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.

Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids

“…Human dermal fibroblasts were reprogrammed into human-induced pluripotent stem cells (hiPSCs), as previously reported [ 34 , 35 , 36 , 37 ]. A healthy donor subject (wild type, WT, 46 XX, age 46) has been recruited for dermal biopsy.…”

Natriuretic peptides are neuroprotective on in vitro models of PD and promote dopaminergic differentiation of hiPSCs-derived neurons via the Wnt/β-catenin signaling

“…5A ). hiPSCs from both donors have been differentiated into DA neurons [ 59 ] as described in the Materials and Methods, resumed in Supplementary Table 1 and schematized in Fig. 5A .…”

“… A hiPSCs obtained from two different healthy donors (Donor A and Donor B) were used. Neural induction (day 1–12) and dopaminergic commitment (day 12–30) have been obtained as previously reported [ 59 ], and described in the Materials and Methods section. Complete dopaminergic neuronal differentiation was reached at day 43 of culture in presence of cAMP.…”

“…The stemness propriety and karyotype analysis have also been assessed (not shown). HiPSCs have been then differentiated into DA neurons by using an embryoid body (EB) formation protocol [ 59 , 72 ], on low attachment cell culture plates, as schematized in Fig. 5A .…”

Effects of Simulated Microgravity on Wild Type and Marfan hiPSCs-Derived Embryoid Bodies

“…Human fibroblasts reprogramming was performed, as previously reported. 37,38 hiPSC colonies were picked 20-25 days post infection on the basis of morphology and expanded by plating on mitomycin C-treated MEFs in hiPSCs medium. Successively, hiPS lines MFS and WT were manually picked, passaged on human embryonic stem cell-qualified Matrigel-coated plates (0.05 mg/mL; BD Biosciences) and cultured under feeder-free condition in mTeSR1 medium (Stem Cell Technologies) with Y-27632 ROCK inhibitor (Stemcell Technologies), maintaining the stability over 20 and more passages.…”