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Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2.

ABSTRACT: Human induced pluripotent stem cells (hiPSCs)-patient specific are an innovative tool to reproduce a model of disease in vitro and summarize the pathological phenotype and the disease etiopathology. Myotonic dystrophy type 2 (DM2) is caused by an unstable (CCTG)n expansion in intron 1 of the CNBP gene, leading to a progressive multisystemic disease with muscle, heart and central nervous dysfunctions. The pathogenesis of CNS involvement in DM2 is poorly understood since no cellular or animal models fully recapitulate the molecular and clinical neurodegenerative phenotype of patients. In this study, we generated for the first time, two DM2 and two wild type hiPSC lines from dermal fibroblasts by polycistronic lentiviral vector (hSTEMCCA-loxP) expressing OCT4, SOX2, KLF4, and cMYC genes and containing loxP-sites, excisable by Cre recombinase. Specific morphological, molecular and immunocytochemical markers have confirmed the stemness of DM2 and wild type-derived hiPSCs. These cells are able to differentiate into neuronal population (NP) expressing tissue specific markers. hiPSCs-derived NP cells maintain (CCTG)n repeat expansion and intranuclear RNA foci exhibiting sequestration of MBNL1 protein, which are pathognomonic of the disease. DM2 hiPSCs represent an important tool for the study of CNS pathogenesis in patients, opening new perspectives for the development of cell-based therapies in the field of personalized medicine and drug screening.

SUBMITTER: Spitalieri P

PROVIDER: S-EPMC6074094 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2.

Spitalieri Paola P Talarico Rosa V RV Murdocca Michela M Fontana Luana L Marcaurelio Marzia M Campione Elena E Massa Roberto R Meola Giovanni G Serafino Annalucia A Novelli Giuseppe G Sangiuolo Federica F Botta Annalisa A

Frontiers in physiology 20180727

Human induced pluripotent stem cells (hiPSCs)-patient specific are an innovative tool to reproduce a model of disease <i>in vitro</i> and summarize the pathological phenotype and the disease etiopathology. Myotonic dystrophy type 2 (DM2) is caused by an unstable (CCTG)n expansion in intron 1 of the <i>CNBP</i> gene, leading to a progressive multisystemic disease with muscle, heart and central nervous dysfunctions. The pathogenesis of CNS involvement in DM2 is poorly understood since no cellular ...[more]

PMID: 30100878

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Project description:IntroductionThe present study had four aims. First, neuronal injury markers, including neurofilament light (NF-L), total tau, glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase (UCH-L1), were compared between individuals with and without adult-onset myotonic dystrophy type 1 (DM1). Second, the impact of age and CTG repeat on brain injury markers was evaluated. Third, change in brain injury markers across the study period was quantified. Fourth, associations between brain injury markers and cerebral white matter (WM) fractional anisotropy (FA) were identified.MethodsYearly assessments, encompassing blood draws and diffusion tensor imaging on a 3T scanner, were conducted on three occasions. Neuronal injury markers were quantified using single molecule array (Simoa).ResultsThe sample included 53 patients and 70 controls. NF-L was higher in DM1 patients than controls, with individuals in the premanifest phases of DM1 (PreDM1) exhibiting intermediate levels ( χ(2)2= 38.142, P < 0.001). Total tau was lower in DM1 patients than controls (Estimate = -0.62, 95% confidence interval [CI] -0.95: -0.28, P < 0.001), while GFAP was elevated in PreDM1 only (Estimate = 30.37, 95% CI 10.56:50.19, P = 0.003). Plasma concentrations of UCH-L1 did not differ between groups. The age by CTG interaction predicted NF-L: patients with higher estimated progenitor allelege length (ePAL) had higher NF-L at a younger age, relative to patients with lower CTG repeat; however, the latter exhibited faster age-related change (Estimate = -0.0021, 95% CI -0.0042: -0.0001, P = 0.045). None of the markers changed substantially over the study period. Finally, cerebral WM FA was significantly associated with NF-L (Estimate = -42.86, 95% CI -82.70: -3.02, P = 0.035).InterpretationWhile NF-L appears sensitive to disease onset and severity, its utility as a marker of progression remains to be determined. The tau assay may have low sensitivity to tau pathology associated with DM1.

Project description:Sleep disturbances such as excessive daytime sleepiness, central and obstructive sleep apneas, restless legs syndrome, and rapid eye movement sleep dysregulation are prominent in patients with myotonic dystrophy type 1 (DM1). Mild intellectual deficits presented in many patients with DM1. In addition, psychosocial issues caused by neuropsychiatric symptoms are a clinical problem. We herein present the cases of four DM1 patients with sleep disturbances and neuropsychiatric symptoms in the preceding stage of clinically significant muscle symptoms. One of the cases exhibited a sleep disorder and neuropsychiatric symptoms before electromyography showed myotonic discharge, suggesting that careful follow-up is also important. Patients 1 and 2 were first referred to our department due to daytime sleepiness. Patients 3 and 4 were objectively suffering from daytime sleepiness of which they were not subjectively aware of. Patients 1, 3, and 4 obtained high apnea-hypopnea index (AHI) scores, which reflected central and/or obstructive apnea, whereas patient 2 had an AHI score of zero. The daytime cerebrospinal fluid (CSF) orexin levels of all patients ranged from the normal lower limit to low, although they were not as low as those observed in narcolepsy with typical cataplexy. Neuropsychological tests of patients 1 and 2 showed frontal lobe dysfunction. Patients 3 and 4 were diagnosed with mild intellectual disability and autism spectrum disorder, respectively. All patients exhibited indifference toward their own symptoms, which may have resulted from the cognitive decline caused by DM1. Based on family history and/or neurological findings such as myotonia, we suspected DM1 as the cause of their sleep disturbances. Molecular analysis using the triplet repeat-primed polymerase chain reaction (TP PCR) method and Southern blotting, which provided a genetic confirmation of the diagnosis of DM1, were performed. These clinical features of sleep disturbances were unrelated to the length of CTG repeats and are caused by unknown molecular mechanisms. Clinicians should take into account that multisystem involvement in DM1 is hugely variable, and thus, a disabling sleep disorder could overshadow muscle impairment in DM1 patients.

Dataset Information

Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2.

Publications

Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2.

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