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1.
Stem Cell Res ; 71: 103172, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37535990

RESUMO

Dilated cardiomyopathy (DCM) is a common heart disorder caused by genetic and non-genetic etiologies, characterized by left ventricular dilatation and contractile dysfunction. Here, we created a human induced pluripotent stem cell line from peripheral blood mononuclear cells using non-integrating vectors from a patient carrying a heterozygous LMNA variant (c.481G > A, p.Glu161Lys, NM_170707.4). The obtained EURACi015-A line, showed the typical morphology of pluripotent cells, normal karyotype and exhibited pluripotency markers and a trilineage differentiation potential. This cell line can be successfully differentiated into cardiomyocytes and endothelial cells. This line represents a human in vitro model to study the genetic basis of DCM.


Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Humanos , Cardiomiopatia Dilatada/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Lamina Tipo A/genética , Células Endoteliais/metabolismo , Leucócitos Mononucleares/metabolismo , Mutação
2.
Comput Struct Biotechnol J ; 21: 1759-1773, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36915380

RESUMO

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are commonly used to model arrhythmogenic cardiomyopathy (ACM), a heritable cardiac disease characterized by severe ventricular arrhythmias, fibrofatty myocardial replacement and progressive ventricular dysfunction. Although ACM is inherited as an autosomal dominant disease, incomplete penetrance and variable expressivity are extremely common, resulting in different clinical manifestations. Here, we propose hiPSC-CMs as a powerful in vitro model to study incomplete penetrance in ACM. Six hiPSC lines were generated from blood samples of three ACM patients carrying a heterozygous deletion of exon 4 in the PKP2 gene, two asymptomatic (ASY) carriers of the same mutation and one healthy control (CTR), all belonging to the same family. Whole exome sequencing was performed in all family members and hiPSC-CMs were examined by ddPCR, western blot, Wes™ immunoassay system, patch clamp, immunofluorescence and RNASeq. Our results show molecular and functional differences between ACM and ASY hiPSC-CMs, including a higher amount of mutated PKP2 mRNA, a lower expression of the connexin-43 protein, a lower overall density of sodium current, a higher intracellular lipid accumulation and sarcomere disorganization in ACM compared to ASY hiPSC-CMs. Differentially expressed genes were also found, supporting a predisposition for a fatty phenotype in ACM hiPSC-CMs. These data indicate that hiPSC-CMs are a suitable model to study incomplete penetrance in ACM.

3.
Stem Cell Res ; 55: 102466, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298432

RESUMO

Arrhythmogenic Cardiomyopathy (ACM) is a genetically based cardiomyopathy associated with ventricular arrhythmias and fibro-fatty substitution of cardiac tissue. It is characterized by incomplete penetrance. We generated human iPSCs by episomal reprogramming of blood cells from three members of the same family: the proband, affected by ACM and carrying the heterozygous plakophillin2 p.N346Lfs*12 mutation, one asymptomatic carrier of the same mutation and one apparently healthy control. hiPSCs were characterized according to standard protocols including karyotyping, pluripotency marker expression and differentiation towards the three germ layers. These hiPSC lines can be used to study the mechanisms of ACM incomplete penetrance in vitro.


Assuntos
Cardiomiopatias , Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Heterozigoto , Humanos , Mutação
4.
EMBO Mol Med ; 13(9): e14365, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34337880

RESUMO

Arrhythmogenic cardiomyopathy (ACM) is hallmarked by ventricular fibro-adipogenic alterations, contributing to cardiac dysfunctions and arrhythmias. Although genetically determined (e.g., PKP2 mutations), ACM phenotypes are highly variable. More data on phenotype modulators, clinical prognosticators, and etiological therapies are awaited. We hypothesized that oxidized low-density lipoprotein (oxLDL)-dependent activation of PPARγ, a recognized effector of ACM adipogenesis, contributes to disease pathogenesis. ACM patients showing high plasma concentration of oxLDL display severe clinical phenotypes in terms of fat infiltration, ventricular dysfunction, and major arrhythmic event risk. In ACM patient-derived cardiac cells, we demonstrated that oxLDLs are major cofactors of adipogenesis. Mechanistically, the increased lipid accumulation is mediated by oxLDL cell internalization through CD36, ultimately resulting in PPARγ upregulation. By boosting oxLDL in a Pkp2 heterozygous knock-out mice through high-fat diet feeding, we confirmed in vivo the oxidized lipid dependency of cardiac adipogenesis and right ventricle systolic impairment, which are counteracted by atorvastatin treatment. The modulatory role of oxidized lipids on ACM adipogenesis, demonstrated at cellular, mouse, and patient levels, represents a novel risk stratification tool and a target for ACM pharmacological strategies.


Assuntos
Displasia Arritmogênica Ventricular Direita , Animais , Arritmias Cardíacas/etiologia , Displasia Arritmogênica Ventricular Direita/genética , Humanos , Lipoproteínas LDL , Camundongos , Fenótipo
5.
Artigo em Inglês | MEDLINE | ID: mdl-32457887

RESUMO

Cardiovascular diseases, including myocardial infarction (MI), represent the main worldwide cause of mortality and morbidity. In this scenario, to contrast the irreversible damages following MI, cardiac regeneration has emerged as a novel and promising solution for in situ cellular regeneration, preserving cell behavior and tissue cytoarchitecture. Among the huge variety of natural, synthetic, and hybrid compounds used for tissue regeneration, alginate emerged as a good candidate for cellular preservation and delivery, becoming one of the first biomaterial tested in pre-clinical research and clinical trials concerning cardiovascular diseases. Although promising results have been obtained, recellularization and revascularization of the infarcted area present still major limitations. Therefore, the demand is rising for alginate functionalization and its combination with molecules, factors, and drugs capable to boost the regenerative potential of the cardiac tissue. The focus of this review is to elucidate the promising properties of alginate and to highlight its benefits in clinical trials in relation to cardiac regeneration. The definition of hydrogels, the alginate characteristics, and recent biomedical applications are herewith described. Afterward, the review examines in depth the ongoing developments to refine the material relevance in cardiac recovery and regeneration after MI and presents current clinical trials based on alginate.

6.
Stem Cell Res ; 41: 101656, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31733438

RESUMO

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.


Assuntos
Sistemas CRISPR-Cas , Neurônios Dopaminérgicos/metabolismo , Edição de Genes , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/biossíntese , Células-Tronco Pluripotentes Induzidas/metabolismo , Reação em Cadeia da Polimerase , Transgenes , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Microscopia de Fluorescência
7.
Stem Cell Res ; 32: 78-82, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30219716

RESUMO

Arrhythmogenic Cardiomyopathy (ACM) is an inherited cardiac disease characterized by arrhythmias and fibro-fatty replacement in the ventricular myocardium. Causative mutations are mainly reported in desmosomal genes, especially in plakophilin2 (PKP2). Here, using a virus-free reprogramming approach, we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of one ACM patient carrying the frameshift heterozygous PKP2 mutation c.2569_3018del50. The iPSC line (EURACi004-A) showed the typical morphology of pluripotent cells, possessed normal karyotype and exhibited pluripotency markers and trilineage differentiation potential, including cardiomyogenic capability. Thus, this line can represent a human in vitro model to study the molecular basis of ACM.


Assuntos
Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Placofilinas/genética , Pele/citologia , Arritmias Cardíacas/metabolismo , Cardiomiopatias/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Fibroblastos/metabolismo , Heterozigoto , Humanos , Mutação/genética
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