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.
Highlights
Myotonic dystrophies (DMs) represent a group of autosomal dominant multisystemic diseases (Harper, 2001), including DM type 1 and type 2 and manifesting highly variability in term of age at onset, severity of the symptoms, and clinical pictures
The results revealed a clear expression of embryonic marker genes in both DM2 and WT Human induced pluripotent stem cells (hiPSCs), compared to those detected in human embryonic stem cells (HUES-3) (Figure 2)
Previous studies have shown that both MBNL1 and MBNL2 have a lowest relative mRNA level in Embryonic Stem cells (ESCs)/hiPSCs compared to other cells and tissues (Han et al, 2013; Venables et al, 2013), confirming a conserved and prominent role for both genes in ESC-differential alternative splicing (AS)
Summary
Myotonic dystrophies (DMs) represent a group of autosomal dominant multisystemic diseases (Harper, 2001), including DM type 1 and type 2 and manifesting highly variability in term of age at onset, severity of the symptoms, and clinical pictures. In contrast to DM1, DM2 has not been associated with developmental abnormalities and does not cause severe childhood symptoms This difference likely explains why no mental retardation similar to that reported in congenital and juvenile forms of DM1 has been described in DM2 patients. We established, for the first time two DM2 and two healthy hiPSC lines as control from human dermal fibroblasts (HDFs), using lentiviral polycistronic vector containing Yamanaka’s four factors (OCT4, SOX2, KLF4, and cMYC) These cells are able to self-renew indefinitely and to differentiate into neuronal population (NP) maintaining the major specific DM2 hallmarks, such CCTG repeat expansion and CCUG-containing intranuclear RNA foci, which are pathognomonic the disease. The development of a hiPSCsbased platform represents an important tool to study the neurodegenerative and neuromuscular nature of the DM2 mutation and to identify molecular targets for drug design in repeat expansion disorders
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