289 Highly efficient RNA-based reprogramming of renal epithelial cells derived from recessive dystrophic epidermolysis bullosa patients into induced pluripotent stem cells

Abstract

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a severe skin blistering disease caused by mutations in type VII collagen, resulting in impairment of the anchoring fibrils at the dermal-epidermal junction. There is no cure for RDEB, and current therapy is limited to wound care. Induced pluripotent stem cells (iPSCs) have immense value in furthering the study of this disease and developing new treatment options. The use of iPSCs, however, is limited by the availability of primary cells for reprogramming, the length of transfection regimens, and low reprogramming efficiency. iPSCs can be generated from a variety of cell sources including skin punch biopsies, blood and urine samples. However, due to the severe fragility of RDEB patients’ skin, it is incredibly difficult to draw blood or obtain skin biopsies from these patients. Exfoliated renal epithelial cells (RECs) found in urine samples can provide an alternative, non-invasive and easily accessible source of RDEB somatic cells for reprogramming. Previously published methods for REC reprogramming rely on electroporation of non-integrating plasmids or the use of the Sendai viral vector. These methods require time-consuming regimens and result in poor efficiency of reprogramming. Here, we report a highly efficient, non-integrating RNA-based method for reprogramming of disease-associated RECs into high quality iPSCs with an efficiency and kinetics that surpass all previously published studies. The approach depends on highly – tuned transfections of RECs with reprogramming modified mRNAs and mature miRNA mimics in combination with optimized culturing conditions. The resulting renal-derived iPSCs exhibit normal karyotypes and display a pluripotent phenotype. Thus, our approach is ideal for generating high quality patient-derived iPSCs from a non-invasive source of somatic cells to be used for modeling RDEB and other diseases and for potential clinical applications.