Pancreatic Differentiation of Stem Cells Reveals Pathogenesis of a Syndrome of Ketosis-Prone Diabetes

Abstract

Human dermal fibroblasts reprogramming to iPSCsFibroblasts were reprogrammed into iPSCs using a non-integration method based on modified mRNA 1 using a StemRNA reprogramming kit (StemGent; Cambridge, MA). Skin fibroblasts were plated at three different densities on irradiated feeder human dermal fibroblasts and transfected with modified mRNA encoding human OCT4, KLF4, SOX2, LIN28, c-MYC, and control GFP. The transfection was repeated daily over 10-14-day periods as iPSC colonies appeared. Newly derived iPSCs were passaged at least three times before banking. qPCR: RNA was extracted using the RNeasy Mini Kit (Qiagen; Hilden, Germany). cDNA was synthesized using KAPA cDNA Mix, and qPCR was performed using KAPA SYBR FAST (Kapa Biosystems; Indianapolis, Indiana) on a Connect CFX light cycler (Bio-Rad; Hercules, CA) with ≤40 PCR cycles. Using qPrimerDepot, primers were designed so the PCR products spanned exon junctions. Primer specificity was checked using CFX Manager Software v3.1 (Applied Biosystems; Foster City, CA) and PCR product agarose gel electrophoresis. Threshold data were analyzed with the CFX Manager Software v3.1 using the Comparative Ct relative quantitation method with TATA-Box binding protein as an internal control. Human induced pluripotent stem cells (iPSCs) maintenanceThe iPSCs were maintained on Matrigel (BD Biosciences; San Jose, CA) in Essential 8 media (E8, StemCell Technologies; Vancouver, Canada). Cells were passaged every 3-5 days at 80% confluency with TrypLE Express (Invitrogen, Carlsbad, CA). After passage, cells were kept in 10 µM Y-27632 (StemGent) for 24 h.