PracticeUpdate Conference Series_WORLDSymposium 2019

RNA Reprogramming Procedure Generates Induced Pluripotent Stem Cells of Patients With Fabry Disease The procedure could be used widely to help reveal the pathogeneticmechanismof various lysosomal diseases. A n RNA reprogramming procedure has been shown to generate induced pluripotent stem cells of patients with Fabry disease. Such RNA The latter then accumulate in body fluids and in the lysosomes of a variety of cell types, including capillary endothelial cells, renal cells (podocytes and tubular, glomerular endothelial, mesangial, and interstitial cells), cardiac cells (cardiomyocytes and fbroblasts), eye cells, and nerve cells.

reprogramming could be useful in uncovering the pathogenesis of numerous lysosomal diseases, fnds a controlled comparative evaluation of the procedure. Mohammad Arif Hossain, PhD, of the Advanced Clinical Research Center in Kawasaki, Kanagawa, Japan, and colleagues focused on reprogramming of RNA. Skin fibroblasts from control patients and those with Fabry disease were transfected with reprogram- ming factors (Oct4, Sox2, Klf4, cMyc, Nanog, Lin28), interferon factors (B18, E3, K3), RNA, and microRNA. Formed induced pluripotent stem cell colonies were obtained after several cell passages and studied morphologically and biochemically. Using this RNA reprogramming method, neither virus nor feeder cells are used. Induced pluripotent stem cells can be obtained efficiently and quickly. Using this method, induced pluripotent stem cells were established successfully from healthy human skin fbroblasts and fbroblasts from patients with Fabry disease. Surface markers and undifferentiated markers were expressed in these induced pluripotent stem cells, as demonstrated by immunostaining and reverse-tran- scriptase polymerase chain reaction. Dr. Hussain explained that Fabry disease is a lyso- somal metabolic disorder with X-linked inheritance caused by a deficiency of α-galactosidase. The deficiency leads to accumulation of globotriao- sylceramide in various organs. Clinical features of Fabry disease are renal, cardiac, and central nerv- ous system involvement. Slow growth (mean height/ weight <50th percentile) in pediatric males affected with Fabry disease is another documented feature of the condition. Patients with partial or complete deficiency of α-galactosidase are unable to effectively degrade glycosphingolipids, globotriaosylceramide, and glycosphingolipid-related compounds, such as gal- abiosylceramide and globotriaosylsphingosine. " Surface markers and undifferentiated markers were expressed in these induced pluripotent stem cells… "

Clinical manifestations in classically affected homozy- gotes with Fabry disease include onset of symptoms during childhood or adolescence, such as pain and paresthesia in the extremities, vessel ectasia (angiokeratoma) in the skin and mucous membranes, hypo- or anhidrosis, abdominal pain, hearing impair- ment, and cornea verticillata. With aging, renal and cardiac impairment may appear. Since enzyme replacement therapy was introduced in 2001, the current biweekly intravenous adminis- tration of recombinant human agalsidase α or β has played a major role in providing comfortable lives for patients with a pathologically missing or functionally impaired GAL gene, leading to an α-galactosidase defcit. Though enzyme replacement therapy limits the sever- ity of the disease and irreversible organ damage, the therapy is accompanied by antibody-mediated side effects. Indeed, patients with high antibody titers tend to show signifcantly higher levels of globotriaosyl- ceramide in urinary samples than patients without antibody production. The precise cellular pathogenesis of Fabry disease remains unknown. The induced pluripotent stem cell technology may be useful for the understanding of pathogenetic mechanism of lysosomal storage disease including Fabry disease. Generation of induced pluripotent stem cells has employed the Sendai virus vector or episomal vector, but these technologies require special environmental handling of viruses. Dr. Hussain concluded that generated induced pluri- potent stem cells from this cohort of patients with Fabry disease were confirmed as such cells using various markers of pluripotency. The RNA reprogramming procedure to generate induced pluripotent stem cells could be used widely with the goal of understanding the pathogenetic mechanisms of various lysosomal diseases, in addi- tion to Fabry disease.

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WORLDSymposium 2019 • PRACTICEUPDATE CONFERENCE SERIES 11