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.

Keywords: OCT4; chitosan nanoparticles; induced pluripotent stem cells; reprogramming; transgene-free stem cells.

Figure 1. Characterization of chitosan NPs

(A) Scanning electron micrographs from S-NPs (left) and L-NPs (right). Bars = 200 nm. (B) Kinetics of HRP release from S-NPs and L-NPs in comparison to free HRP, as measured by enzyme activity. Results are given as mean ± SD from three experiments performed in triplicate. Similar release profiles were obtained by measuring protein content.

Figure 2. OCT4 expression and purification from Sf9 cells

(A) Efficient Sf9 cell infection with recombinant OCT4-encoding baculoviruses was monitored by expression of GFP five days post-infection. (B) Recombinant OCT4 is enriched in nuclear fractions of Sf9 cells and can be purified by GST affinity chromatography, as shown by silver staining (left) and immunoblot analysis using an OCT4 antibody (right). FT: column flow-through, N: nuclear fraction, C: cytosolic fraction, E: column eluates.

Figure 3. S-NP encapsulation stabilizes OCT4 DNA-binding activity

(A) EMSA analysis showing OCT4 stabilization by S-NPs but not L-NPs. Unloaded S-NPs, soluble OCT4 protein as well as OCT4 encapsulated in S-NPs and L-NPs were subjected to EMSA analysis. The DNA-binding activity of OCT4 was analyzed using an oligonucleotide containing the OCT4 consensus motif. BSA was used as a negative control. (B–D) OCT4-loaded S-NPs stabilize OCT4 DNA-binding activity for 7 weeks at 4°C (B), for 14 days at RT (C), and for 24 h under cell culture conditions at 37°C in the presence of serum (D), whereas the DNA-binding activity of soluble OCT4 is rapidly lost under these conditions. The slowly migrating protein/DNA complex of soluble OCT4 shown in (D) is presumably caused by aggregation of the OCT4 protein under cell culture conditions. The protein amount used per lane for the EMSAs corresponds to 30 ng (A), 100 ng (B) and 250 ng (C, D). The OCT4/DNA complexes and unbound oligonucleotide are marked by closed and open arrowheads, respectively.

Figure 4. Effects of NLS density on S-NP cell surface binding, uptake and nuclear delivery

(A–C) Non-modified S-NPs or S-NPs tagged with low (L), intermediate (I) or high (H) NLS densities were incubated at the indicated concentrations with human fibroblasts. After 24 h chitosan NPs were stained as detailed in Material and Methods. The recovered amount of (A) cell-associated (i.e. surface-bound and internalized) NPs, (B) NPs bound to cell surface or (C) NPs taken up intracellularly was calculated from a standard curve by fluorometry. (D) Effects of NLS density on S-NP nuclear delivery as assessed by FRET fluoroscopy. Human fibroblasts were treated for 24 h with 250 μg/mL of the indicated versions of S-NPs. Measurement of FRET efficiency indicates the strongest colocalization of the nuclear DNA dye with SN-Ps lacking an NLS. Results are given as means ± SD.

Figure 5. OCT4-loaded S-NPs but not soluble OCT4 protein are imported into cells and partially localize in the cell nucleus

Human primary fibroblasts were treated with 50 μg of each recombinant soluble OCT4 (A–C) or OCT4 encapsulated in S-NPs (D–F). After 24 h cells were stained with OCT4 antibodies (green) or for chitosan NPs using WGA-Alexafluor 488 (red). Nuclear DNA was stained with Hoechst 33258 (blue). Merged images demonstrate that exogenous soluble OCT4 is excluded from cells and adheres to the cell membrane. In contrast, OCT4-loaded S-NPs are localized intracellularly, showing a partial overlap with Hoechst nuclear staining.

Figure 6. Z-stack imaging series of human fibroblasts treated with OCT4-loaded S-NPs

Cells were treated for 24 h with 50 μg of the SN-Ps and then stained with OCT4 antibody (green), WGA-Alexafluor 488 (red) and Hoechst 33258 (blue). Z-stack images through the cell nucleus were collected at 1-μm steps by confocal laser scanning microscopy. The yellow fluorescence of the merged images indicates the colocalization of OCT4 and S-NPs in perinuclear and nuclear regions. The depth in micrometers at which images were taken is indicated. Scale bar = 10 μM.