Listing
April 2021

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

JCI Insight 2021 04 8;6(7). Epub 2021 Apr 8.

Stanford Cardiovascular Institute.

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. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.
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http://dx.doi.org/10.1172/jci.insight.142000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119193PMC
April 2021

Generation of Vascular Smooth Muscle Cells From Induced Pluripotent Stem Cells: Methods, Applications, and Considerations.

Circ Res 2021 Mar 4;128(5):670-686. Epub 2021 Mar 4.

Stanford Cardiovascular Institute (M.S., T.Q., M.P.F., J.C.W.).

The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.318049DOI Listing
March 2021

Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity.

Front Pharmacol 2021 10;12:613837. Epub 2021 Mar 10.

Stanford Cardiovascular Institute, Stanford University, Stanford, CA, United States.

Evaluation of potential vascular injury is an essential part of the safety study during pharmaceutical development. Vascular liability issues are important causes of drug termination during preclinical investigations. Currently, preclinical assessment of vascular toxicity primarily relies on the use of animal models. However, accumulating evidence indicates a significant discrepancy between animal toxicity and human toxicity, casting doubt on the clinical relevance of animal models for such safety studies. While the causes of this discrepancy are expected to be multifactorial, species differences are likely a key factor. Consequently, a human-based model is a desirable solution to this problem, which has been made possible by the advent of human induced pluripotent stem cells (iPSCs). In particular, recent advances in the field now allow the efficient generation of a variety of vascular cells (e.g., endothelial cells, smooth muscle cells, and pericytes) from iPSCs. Using these cells, different vascular models have been established, ranging from simple 2D cultures to highly sophisticated vascular organoids and microfluidic devices. Toxicity testing using these models can recapitulate key aspects of vascular pathology on molecular (e.g., secretion of proinflammatory cytokines), cellular (e.g., cell apoptosis), and in some cases, tissue (e.g., endothelium barrier dysfunction) levels. These encouraging data provide the rationale for continuing efforts in the exploration, optimization, and validation of the iPSC technology in vascular toxicology.
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http://dx.doi.org/10.3389/fphar.2021.613837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006367PMC
March 2021

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells.

J Vis Exp 2021 03 14(169). Epub 2021 Mar 14.

Stanford Cardiovascular Institute, Stanford University School of Medicine; Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine; Department of Radiology, Stanford University School of Medicine;

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.
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http://dx.doi.org/10.3791/61879DOI Listing
March 2021

ALDH1A3 Coordinates Metabolism With Gene Regulation in Pulmonary Arterial Hypertension.

Circulation 2021 May 25;143(21):2074-2090. Epub 2021 Mar 25.

Vera Moulton Wall Center for Pulmonary Vascular Diseases (D.L., J-R.M., S.O., L.W., T.N., E.Y., M.R.), Stanford University School of Medicine, CA.

Background: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC.

Methods: RNA sequencing was applied to pulmonary arterial SMC (PASMC) from PAH patients with and without a BMPR2 (bone morphogenetic receptor 2) mutation versus control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (, an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC.

Results: mRNA and protein were increased in PAH versus control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with deleted in SMC did not develop hypoxia-induced pulmonary arterial muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl coenzyme A to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at NFYA (nuclear transcription factor Y subunit α) binding sites via the acetyltransferase KAT2B (lysine acetyltransferase 2B) and permitted NFY-mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of and in PAH PASMC was β-catenin dependent.

Conclusions: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not endothelial cells, is an important therapeutic consideration.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.048845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8289565PMC
May 2021

Generation of two heterozygous MYBPC3 mutation-carrying human iPSC lines, SCVIi001-A and SCVIi002-A, for modeling hypertrophic cardiomyopathy.

Stem Cell Res 2021 05 11;53:102279. Epub 2021 Mar 11.

