Disease modeling of neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), is hindered by limited accessibility of affected cells. This problem can be overcome by generation of human induced pluripotent stem cells (hiPSC), which can be then differentiated into required cells. Here, we describe the detailed protocol of hiPSC establishment from peripheral blood mononuclear cells (PBMC) of two ALS patients with detected expansion of G4C2 (GGGGCC) repeats in the first intron of C9ORF72 gene, known to be linked with the most common form of familial ALS.
View Article and Find Full Text PDFCardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) offer an attractive platform to evaluate the mechanisms of cardiovascular-related incidents and to develop and test new drugs for heart diseases. This work focuses on the comparison of two hiPSC-CM differentiation protocols: the GiWi method based on temporal modulation of the Wnt/β-catenin pathway and the commercially available PSC Cardiomyocyte Differentiation Kit. We underlined the need to optimize several parameters such as cell density or small molecule concentration (CHIR-99021, IWR-1) to obtain functional hiPSC-CMs.
View Article and Find Full Text PDFDuchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD.
View Article and Find Full Text PDFBackground: Adult muscle-resident myogenic stem cells, satellite cells (SCs), that play non-redundant role in muscle regeneration, are intrinsically impaired in Duchenne muscular dystrophy (DMD). Previously we revealed that dystrophic SCs express low level of anti-inflammatory and anti-oxidative heme oxygenase-1 (HO-1, HMOX1). Here we assess whether targeted induction of HMOX1 affect SC function and alleviates hallmark symptoms of DMD.
View Article and Find Full Text PDFBecker muscular dystrophy (BMD) is an X-linked recessive disorder caused by in-frame deletions in the dystrophin gene (DMD), leading to progressive muscle degeneration and weakness. We generated a human induced pluripotent stem cell (hiPSC) line from a BMD patient. BMD hiPSCs were then engineered by CRISPR/Cas9-mediated knock-in of missing exons 3-9 of DMD gene.
View Article and Find Full Text PDFAims: Duchenne muscular dystrophy (DMD)-associated cardiomyopathy is a serious life-threatening complication, the mechanisms of which have not been fully established, and therefore no effective treatment is currently available. The purpose of the study was to identify new molecular signatures of the cardiomyopathy development in DMD.
Methods And Results: For modelling of DMD-associated cardiomyopathy, we prepared three pairs of isogenic control and dystrophin-deficient human induced pluripotent stem cell (hiPSC) lines.
Background: Cardiac-abundant microRNA-378a (miR-378a) is associated with postnatal repression of insulin-like growth factor 1 receptor (IGF-1R) controlling physiological hypertrophy and survival pathways. IGF-1/IGF-1R axis has been proposed as a therapeutic candidate against the pathophysiological progress of diabetic cardiomyopathy (DCM). We ask whether hyperglycemia-driven changes in miR-378a expression could mediate DCM progression.
View Article and Find Full Text PDFBackground: Impaired muscle regeneration is a hallmark of Duchenne muscular dystrophy (DMD), a neuromuscular disorder caused by mutations in the DMD gene encoding dystrophin. The lack of heme oxygenase-1 (HO-1, Hmox1), a known anti-inflammatory and cytoprotective enzyme, was shown to aggravate DMD pathology.
Methods: We evaluated the role of HO-1 overexpression in human induced pluripotent stem cell (hiPSC)-derived skeletal muscle cells (hiPSC-SkM) in vitro and in the regeneration process in vivo in wild-type mice.
miR-15a/16-1 cluster, composed of MIR15A and MIR16-1 genes located in close proximity on chromosome 13 was described to regulate post-natal cell cycle withdrawal of cardiomyocytes in mice. In humans, on the other hand, the level of miR-15a-5p and miR-16-p was negatively associated with the severity of cardiac hypertrophy. Therefore, to better understand the role of these microRNAs in human cardiomyocytes in regard to their proliferative potential and hypertrophic growth, we generated hiPSC line with complete deletion of miR-15a/16-1 cluster using CRISPR/Cas9 gene editing.
View Article and Find Full Text PDFDuchenne muscular dystrophy (DMD), originating from the lack of functional dystrophin, clinically manifests as devastating disease of skeletal muscles with progressive cardiac involvement. HMOX1 promoter polymorphism may reflect different activity of heme oxygenase-1 (HO-1) that may be critical for DMD progression. Here we generated human induced pluripotent stem cell (hiPSC) lines from healthy donors-derived peripheral blood mononuclear cells with different variants of HMOX1 promoter (GT repeats), and engineered by CRISPR/Cas9-mediated deletion of exon 50 of DMD gene.
View Article and Find Full Text PDFThe advent of methods for efficient generation and cardiac differentiation of pluripotent stem cells opened new avenues for disease modelling, drug testing, and cell therapies of the heart. However, cardiomyocytes (CM) obtained from such cells demonstrate an immature, foetal-like phenotype that involves spontaneous contractions, irregular morphology, expression of embryonic isoforms of sarcomere components, and low level of ion channels. These and other features may affect cellular response to putative therapeutic compounds and the efficient integration into the host myocardium after in vivo delivery.
