Induced pluripotent stem cells as genetic disease models

Duarte, Ana Joana; Bragança, José; Amaral, Olga

http://hdl.handle.net/10400.18/5344

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Authors

Duarte, Ana Joana

Bragança, José

Amaral, Olga

Abstract(s)

Lysosomal storage disorders (LSDs) are a group of genetic diseases characterised by lysosomal dysfunction. Some of the commonest LSDs are currently treated by enzyme replacement therapy. However, particularly in cases of advanced disease or late onset, results are discouraging. The lack of good ex vivo models hinders R&D and delays the understanding of the human pathophysiologic mechanisms. Thus, using iPSCs methods to generate the cell-targets to reproduce the disease, might help create ideal models for studying pathogenic mechanisms and to find new or more effective therapeutic strategies. iPSCs generated from somatic cells from patients are a necessary source for patient-specific studies since they maintain the patient’s genetic background. Material and Methods: Using commercially obtained skin fibroblasts, as a control, guarantees better consistency in technical conditions. In this study we used two different methods to achieve forced expression of Oct4, Sox2, Klf4 and c-Myc: a non-integrative polycistronic plasmid vector and the Sendai virus method Transformation conditions with different vehicles of delivery were tested: different reagents, concentration ratios and timings were compared. Posterior validation of cells pluripotent state is currently underway. Results: Fibroblasts are very difficult to transform but colonies were observed at around three weeks post-transfection using plasmid DNA. The Sendai virus method proved to be easier and faster. Aims: Currently we are generating iPSCs from human skin fibroblasts and intend to obtain a good cellular model for LSDs.