title: Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.
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.
authors.affiliation: Laboratory of Pharmaceutical Technology and Biopharmaceutics, University of Bonn, 53121 Bonn, Germany.|Department of Chemistry, The American University in Cairo, 11835 Cairo, Egypt.
authors.forename: Salma
authors.initials: S
authors.lastname: Tammam
authors.affiliation: Interfaculty Institute for Biochemistry, University of Tuebingen, 72076 Tuebingen, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Malak
authors.affiliation: Interfaculty Institute for Biochemistry, University of Tuebingen, 72076 Tuebingen, Germany.
authors.forename: Daphne
authors.initials: D
authors.lastname: Correa
authors.affiliation: Interfaculty Institute for Biochemistry, University of Tuebingen, 72076 Tuebingen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Rothfuss
authors.affiliation: Department of Chemistry, The American University in Cairo, 11835 Cairo, Egypt.
authors.forename: Hassan M E
authors.initials: HM
authors.lastname: Azzazy
authors.affiliation: Laboratory of Pharmaceutical Technology and Biopharmaceutics, University of Bonn, 53121 Bonn, Germany.|Laboratory of Pharmaceutical Engineering, University of Franche-Comté, 25000 Besançon, France.
authors.forename: Alf
authors.initials: A
authors.lastname: Lamprecht
authors.affiliation: Interfaculty Institute for Biochemistry, University of Tuebingen, 72076 Tuebingen, Germany.|German Cancer Consortium (DKTK) and German Cancer Research Center, 69120 Heidelberg, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Schulze-Osthoff
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D065150:Cellular Reprogramming; D048271:Chitosan / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D007313:Insecta; D000090062:Kruppel-Like Factor 4; D053758:Nanoparticles / Q000737:chemistry; D019913:Nuclear Localization Signals; D050814:Octamer Transcription Factor-3 / Q000235:genetics*; D011485:Protein Binding; D017510:Protein Folding; D061987:Sf9 Cells
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D019913:Nuclear Localization Signals; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D048271:Chitosan
keywords: OCT4; chitosan nanoparticles; induced pluripotent stem cells; reprogramming; transgene-free stem cells