Generation of hiPSCs from Type-I Diabetic Fibroblasts

Adult human Type-I diabetic (T1D) donor fibroblasts obtained with patient informed consent, were purchased from DV Biologics, and cultured in fibroblast culture medium (I-Gro medium, DV Biologics). Episomal expression of SOX2, OCT4, KLF4, c-MYC, NANOG, LIN28, SV40LT was performed by nucleofection of 1x10⁶ diabetic fibroblast cells with 2 μg each of three plasmids, pCEP4-EO2S-EN2L, pCEP4-EO2S-ET2K, and pCEP4-EO2S-EM2K^{27 (link),28 (link)}. Single fibroblast cells were obtained with Accutase, and nucleofected using the human dermal fibroblast nucleofector kit (Lonza, VPD-1001) and Amaxa nucleofector program U-023. Nucleofected cells were transferred onto irradiated MEF in fibroblast growth medium supplemented with 10 μM Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor Y27362 (Stemgent). The next day, 2 mL of DMEM/F-12 supplemented with 20% KOSR, 0.1 mM MEM NEAA, 1 mM L-Glutamine, 0.1 mM β-mercaptoethanol, 50 ng/mL bFGF, 10 μM Y27362, 5 μg/mL ascorbic acid, and 3 μM CHIR99021 was added. Half of the medium was replaced with fresh medium without Y27362 every other day, until hiPSC colonies appeared. Individual hiPSC colonies were manually isolated, expanded onto vitronectin-coated plates in E8 medium, or further expanded and cryopreserved.