Generation of induced pluripotent stem cell line from a patient suffering from arterial calcification due to deficiency of CD73 (ACDC).

Arterial calcification due to deficiency of CD73 (ACDC) is an adult onset, rare genetic vascular disorder signified by calcium deposition in lower extremity arteries and joints of hands and feet. Mutations in NT5E gene has been shown to be responsible for the inactivation of enzyme CD73 causing calcium buildup. Here, we report a iPSC line generated from a patient showing signs of ACDC and carrying a missense mutation in NT5E (c.1126A→G,p.T376A) gene. This iPSC line shows normal morphology, pluripotency, karyotype, and capability to differentiate into three germ layers, making it useful for disease modeling and investigating pathological mechanisms of ACDC.

Generation of two induced pluripotent stem cell lines from hereditary amyloidosis patients with polyneuropathy carrying heterozygous transthyretin (TTR) mutation.

Hereditary transthyretin amyloidosis with polyneuropathy (ATTR-PN) results from specific TTR gene mutations. In this study, we generated two induced pluripotent stem cell (iPSC) lines derived from ATTR-PN patients with heterozygous TTR gene mutations (Ala97Ser and Phe64Leu). These iPSC lines exhibited normal morphology, karyotype, high pluripotency marker expression, and differentiation into cells representing all germ layers. The generation of these iPSC lines serve as a valuable tool for investigating the mechanisms of ATTR-PN across various cell types and facilitating patient-specific in vitro amyloidosis modeling.

Generation of two induced pluripotent stem cell lines from patients with cardiac amyloidosis carrying heterozygous transthyretin (TTR) mutation.

Specific mutations in the TTR gene are responsible for the development of variant (hereditary) ATTR amyloidosis. Here, we generated two human induced pluripotent stem cell (iPSC) lines from patients diagnosed with Transthyretin Cardiac Amyloidosis (ATTR-CM) carrying heterozygous mutation in the TTR gene (i.e., p.Val30Met). The patient-derived iPSC lines showed expression of high levels of pluripotency markers, trilineage differentiation capacity, and normal karyotype. The generation of these iPSC lines represents a great tool for modeling patient-specific amyloidosis in vitro, allowing the investigation of the pathological mechanisms related to the disease in different cell types and tissues.

Cardiovascular effects of immunosuppression agents.

Immunosuppressive medications are widely used to treat patients with neoplasms, autoimmune conditions and solid organ transplants. Key drug classes, namely calcineurin inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and purine synthesis inhibitors, have direct effects on the structure and function of the heart and vascular system. In the heart, immunosuppressive agents modulate cardiac hypertrophy, mitochondrial function, and arrhythmia risk, while in vasculature, they influence vessel remodeling, circulating lipids, and blood pressure. The aim of this review is to present the preclinical and clinical literature examining the cardiovascular effects of immunosuppressive agents, with a specific focus on cyclosporine, tacrolimus, sirolimus, everolimus, mycophenolate, and azathioprine.

Generation of two induced pluripotent stem cell lines carrying the phospholamban R14del mutation for modeling ARVD/C.

The phospholamban (PLN) R14del mutation is associated with arrhythmogenic right ventricular dysplasia (ARVD/C). ARVD/C is a cardiac disease characterized by arrhythmias and structural abnormalities in the right ventricle. Because PLN is a regulator of calcium release, this mutation can have deleterious effects on tissue integrity and contraction. This mutation is a trinucleotide (AGA) deletion that leads to an arginine deletion at position 14 of the PLN structure. Here we show two lines carrying this mutation with typical iPSC morphology, pluripotency, karyotype, ability to differentiate into the three germ layers in vitro, and readily availability for studying pathological mechanisms or ARVD/C.

Coronary Artery Vasospasm Requiring Cardiac Autotransplantation Yet Controlled With Tobacco.

Coronary artery vasospasm is typically managed through avoidance of triggers and with symptomatic treatments with calcium channel blockers and long-acting nitrates. Here, we report a rare case of medically refractory coronary artery vasospasm associated with genetic predispositions that initially required cardiac autotransplantation followed paradoxically by nicotine for long-term symptomatic control. (Level of Difficulty: Intermediate.).

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.

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.

Modeling Secondary Iron Overload Cardiomyopathy with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model.

The growing number of victims of "stem cell tourism," the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy. External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.

RATIONALE: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. OBJECTIVE: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. METHODS AND RESULTS: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonß1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonß1 treatment. CONCLUSIONS: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

Modeling inherited cardiac disorders.

