Increased genomic integrity of an improved protein-based mouse induced pluripotent stem cell method compared with current viral-induced strategies.
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.
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA; Harvard Medical School, Boston, Massachusetts, USA; Molecular Neurobiology Laboratory, McLean Hospital/Harvard Medical School, Belmont, Massachusetts, USA; Harvard Stem Cell Institute, Boston, Massachusetts, USA; Department of Animal Biotechnology, College of Animal Bioscience and Technology, Konkuk University, Seoul, Korea; Biotechnology Process Engineering Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon, Korea; Advanced Cell Technology, Marlborough, Massachusetts, USA.
Hansoo
H
Park
Dohoon
D
Kim
Chun-Hyung
CH
Kim
Ryan E
RE
Mills
Mi-Yoon
MY
Chang
Rebecca Cheryl
RC
Iskow
Sanghyeok
S
Ko
Jung-Il
JI
Moon
Hyun Woo
HW
Choi
Paulo Sng
PS
Man Yoo
Jeong Tae
JT
Do
Min-Joon
MJ
Han
Eun Gyo
EG
Lee
Joon Ki
JK
Jung
Chengsheng
C
Zhang
Robert
R
Lanza
Kwang-Soo
KS
Kim
D000818:Animals; D042822:Genomic Instability; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus; D051379:Mice; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014161:Transduction, Genetic
C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors
Copy number variant; Genomic integrity; Induced pluripotent stem cell; Protein-based iPSC; Reprogramming method