View Article and Find Full Text PDFAlthough Duchenne muscular dystrophy (DMD) primarily affects muscle tissues, the alterations to systemic metabolism manifested in DMD patients contribute to the severe phenotype of this fatal disorder. We propose that microRNA-378a (miR-378) alters carbohydrate and lipid metabolism in dystrophic mdx mice. In our study, we utilized double knockout animals which lacked both dystrophin and miR-378 (mdx/miR-378).
View Article and Find Full Text PDFSpinal Muscular Atrophy (SMA) is a genetic neuromuscular disease caused by mutations inSMN1 gene encoding survival motor neuron (SMN) protein. Lack of this protein leads to progressive loss of motor neurons and therefore to gradual loss of signal transmission between motor neurons and skeletal muscle cells. As a consequence, patients develop muscle atrophy and lose the ability to move independently, what is also related to problems with breathing and swallowing.
View Article and Find Full Text PDFThe rising prevalence of diabetes is threatening global health. It is known not only for the occurrence of severe complications but also for the SARS-Cov-2 pandemic, which shows that it exacerbates susceptibility to infections. Current therapies focus on artificially maintaining insulin homeostasis, and a durable cure has not yet been achieved.
View Article and Find Full Text PDFLAMA2-related muscular dystrophy (LAMA2-MD) results from mutations in LAMA2 gene, encoding laminin α-2. It is a congenital disease characterized by muscle wasting, with the most severe version being diagnosed within first few months after birth. To generate LAMA2-DM in vitro model, we excised exon 3 from the LAMA2 gene in our previously derived healthy human induced pluripotent stem cells (hiPSCs).
View Article and Find Full Text PDFmicroRNA-378a (miR-378a) is one of the most highly expressed microRNAs in the heart. However, its role in the human cardiac tissue has not been fully understood. It was observed that miR-378a protects cardiomyocytes from hypertrophic growth by regulation of IGF1R and the expression of downstream kinases.
View Article and Find Full Text PDFDoxorubicin (DOX) is a widely used anticancer drug. However, its clinical use is severely limited due to drug-induced cumulative cardiotoxicity, which leads to progressive cardiomyocyte dysfunction and heart failure. Enormous efforts have been made to identify potential strategies to alleviate DOX-induced cardiotoxicity; however, to date, no universal and highly effective therapy has been introduced.
View Article and Find Full Text PDFDuchenne muscular dystrophy (DMD), caused by a lack of functional dystrophin, is characterized by progressive muscle degeneration. Interestingly, dystrophin is also expressed in endothelial cells (ECs), and insufficient angiogenesis has already been hypothesized to contribute to DMD pathology, however, its status in mice, a model of DMD, is still not fully clear. Our study aimed to reveal angiogenesis-related alterations in skeletal muscles of mice compared to wild-type (WT) counterparts.
View Article and Find Full Text PDFHeme oxygenase-1 (HO-1, encoded by ) is a cytoprotective enzyme degrading heme into CO, Fe, and biliverdin. HO-1 was demonstrated to affect cardiac differentiation of murine pluripotent stem cells (PSCs), regulate the metabolism of murine adult cardiomyocytes, and influence regeneration of infarcted myocardium in mice. However, the enzyme's effect on human cardiogenesis and human cardiomyocytes' electromechanical properties has not been described so far.
View Article and Find Full Text PDFDevelopment of new drugs is of high interest for the field of cardiac and cardiovascular diseases, which are a dominant cause of death worldwide. Before being allowed to be used and distributed, every new potentially therapeutic compound must be strictly validated during preclinical and clinical trials. The preclinical studies usually involve the in vitro and in vivo evaluation.
View Article and Find Full Text PDFG-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear.
View Article and Find Full Text PDFCell therapies are extensively tested to restore heart function after myocardial infarction (MI). Survival of any cell type after intracardiac administration, however, may be limited due to unfavorable conditions of damaged tissue. Therefore, the aim of this study was to evaluate the therapeutic effect of adipose-derived stromal cells (ADSCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing either the proangiogenic SDF-1α or anti-inflammatory heme oxygenase-1 (HO-1) in a murine model of MI.
View Article and Find Full Text PDFInadequate supply of oxygen (O) is a hallmark of many diseases, in particular those related to the cardiovascular system. On the other hand, tissue hypoxia is an important factor regulating (normal) embryogenesis and differentiation of stem cells at the early stages of embryonic development. In culture, hypoxic conditions may facilitate the derivation of embryonic stem cells (ESCs) and the generation of induced pluripotent stem cells (iPSCs), which may serve as a valuable tool for disease modeling.
View Article and Find Full Text PDFSystemically delivered adeno-associated viral vector serotype 9 (AAV9) effectively transduces murine heart, but provides transgene expression also in liver and skeletal muscles. Improvement of the selectivity of transgene expression can be achieved through incorporation of target sites (TSs) for miRNA-122 and miRNA-206 into the 3' untranslated region (3' UTR) of the expression cassette. Here, we aimed to generate such miRNA-122- and miRNA-206-regulated AAV9 vector for a therapeutic, heart-specific overexpression of heme oxygenase-1 (HO-1).
View Article and Find Full Text PDFElevated expression of heme oxygenase-1 (HO-1, encoded by ) is observed in various types of tumors. Hence, it is suggested that HO-1 may serve as a potential target in anticancer therapies. A novel approach to inhibit HO-1 is related to the synthetic lethality of this enzyme and fumarate hydratase (FH).
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