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.

A biomaterial approach to cell reprogramming and differentiation

“…More recently, chitosan nanoparticles (CNPs) were employed to deliver recombinant Oct4 to human fibroblasts, demonstrating a potential of CNPs to be a promising tool for the generation of transgene-free hiPSCs (Figure 4E) [42]. A nanoparticle formulation provides protection for the loaded recombinant Oct4, while naked recombinant Oct4 is known to become rapidly degraded in cell culture conditions.…”

Adult Neural Stem Cells: Basic Research and Production Strategies for Neurorestorative Therapy

“…In such a fashion, a culture of stable iPSCs that can sustain for over 35 passages was obtained from fibroblasts [ 127 ]. The efficiency of a protein delivery to the cell can be promoted by the implementation of chitosan nanoparticles [ 128 ] which increases the internalization of the complex by cell and transfer to the nuclear membrane. Despite the obvious attractiveness of this approach, it should be noted that the efficiency of reprogramming with help of recombinant proteins is very low, and the production of functionally active recombinant proteins is labor extensive and quite costly.…”

“…(b) Logical schemes of reprogramming. From top to bottom: (1) scheme in which the IPSC stage is present [ 94 , 126 , 128 , 129 ]. (2)-(3) Schemes in which the IPSC stage is absent are divided into three main stages: preparatory (destabilizing the genome and increasing functional plasticity); the stage of redifferentiation in the NSC, and the stage of terminal neuroglial differentiation (explanations in the text).…”

Targeting Activated Hepatic Stellate Cells Using Collagen-Binding Chitosan Nanoparticles for siRNA Delivery to Fibrotic Livers

“…For drug targeting to aHSCs, NPs should initially encapsulate the active molecule without compromising its therapeutic activity. This is of particular importance when sensitive macromolecular compounds, such as siRNA or proteins, are to be delivered [ 12 , 13 ]. Following in-vivo administration, NPs should then localize in the space of the Disse, where the aHSCs reside.…”

“…Chitosan is a biodegradable polymer that has been widely employed in the development of NPs for drug delivery, in particular for protein and nucleic acid delivery. This is mainly attributed to the NPs’ hydrophilicity as well as to the fact that their formulation by ionotropic gelation is devoid of organic solvents and the use of heat [ 2 , 12 , 19 , 20 , 21 , 22 , 23 ]. Additionally, and highly important in the context of fibrotic diseases, chitosan has been reported to bind and interact with collagen [ 24 ].…”