Passage Number is a Major Contributor to Genomic Structural Variations in Mouse iPSCs

Abstract

Emergence of genomic instability is a practical issue in preparing neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs). However, it is still not fully understood what the origins and mechanisms for formation are for the genomic alternations observed. Here, we studied the extent of genomic variation on the scale of individual cells originating from the same animal. We used mouse NSCs grown from embryonic cells and iPSCs generated from embryonic brain cells, B cells or fibroblasts, and performed comparative analysis with cultures of fibroblasts from the same mouse. In the first passage of these cell lines, aneuploidies were only observed for chromosomes 6, 11, 12, 19 and Y, which is overall at a rate lower than previously reported; de novo copy number variations (CNVs) were observed in 4.3% of neural iPSCs, 29% of B cell iPSCs, 10% of fibroblast iPSCs, and 1.3% of neurospheres. In contrast, propagation of these first passage cells to a later passage induced additional aneuploidies and CNVs. Breakpoint sequencing analysis suggested that the majority of the detected CNVs arose by replicative mechanisms. Interestingly, we detected identical de novo CNVs in different single cell colonies that appeared to have arisen independently from each other, which suggests a novel CNV formation mechanism in these cells. Our findings provide insights into mechanisms of CNV formation during reprogramming, and suggest that replicative mechanisms for CNV formation accompany mitotic divisions.

Influence ofATM-Mediated DNA Damage Response on Genomic Variation in Human Induced Pluripotent Stem Cells

“…An initial characterization of the CNVs demonstrated substantial variability in the number of CNVs across the various lines and passages ( Fig. 2A and Supplementary Table S1), consistent with what has been previously reported in similar analyses [4][5][6]26]. Most iPSC CNVs were novel-only a minority was also present in parental fibroblast (Fig.…”

Somatic mosaicism: implications for disease and transmission genetics

“…For example, isochromosome 12p is often undetectable in blood, but examination of cells of ectodermal origin frequently reveals the mosaic aneuploidy [93]. Culturing cell lines derived from individuals present similar challenges, not only because cells accumulate mutations as they undergo mitosis [94], but also because culture conditions exert selective forces. To overcome these limitations, other or multiple sources of primarily obtained DNA can be examined.…”

iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations

“…To understand the scope of acquired genetic changes that occur during iPSC generation, previous studies have compared single nucleotide variants (SNVs), copy number variations (CNVs), and chromosomal rearrangements in iPSCs to donor somatic cells or embryonic stem cells (ESCs) using various assays, including SNP array and next-generation sequencing methods (1)(2)(3)(4)(5)(6). Whole genome or exome sequencing (WGS/ WES) of parental and iPSC pairs have demonstrated that there are, on average, 6-12 coding SNVs per iPSC line (1,(7)(8)(9) with varying theories regarding when these SNVs arose in the iPSCs.…”