Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells

// Salma Tammam 1, 2, # , Peter Malak 3, # , Daphne Correa 3 , Oliver Rothfuss 3 , Hassan M.E. Azzazy 2 , Alf Lamprecht 1, 4, * , Klaus Schulze-Osthoff 3, 5, * 1 Laboratory of Pharmaceutical Technology and Biopharmaceutics, University of Bonn, 53121 Bonn, Germany 2 Department of Chemistry, The American University in Cairo, 11835 Cairo, Egypt 3 Interfaculty Institute for Biochemistry, University of Tuebingen, 72076 Tuebingen, Germany 4 Laboratory of Pharmaceutical Engineering, University of Franche-Comte, Besancon 25000, France 5 German Cancer Consortium (DKTK) and German Cancer Research Center, 69120 Heidelberg, Germany # Co-first authors, these authors contributed equally to this work * These authors have contributed equally and share senior authorship Correspondence to: Klaus Schulze-Osthoff, email: kso@uni-tuebingen.de Keywords: induced pluripotent stem cells, OCT4, transgene-free stem cells, chitosan nanoparticles, reprogramming Received: March 03, 2016      Accepted: April 16, 2016      Published: May 10, 2016 ABSTRACT Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.