RESUMEN
Spinal muscular atrophy is a progressive motor neuron disorder caused by deletions or point mutations in the SMN1 gene. It is not known why motor neurons are particularly sensitive to a decrease in SMN protein levels and what factors besides SMN2 underlie the high clinical heterogeneity of the disease. Here we studied the methylation patterns of genes on sequential stages of motor neuron differentiation from induced pluripotent stem cells derived from the patients with SMA type I and II. The genes involved in the regulation of pluripotency, neural differentiation as well as those associated with spinal muscular atrophy development were included. The results show that the PAX6, HB9, CHAT, ARHGAP22, and SMN2 genes are differently methylated in cells derived from SMA patients compared to the cells of healthy individuals. This study clarifies the specificities of the disease pathogenesis and extends the knowledge of pathways involved in the SMA progression.
Asunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Neuronas Motoras/fisiología , Atrofia Muscular Espinal/genética , Diferenciación Celular , Células Cultivadas , Metilación de ADN , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Neurogénesis , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Spinal muscular atrophy (SMA) is a genetic disease, which characterized by the degeneration of motor neurons in the spinal cord and further striated muscle atrophy. The research of the processes in diseased neurons is complicated due to the impossibility of obtaining them safely from patients. Thus, we generated SMA type III induced pluripotent stem cell lines via using non-integrated episomal plasmid vectors. The resulting cell line expresses the major pluripotency markers and can differentiate in vitro into derivatives of three germ layers. The iPSC line can be used for further studies by providing in vitro the relevant cell types.
Asunto(s)
Células Madre Pluripotentes Inducidas , Atrofia Muscular Espinal , Atrofias Musculares Espinales de la Infancia , Línea Celular , Humanos , Neuronas Motoras , Atrofia Muscular Espinal/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe and rapidly progressive hereditary muscular disease with X-linked recessive inheritance, occurring mainly in males. A complete loss of dystrophin resulted from out-of-frame deletion mutations in the DMD gene leads to Duchenne muscular dystrophy. DMD induced pluripotent stem cells (iPSCs) are a suitable cell model to study muscle development and disease mechanisms underlying muscular dystrophy and to screen novel compounds with potential therapeutic effects. We generated iPSCs from a DMD patient using non-integrating episomal plasmid vectors. The obtained iPSC lines showed ESC-like morphology, expression pluripotency markers, displayed a normal karyotype and possessed trilineage differentiation potential.
Asunto(s)
Células Madre Pluripotentes Inducidas , Distrofia Muscular de Duchenne , Diferenciación Celular , Distrofina/genética , Humanos , Masculino , Distrofia Muscular de Duchenne/genéticaRESUMEN
Uterine leiomyoma (UL) is the most common benign tumor in women of reproductive age. Gene therapy using suicidal genes appears to be a promising approach for UL treatment. One of key factors for success of gene therapy is the right choice of genetic construct carrier. A promising group of non-viral carriers for cell delivery of expression vectors is cationic Cys-flanked peptides which form tight complexes with DNA due to electrostatic interactions and the presence of interpeptide disulfide bonds. The paper reports a comparative study of the physico-chemical, toxic, and transfectional properties of the DNA-peptide complexes obtained by matrix polymerization or oxidative polycondensation of Cys-flanked peptides using the chain growth terminator 2-amino ethanethiol. We have demonstrated the therapeutic effect of the delivery of the pPTK-1 plasmid carrying the herpes simplex virus type 1 (HSV-1) thymidine kinase gene into PANC-1, and HEK-293T cell culture as well as into primary UL cells. It has been shown that the carriers obtained by oxidative polycondensation transform primary UL cells more efficiently than those produced by matrix polymerization. Treatment with ganciclovir resulted in the death of up to 40% of UL cells transfected with the pPTK-1 plasmid. The perspectives of use of the polyR6 carrier produced by oxidative polycondensation as a tool for the development of modular peptide carriers for the purposes of UL gene therapy were discussed.
Asunto(s)
Genes Transgénicos Suicidas , Terapia Genética , Vectores Genéticos , Leiomioma , Timidina Quinasa , Femenino , Células HEK293 , Humanos , Leiomioma/terapia , Péptidos , Simplexvirus/enzimología , Timidina Quinasa/genéticaRESUMEN
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletion or mutation in SMN1 gene. SMA human induced pluripotent stem cells (iPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. We generated iPSCs from a SMA type I patient and SMA type II patient by using non-integrating episomal plasmid vectors. The resulting iPSCs are episomal-free, express pluripotency markers, display a normal karyotype, retain the mutation (homozygous deletion of SMN1) and are able to differentiate into the three germ layers.