Stanford Cardiovascular Institute, Stanford University School of Medicine, CA, USA; Division of Cardiovascular Medicine, Depart of Medicine, Stanford University School of Medicine, CA, USA; Department of Radiology, Stanford University School of Medicine, CA, USA. Electronic address:

Hypertrophic cardiomyopathy (HCM) is an inherited heart disease that can cause sudden cardiac death and heart failure. HCM often arises from mutations in sarcomeric genes, among which the MYBPC3 is the most frequently mutated. Here we generated two human induced pluripotent stem cell (iPSC) lines from a HCM patient who has a familial history of HCM and his daughter who carries the pathogenic non-coding mutation. All lines show the typical morphology of pluripotent cells, a high expression of pluripotency markers, normal karyotype, and in vitro capacity to differentiate into all three germ layers. These lines provide a valuable resource for studying the molecular basis of HCM and drug screening for HCM.
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http://dx.doi.org/10.1016/j.scr.2021.102279DOI Listing
May 2021

Generation of three induced pluripotent stem cell lines, SCVIi003-A, SCVIi004-A, SCVIi005-A, from patients with ARVD/C caused by heterozygous mutations in the PKP2 gene.

Stem Cell Res 2021 05 12;53:102284. Epub 2021 Mar 12.

Stanford Cardiovascular Institute, Stanford University, School of Medicine, United States; Division of Cardiovascular Medicine, Depart of Medicine, Stanford University, School of Medicine, United States; Department of Radiology, Stanford University, School of Medicine, United States. Electronic address:

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease which can cause life-threatening ventricular arrhythmias and cardiac dysfunction. The autosomal dominant form of ARVD/C is caused by mutations in the cardiac desmosome, such as those in the plakoglobin plakophilin-2 (PKP2) gene. Here, we generated three human induced pluripotent stem cell (iPSC) lines from the peripheral blood mononuclear cells (PBMCs) of three ARVD/C patients carrying pathogenic variants in their PKP2 genes (c.2065_2070delinsG; c.235C>T; c.1725_1728dup). All lines show the typical morphology of pluripotent stem cells, demonstrate high expression of pluripotent markers, display normal karyotype, and differentiate into all three germ layers in vitro. These lines are valuable resources for studying the pathological mechanisms of ARVD/C caused by PKP2 mutation.
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http://dx.doi.org/10.1016/j.scr.2021.102284DOI Listing
May 2021

Race and Genetics in Congenital Heart Disease: Application of iPSCs, Omics, and Machine Learning Technologies.

Front Cardiovasc Med 2021 17;8:635280. Epub 2021 Feb 17.

Stanford Cardiovascular Institute, Stanford University, Stanford, CA, United States.

Congenital heart disease (CHD) is a multifaceted cardiovascular anomaly that occurs when there are structural abnormalities in the heart before birth. Although various risk factors are known to influence the development of this disease, a full comprehension of the etiology and treatment for different patient populations remains elusive. For instance, racial minorities are disproportionally affected by this disease and typically have worse prognosis, possibly due to environmental and genetic disparities. Although research into CHD has highlighted a wide range of causal factors, the reasons for these differences seen in different patient populations are not fully known. Cardiovascular disease modeling using induced pluripotent stem cells (iPSCs) is a novel approach for investigating possible genetic variants in CHD that may be race specific, making it a valuable tool to help solve the mystery of higher incidence and mortality rates among minorities. Herein, we first review the prevalence, risk factors, and genetics of CHD and then discuss the use of iPSCs, omics, and machine learning technologies to investigate the etiology of CHD and its connection to racial disparities. We also explore the translational potential of iPSC-based disease modeling combined with genome editing and high throughput drug screening platforms.
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http://dx.doi.org/10.3389/fcvm.2021.635280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925393PMC
February 2021

Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes.

STAR Protoc 2021 Mar 9;2(1):100334. Epub 2021 Feb 9.

Utrecht Regenerative Medicine Center, Circulatory Health Laboratory, University Utrecht, Department of Cardiology, University Medical Center Utrecht, 3508 GA Utrecht, the Netherlands.

