Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts

Generation 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...

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Published in:Animal cells and systems 2018, 22(2), , pp.132-139
Main Authors: Bang, Jin Seok, Choi, Na Young, Lee, Minseong, Ko, Kisung, Lee, Hye Jeong, Park, Yo Seph, Jeong, Dahee, Chung, Hyung-Min, Ko, Kinarm
Format: Article
Language:English
Subjects:
c-Myc protein
Cell density
Density
Deoxyribonucleic acid
DNA
Episomal vector
Fibroblasts
Gene transfer
Integration
integration-free
iPSC generation
KLF4 protein
Mutagenesis
Myc protein
Oct-4 protein
Optimization
p53 Protein
Pluripotency
Proteins
reprogramming
Stem cells
Transcription factors
Transfection
Viruses
생물학
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Summary:Generation 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.
ISSN:1976-8354
2151-2485
DOI:10.1080/19768354.2018.1451367