Generation of iPS cells derived from skin fibroblasts of patients with Fabry disease using RNA-reprogramming

Abstract

Fabry disease is a lysosomal metabolic disorder with X-linked inheritance caused by a deficiency of alpha-galactosidase which leads to accumulation of globotriaosylceramide (Gb3) in various organs. Clinical features of Fabry disease are renal, cardiac and CNS involvement. However, precise cellular pathogenesis in Fabry disease is still unknown. The iPS technology may be useful for the understanding of pathogenetic mechanism of lysosomal storage disease including Fabry disease. The generation of iPS cells generally have been carried out by the use of Sendai virus vector or episomal vector, but these technologies need the environmental attention and equipment for handling viruses. Therefore, we focused on RNA-reprogramming procedure. Skin fibroblasts from control and Fabry disease were transfected with reprogramming factors (Oct4, Sox2, Klf4, cMyc, Nanog, Lin28), interferon factors (B18, E3, K3), RNA and microRNA and formed iPS colonies were obtained after several cell passages and surveyed for morphological and biochemical studies. Using this method, no virus is used and feeder cells are not required, and also iPS cells can be efficiently obtained in a short period of time. Using this method, the iPS cells were successfully established from healthy human skin fibroblasts and skin fibroblasts derived from three Fabry male sibling patients in same family. Expression of surface markers and undifferentiated markers in these iPS cells showed positive, demonstrated by immunostaining and RT-PCR. Generated iPS cells from these Fabry patients were confirmed as iPS cells by various pluripotency markers. Our RNA reprograming procedure to generate iPS cells could be more widely used for understanding of pathogenetic mechanism of various lysosomal diseases including Fabry disease.