While allogenic islet grafting has shown significant promise for the treatment of T1D, donor tissue scarcity and the need for aggressive immunosuppression has limited widespread use of this therapy. Consequently, recent studies have focused on deriving β-like cells from autologoussources (e.g., exocrine pancreas, iPSCs) via reprogramming and/or lengthy differentiation protocols. However, current approaches to β-cell derivation face several hurdles, including limited and/or functionally impaired cell sources, tedious pre-processing steps, and heavy reliance on viral vectors (i.e., for cell transformations), among others. Here we report on the development ofa non-viral approach to β-cell derivation from human skin fibroblasts via direct reprogramming.Human fibroblasts were isolated from surgical discard tissue. Electroporation was then used to screen a pool of transcription factor genes that included Pdx1, Ngn3, MafA, Pax4, and Tcf3, to identify optimum permutations that can drive β-cell-directed conversions in human skin fibroblast cultures. Transfection extent, efficiencies, and transcriptive activity, were evaluated by qRT-PCR,fluorescence microscopy, and RNA sequencing. Transfected fibroblasts were maintained indifferentiation media for ~3 weeks. β-cell-directed transformations were evaluated in terms of morphology and C-peptide expression. Our results indicate that while transfection with Tcf3 alone did not lead to significant changes in cell morphology and phenotype compared to sham, co-transfection of Tcf3 with Pdx1+Ngn3+MafA (PNM) or Pdx1+Ngn3+MafA+Pax4 (PNMP) led to cell clustering, as well as C-peptide expression as early as day 15 post-transfection, which was absent in fibroblasts transfected only with PNM or PNMP. Altogether, our findings indicate that Tcf3 maybe a key factor in enabling direct conversion of human dermal fibroblasts into hormone-expressingβ-like cells for potential applications in β-cell replacement therapies.


L.R. Lemmerman: None. M. Rincon-Benavides: None. S.A. Tersey: None. B.N. Blackstone: None. H.M. Powell: None. N. Higuita-Castro: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. D. Gallego-Perez: None.

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