Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells
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Salma Tammam1,2,#, Peter Malak3,#, Daphne Correa3, Oliver Rothfuss3, Hassan M.E. Azzazy2, Alf Lamprecht1,4,*, Klaus Schulze-Osthoff3,5,*
1Laboratory of Pharmaceutical Technology and Biopharmaceutics, University of Bonn, 53121 Bonn, Germany
2Department of Chemistry, The American University in Cairo, 11835 Cairo, Egypt
3Interfaculty Institute for Biochemistry, University of Tuebingen, 72076 Tuebingen, Germany
4Laboratory of Pharmaceutical Engineering, University of Franche-Comté, Besançon 25000, France
5German 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
Klaus Schulze-Osthoff, email: [email protected]
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
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.
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