Advances in the understanding and treatment of cardiac disorders have been thwarted by the inability to study beating human cardiac cells in vitro. Induced pluripotent stem cells (iPSCs) bypass this hurdle by enabling the creation of patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). These cells provide a unique platform to study cardiac diseases in vitro, especially hereditary cardiac conditions. To date, iPSC-CMs have been used to successfully model arrhythmic disorders, showing excellent recapitulation of cardiac channel function and electrophysiologic features of long QT syndrome types 1, 2, 3, and 8, and catecholaminergic polymorphic ventricular tachycardia (CPVT). Similarly, iPSC-CM models of dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) have shown robust correlation of predicted morphologic, contractile, and electrical phenotypes. In addition, iPSC-CMs have shown some features of the respective phenotypes for arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), LEOPARD syndrome, Pompe's disease, and Friedriech's ataxia. In this review, we examine the progress of utilizing iPSC-CMs as a model for cardiac conditions and analyze the potential for the platform in furthering the biology and treatment of cardiac disorders.

Cardiac stem cell biology: glimpse of the past, present, and future.

Cardiac regeneration strategies and de novo generation of cardiomyocytes have long been significant areas of research interest in cardiovascular medicine. In this review, we outline a variety of common cell sources and methods used to regenerate cardiomyocytes and highlight the important role that key Circulation Research articles have played in this flourishing field.

Embryonic stem cell biology: insights from molecular imaging.

Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.

Laminin alpha-1, alpha-3, and alpha-5 chain expression in human prepubertal [correction of prepubetal] benign prostate glands and adult benign and malignant prostate glands.

BACKGROUND: Laminins (Lns) are a family of extracellular matrix glycoproteins located in the basement membrane (BM) of epithelial cells. They exist as heterotrimers composed of an alpha, beta, and gamma chain. Presently, five alpha (alpha1-5), three beta (beta1-3), and three gamma (gamma1-3) chains have been identified with different combinations of these chains resulting in 14 laminin heterotrimers thus far identified (1, 3-5). METHODS: In this study, using immunohistochemistry with chain-specific antibodies, we characterized the expression of the alpha1 (Lns-1/3), alpha3 (Lns 5,6,7), and alpha5 (Lns 10/11) chains in fetal, newborn, infant, prepubertal, and adult benign and malignant prostate glands. RESULTS: In general, alpha1 expression was higher in normal fetal prostate glands and declined by full-term birth, whereas the alpha3 and alpha5 chains remained highly expressed in the adult normal glands. In carcinoma alpha1 (Lns 1/3) and alpha5 (Lns 5,6,7) are lost, whereas alpha5 (Lns 10/11) persists. CONCLUSIONS: Alpha 1 (Lns 1/3) is prominent in BM, but is replaced by a laminin matrix rich in alpha3 (Lns 5,6,7) and alpha5 (Lns 10/11) in benign adult prostate glands. In carcinoma, both alpha1 (Lns-1/3) and alpha3 (Lns 5,6,7) are not expressed with persistence of a BM rich in alpha5 (Lns 10/11).

Different phenotypes in human prostate cancer: alpha6 or alpha3 integrin in cell-extracellular adhesion sites.

The distribution of alpha6/alpha3 integrin in adhesion complexes at the basal membrane in human normal and cancer prostate glands was analyzed in 135 biopsies from 61 patients. The levels of the polarized alpha6/alpha3 integrin expression at the basal membrane of prostate tumor glands were determined by quantitative immunohistochemistry. The alpha6/alpha3 integrin expression was compared with Gleason sum score, pathological stage, and preoperative serum prostate-specific antigen (PSA). The associations were assessed by statistical methods. Eighty percent of the tumors expressed the alpha6 or alpha3 integrin and 20% was integrin-negative. Gleason sum score, but not serum PSA, was associated with the integrin expression. Low Gleason sum score correlated with increased integrin expression, high Gleason sum score with low and negative integrin expression. Three prostate tumor phenotypes were distinguished based on differential integrin expression. Type I coexpressed both alpha6 and alpha3 subunits, type II exclusively expressed alpha6 integrin, and type III expressed alpha3 integrin only. Fifteen cases were further examined for the codistribution of vinculin, paxillin, and CD 151 on frozen serial sections using confocal laser scanning microscopy. The alpha6/alpha3 integrins, CD151, paxillin, and vinculin were present within normal glands. In prostate carcinoma, alpha6 integrin was colocalized with CD 151, but not with vinculin or paxillin. In tumor phenotype I, the alpha6 subunit did not colocalize with the alpha subunit indicating the existence of two different adhesion complexes. Human prostate tumors display on their cell surface the alpha6beta1 and/or alpha3beta1 integrins. Three tumor phenotypes associated with two different adhesion complexes were identified, suggesting a reorganization of cell adhesion structures in prostate cancer.