Efficient Production of Retroviruses Using PLGA/bPEI-DNA Nanoparticles and Application for Reprogramming Somatic Cells

Abstract

Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate.

Multilayered Nanoparticles for Gene Delivery Used to Reprogram Human Foreskin Fibroblasts to Neurospheres

“…[1][2][3][4][5] In recent years, nonviral methods for delivery have received particular attention due to immunogenic and toxic responses associated with viral vectors, which limit practical use and tenability. [6][7][8] One important limitation of nonviral gene delivery is the inefficient delivery of functional nucleic acids to the nucleus.…”

Application of biomaterials to advance induced pluripotent stem cell research and therapy

“…For example, nano‐/microparticles formulated by poly(lactic‐co‐glycolic acid) (PLGA) offer high stability, potential for narrow size distribution, tunability, and an excellent safety profile (FDA‐approved) (Danhier et al , ; Ankrum et al , ). It has been adopted as a gene delivery vehicle for multiple transfection applications (Seo et al , ; Tian et al , ). PLGA degrades into lactic acid and glycolic acid (natural metabolites found in the body).…”

Human Induced Pluripotent Stem Cell and Nanotechnology-Based Therapeutics

“…On the other hand, the positively charged amine groups of branch-chained polyethyleneimine (bPEI) contribute to high cationic-charge-density of the polymer and make bPEI an ideal carrier for nucleic acid. Seo and coworkers developed PLGA/bPEI NPs in transfection and virus production (52). The addition of positively charged bPEI can interact with the negatively charged nucleic acid and increase the absorption of nucleic acid onto the PLGA/bPEI nanoparticles.…”