RESUMEN
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.
RESUMEN
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.
Asunto(s)
Glucógeno Fosforilasa de Forma Muscular , Enfermedad del Almacenamiento de Glucógeno Tipo V , Células Madre Pluripotentes Inducidas , Humanos , Enfermedad del Almacenamiento de Glucógeno Tipo V/genética , Células Madre Pluripotentes Inducidas/metabolismo , Glucógeno/metabolismo , TecnologíaRESUMEN
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.
Asunto(s)
Línea Celular , Enfermedad del Almacenamiento de Glucógeno Tipo V , Células Madre Pluripotentes Inducidas , Humanos , Factor 4 Similar a Kruppel , Leucocitos Mononucleares , Mutación/genéticaRESUMEN
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.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/trasplante , Medicina de Precisión/tendencias , Humanos , Medicina de Precisión/métodosRESUMEN
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.
Asunto(s)
Aconitato Hidratasa/genética , Línea Celular/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Atrofia Óptica Autosómica Dominante/genética , Aconitato Hidratasa/metabolismo , Diferenciación Celular , Línea Celular/citología , Reprogramación Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Masculino , Mutación Missense , Atrofia Óptica Autosómica Dominante/metabolismo , Atrofia Óptica Autosómica Dominante/fisiopatología , Mutación PuntualRESUMEN
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.
Asunto(s)
Técnicas de Reprogramación Celular/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Músculo Esquelético/citología , Regeneración/fisiología , Ingeniería de Tejidos/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Medicina de Precisión/métodos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Andamios del TejidoRESUMEN
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.