Abstract
ABSTRACTGeneration of induced pluripotent stem cells (iPSCs) by defined factors (OCT4, SOX2, C-MYC, and KLF4) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (OCT4, SOX2, C-MYC, and KLF4) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (OCT4/p53, SOX2/KLF4, L-MYC/LIN28A) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors.
Highlights
Induction of pluripotency using retro- or lentiviral vectors to express reprogramming factors (OCT4, SOX2, KLF4, and C-MYC) is successful in various human cell types, including fibroblasts, urine-derived cells, and peripheral blood cells (Takahashi et al 2007; Staerk et al 2010; Zhou et al 2011)
Several methods have been developed for generation of integration-free induced pluripotent stem cells, such as the piggyBac system, a minicircle vector, synthetic mRNA, Sendai virus, recombinant proteins, and episomal vectors (Fusaki et al 2009; Kim et al 2009; Woltjen et al 2009; Yu et al 2009; Jia et al 2010; Okita et al 2011)
BJ cells were obtained from ATCC and were cultured in mouse embryonic fibroblast (MEF) medium consisting of Dulbecco’s Modified Eagle’s Media (DMEM; Welgene) supplemented with 10% fetal bovine serum (FBS; Corning), 0.1 mM non-essential amino acids (NEAA; Gibco), and 1% penicillin/streptomycin (P/S; Welgene)
Summary
Induction of pluripotency using retro- or lentiviral vectors to express reprogramming factors (OCT4, SOX2, KLF4, and C-MYC) is successful in various human cell types, including fibroblasts, urine-derived cells, and peripheral blood cells (Takahashi et al 2007; Staerk et al 2010; Zhou et al 2011). Viralbased methods were used, which are efficient, straightforward, reliable, and adoptable These methods required viral vectors that integrate into the host genome, which may lead to gene mutations and tumor formation and limit the use of such cells in therapeutic applications. To circumvent these problems, several methods have been developed for generation of integration-free induced pluripotent stem cells (iPSCs), such as the piggyBac system, a minicircle vector, synthetic mRNA, Sendai virus, recombinant proteins, and episomal vectors (Fusaki et al 2009; Kim et al 2009; Woltjen et al 2009; Yu et al 2009; Jia et al 2010; Okita et al 2011). Due to differences in vector uptake by nucleofection, gene expression levels each cell are highly variable (Drozd et al 2015)
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