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Increased Genomic Integrity of an Improved Protein-Based Mouse Induced Pluripotent Stem Cell Method Compared With Current Viral-Induced Strategies
- Source :
- Stem Cells Translational Medicine. 3:599-609
- Publication Year :
- 2014
- Publisher :
- Oxford University Press (OUP), 2014.
-
Abstract
- It has recently been shown that genomic integrity (with respect to copy number variants [CNVs]) is compromised in human induced pluripotent stem cells (iPSCs) generated by viral-based ectopic expression of specific transcription factors (e.g., Oct4, Sox2, Klf4, and c-Myc). However, it is unclear how different methods for iPSC generation compare with one another with respect to CNV formation. Because array-based methods remain the gold standard for detecting unbalanced structural variants (i.e., CNVs), we have used this approach to comprehensively identify CNVs in iPSC as a proxy for determining whether our modified protein-based method minimizes genomic instability compared with retro- and lentiviral methods. In this study, we established an improved method for protein reprogramming by using partially purified reprogramming proteins, resulting in more efficient generation of iPSCs from C57/BL6J mouse hepatocytes than using protein extracts. We also developed a robust and unbiased 1 M custom array CGH platform to identify novel CNVs and previously described hot spots for CNV formation, allowing us to detect CNVs down to the size of 1.9 kb. The genomic integrity of these protein-based mouse iPSCs (p-miPSCs) was compared with miPSCs developed from viral-based strategies (i.e., retroviral: retro-miPSCs or lentiviral: lenti-miPSCs). We identified an increased CNV content in lenti-miPSCs and retro-miPSCs (29∼53 CNVs) compared with p-miPSCs (9∼10 CNVs), indicating that our improved protein-based reprogramming method maintains genomic integrity better than current viral reprogramming methods. Thus, our study, for the first time to our knowledge, demonstrates that reprogramming methods significantly influence the genomic integrity of resulting iPSCs.
- Subjects :
- Genetics
Genome instability
Induced Pluripotent Stem Cells
Lentivirus
Cell Biology
General Medicine
Computational biology
Biology
Genomic Instability
Kruppel-Like Factor 4
Mice
HEK293 Cells
SOX2
Transduction, Genetic
KLF4
Animals
Humans
Ectopic expression
Copy-number variation
Induced pluripotent stem cell
Reprogramming
Transcription factor
Transcription Factors
Embryonic Stem Cells/Induced Pluripotent Stem (iPS) Cells
Developmental Biology
Subjects
Details
- ISSN :
- 21576580 and 21576564
- Volume :
- 3
- Database :
- OpenAIRE
- Journal :
- Stem Cells Translational Medicine
- Accession number :
- edsair.doi.dedup.....1f8ca94cf019147078d852bbdfd8c93c