Browsing by Author "Madeira, Cristiana Raquel Rodrigues"
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- Generation of a novel Cockayne syndrome B neuronal culture model from patient-derived induced pluripotent stem cellsPublication . Madeira, Cristiana Raquel Rodrigues; Matos, Carlos; Calado, SofiaCockayne syndrome type B (CSB) is a rare genetic, multisystem disorder, which results from accumulation of DNA damage that arises when the associated gene, ERCC6, is dysfunctional. This leads to premature aging and neurodegeneration. Studies on CSB pathophysiology are lacking, and pre-clinical research of potential therapeutic strategies is nonexistent. Patients with CSB are left without any chance of disease cure or delay, experiencing premature death. Patient-derived induced pluripotent stem cells (iPSCs) offer a powerful tool to investigate disease mechanisms while generating possibilities for treatment. As a result, our goal was to generate iPSCs from the fibroblasts of CSB patient, as an in vitro model, and differentiate them into neural cell cultures that exhibit CSB-related neuropathological alterations. To achieve this goal, human patient fibroblasts were reprogramed into CSB-iPSCs with the CytoTune iPS 2.0 Sendai Reprogramming Kit. CSB-iPSCs and fibroblasts genetic profile analysis confirmed relatedness between cell lines. The ERCC6 gene was sequenced demonstrating to have the expected mutation, and Western blot analysis further confirmed the presence of a pathogenic mutation. PCR showed that CSB-iPSCs are free of reprograming virus at passage 10 and cultured cells did not contain mycoplasma contamination. Through immunohistochemistry and flow cytometry, CSB-iPSCs demonstrated to express pluripotency markers and capacity to differentiate into the three germ layers. CSB fibroblasts displayed an accumulation of dysfunctional mitochondria, and their DNA repair capacity was affected, upon UV light exposure, as observed through flow cytometry and immunocytochemistry, respectively. This suggests that CSB cells have a dysfunctional transcription-coupled nucleotide excision repair (TC-NER) and mtDNA repair. CSB-iPSCs were successfully differentiated into neurons and CSB-Neurons appeared to have longer neuronal prolongations that neurons derived from a healthy donor, indicating premature maturation. Our study suggests that the in vitro CSB model generated can help understand this disorder and contribute to the pre-clinical testing of disease-modifying therapies.