Increased Genomic Integrity of an Improved Protein-Based Mouse Induced Pluripotent Stem Cell Method Compared With Current Viral-Induced Strategies

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.

Methods of Reprogramming to Induced Pluripotent Stem Cell Associated with Chromosomal Integrity and Delineation of a Chromosome 5q Candidate Region for Growth Advantage

“…The corresponding figure for cytogenetic aberrations in our lines reprogrammed by SeV was 1 out of 16 and clearly below the reported average for iPSC using integrative methods. Furthermore, the rate of cytogenetic aberrations at early passages and after reprogramming with SeV in our study is in line with studies using episomal-based reprogramming [16]. After prolonged culture, we detected one acquired aberration in a SeV-reprogrammed line.…”

“…The reason for differences in the types of karyotypic abnormalities in iPSC versus ESC is yet unclear and it may be hypothesized that it is related to the methods used for reprogramming [15]. Furthermore, only a few studies have compared the frequency of genomic rearrangements in iPSC with respect to different methods of reprogramming [16]. The techniques for derivation of iPSCs can be broadly categorized into integrative and nonintegrative delivery of reprogramming factors.…”

p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming

“…To minimize the genomic instabilities of iPS cells, strategies of generating integration-free iPS cells have been developed. However, iPS cells generated either with episomal vector or protein-base method were still found to carry a large number of de novo genetic variants323. One of the most important reasons for the de novo genetic variants in iPS cells is that reprogramming process can trigger DNA damage response.…”

Generation of murine induced pluripotent stem cells by using high-density distributed electrodes network

“…Relatively high expression of murine oct4 and sox2 genes in the group of iPSCs has been observed in this work, In the previous study, gene expression level was about 25-10 4 of murine oct4 and sox2 in a group of MEFs. 25,26 Our experimental results showed that a single MEFs achieved over 10 5 in gene expression level of murine oct4 and sox2, as shown in Table II. It also demonstrates that the application of the HDEN device achieved higher gene expression level in single host cell.…”