Since the discovery of human induced pluripotent stem cells (hiPSCs), numerous strategies have been established to efficiently derive cardiomyocytes from hiPSCs (hiPSC-CMs). Here, we describe a cost-effective strategy for the subsequent massive expansion (>250-fold) of high-purity hiPSC-CMs relying on two aspects: removal of cell-cell contacts and small-molecule inhibition with CHIR99021. The protocol maintains CM functionality, allows cryopreservation, and the cells can be used in downstream assays such as disease modeling, drug and toxicity screening, and cell therapy. For complete details on the use and execution of this protocol, please refer to Buikema (2020).
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http://dx.doi.org/10.1016/j.xpro.2021.100334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881265PMC
March 2021

Air pollution exposure is linked with methylation of immunoregulatory genes, altered immune cell profiles, and increased blood pressure in children.

Sci Rep 2021 Feb 18;11(1):4067. Epub 2021 Feb 18.

Sean N. Parker Center for Allergy and Asthma Research at Stanford University, Stanford, CA, 94305, USA.

Ambient air pollution exposure is associated with cardiovascular dysregulation and immune system alterations, yet no study has investigated both simultaneously in children. Understanding the multifaceted impacts may provide early clues for clinical intervention prior to actual disease presentation. We therefore determined the associations between exposure to multiple air pollutants and both immunological outcomes (methylation and protein expression of immune cell types associated with immune regulation) and cardiovascular outcomes (blood pressure) in a cohort of school-aged children (6-8 years; n = 221) living in a city with known elevated pollution levels. Exposure to fine particular matter (PM), carbon monoxide (CO), and ozone (O) was linked to altered methylation of most CpG sites for genes Foxp3, IL-4, IL-10 and IFN-g, all involved in immune regulation (e.g. higher PM exposure 1 month prior to the study visit was independently associated with methylation of the IL-4 CpG24 site (est = 0.16; P = 0.0095). Also, immune T helper cell types (Th1, Th2 and Th17) were associated with short-term exposure to PM, O and CO (e.g. Th1 cells associated with PM at 30 days: est = - 0.34, P < 0.0001). Both B cells (est = - 0.19) and CD4+ cells (est = 0.16) were associated with 1 day NO2 exposure (P ≤ 0.031), whereas CD4+ and CD8+ cells were associated with chronic exposure to PAH, NOx and/or NO (P ≤ 0.038 for all). Finally, diastolic BP (DBP) was inversely associated with long-term exposures to both CO and PAH, and both systolic and pulse pressure were associated with short-term NO and chronic NOx exposure. Our findings demonstrate links between air pollution exposure and methylation of immunoregulatory genes, immune cell profiles and blood pressure, suggesting that even at a young age, the immune and cardiovascular systems are negatively impacted by exposure to air pollution.
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http://dx.doi.org/10.1038/s41598-021-83577-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893154PMC
February 2021

CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.

Sci Rep 2021 Feb 4;11(1):3026. Epub 2021 Feb 4.

Stanford University, Stanford, CA, USA.

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.
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http://dx.doi.org/10.1038/s41598-021-81860-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862643PMC
February 2021

Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes.

Cell Stem Cell 2021 05 1;28(5):938-954.e9. Epub 2021 Feb 1.

Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19014, USA; Department of Cell and Developmental Biology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19014, USA; Penn Cardiovascular Institute, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19014, USA; Institute for Regenerative Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19014, USA. Electronic address:

Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types.
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http://dx.doi.org/10.1016/j.stem.2020.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106635PMC
May 2021

Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness.

Cell Chem Biol 2021 May 26;28(5):625-635.e5. Epub 2021 Jan 26.

Sanford-Burnham-Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA; University of California, San Diego, San Diego, CA 92093, USA; Cardiovascular Institute, Stanford University, 240 Pasteur Drive, Palo Alto, CA 94305, USA; Department of Medicine, Stanford University, 240 Pasteur Drive, Palo Alto, CA 94305, USA. Electronic address:

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through β-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.
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http://dx.doi.org/10.1016/j.chembiol.2021.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140986PMC
May 2021
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