RESUMO
McArdle disease is a rare autosomal recessive disorder caused by mutations in the PYGM gene. This gene encodes for the skeletal muscle isoform of glycogen phosphorylase (myophosphorylase), the first enzyme in glycogenolysis. Patients with this disorder are unable to obtain energy from their glycogen stored in skeletal muscle, prompting an exercise intolerance. Currently, there is no treatment for this disease, and the lack of suitable in vitro human models has prevented the search for therapies against it. In this article, we have established the first human iPSC-based model for McArdle disease. For the generation of this model, induced pluripotent stem cells (iPSCs) from a patient with McArdle disease (harbouring the homozygous mutation c.148C>T; p.R50* in the PYGM gene) were differentiated into myogenic cells able to contract spontaneously in the presence of motor neurons and generate calcium transients, a proof of their maturity and functionality. Additionally, an isogenic skeletal muscle model of McArdle disease was created. As a proof-of-concept, we have tested in this model the rescue of PYGM expression by two different read-through compounds (PTC124 and RTC13). The developed model will be very useful as a platform for testing drugs or compounds with potential pharmacological activity.
Assuntos
Glicogênio Fosforilase Muscular , Doença de Depósito de Glicogênio Tipo V , Células-Tronco Pluripotentes Induzidas , Humanos , Doença de Depósito de Glicogênio Tipo V/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Glicogênio/metabolismo , TecnologiaRESUMO
Both volumetric muscle loss (VML) and muscle degenerative diseases lead to an important decrease in skeletal muscle mass, condition that nowadays lacks an optimal treatment. This issue has driven towards an increasing interest in new strategies in tissue engineering, an emerging field that can offer very promising approaches. In addition, the discovery of induced pluripotent stem cells (iPSCs) has completely revolutionized the actual view of personalized medicine, and their utilization in skeletal muscle tissue engineering could, undoubtedly, add myriad benefits. In this review, we want to provide a general vision of the basic aspects to consider when engineering skeletal muscle tissue using iPSCs. Specifically, we will focus on the three main pillars of tissue engineering: the scaffold designing, the selection of the ideal cell source and the addition of factors that can enhance the resemblance with the native tissue.
Assuntos
Técnicas de Reprogramação Celular/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Músculo Esquelético/citologia , Regeneração/fisiologia , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Medicina de Precisão/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Alicerces TeciduaisRESUMO
The implementation of induced pluripotent stem cells (iPSCs) in biomedical research more than a decade ago, resulted in a huge leap forward in the highly promising area of personalized medicine. Nowadays, we are even closer to the patient than ever. To date, there are multiple examples of iPSCs applications in clinical trials and drug screening. However, there are still many obstacles to overcome. In this review, we will focus our attention on the advantages of implementing induced pluripotent stem cells technology into the clinics but also commenting on all the current drawbacks that could hinder this promising path towards the patient.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/transplante , Medicina de Precisão/tendências , Humanos , Medicina de Precisão/métodosRESUMO
McArdle disease is a rare autosomal recessive condition caused by mutations in the PYGM gene. This gene encodes the skeletal muscle isoform of glycogen phosphorylase or myophosphorylase. Patients with McArdle disease have an inability to obtain energy from their muscle glycogen stores, which manifests as a marked exercise intolerance. Nowadays, there is no cure for this disorder and recommendations are intended to prevent and mitigate symptoms. There is great heterogeneity among the pathogenic variants found in the PYGM gene, and there is no obvious correlation between genotypes and phenotypes. Here, we present the generation of the first human iPSC-based skeletal muscle model harbouring the second most frequent mutation in PYGM in the Spanish population: NM_005609.4: c.2392T>C (p.Trp798Arg). To this end, iPSCs derived from a McArdle patient and a healthy control were both successfully differentiated into skeletal muscle cells using a small molecule-based protocol. The created McArdle skeletal muscle model was validated by confirming distinctive biochemical aspects of the disease such as the absence of myophosphorylase, the most typical biochemical feature of these patients. This model will be very valuable for use in future high-throughput pharmacological screenings.
RESUMO
Peripheral blood mononuclear cells (PBMCs) from a McArdle patient carrying a homozygous mutation in the PYGM gene: c.2392 T > C; p.Trp798Arg were used for the generation of the human iPSC line, IISHDOi007-A. For the delivery of the reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc, a non-integrative methodology that implies the use of Sendai virus has been applied.
Assuntos
Linhagem Celular , Doença de Depósito de Glicogênio Tipo V , Células-Tronco Pluripotentes Induzidas , Humanos , Fator 4 Semelhante a Kruppel , Leucócitos Mononucleares , Mutação/genéticaRESUMO
Human iPSC line, IISHDOi006-A, was obtained from fibroblasts of a patient with Dominant Optic Atrophy (DOA) carrying a heterozygous mutation in the gene ACO2: c.1999G>A; p.Glu667Lys. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using a non-integrative methodology that involves the use of Sendai virus.
Assuntos
Aconitato Hidratase/genética , Linhagem Celular/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Atrofia Óptica Autossômica Dominante/genética , Aconitato Hidratase/metabolismo , Diferenciação Celular , Linhagem Celular/citologia , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Masculino , Mutação de Sentido Incorreto , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/fisiopatologia , Mutação PuntualRESUMO
A mouse iPSC line, IISHDOi005-A, generated from fibroblasts obtained from a mouse C57BL/6J with an age of 1â¯year and a half, has been obtained. For this purpose, reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using Sendai virus.