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.

Keywords: Chromosomal aberrations; DNA copy number variations; DNA replication; Induced pluripotent stem cells; Neural stem cells.

Figure 1

Schematic flow chart illustrating the procedure of experiments in this study.

Figure 2. High-density aCGH results for CNVs identified

Samples whose breakpoint sequence was characterized at the nucleotide level are highlighted in red. The breakpoint sequences as well as junction PCR results can be found in Figures 3 and S2. The sample type and coordinates (NCBI37/mm9) of regions affected are listed Table 2. Dash one in the name (for example, F26-1) indicates that this is the first CNV in this sample; dash two indicates the second CNV, etc. Note that G10-1 has the same breakpoint sequence as G23; G5 and G10 have apparently the same breakpoint according to aCGH.

Figure 3. Breakpoint junction sequences characterized

The breakpoint sequences are aligned to reference sequences at the distal and proximal ends of the rearrangement. The red to blue color transition indicates the precise locus of the breakpoint junction. Microhomology is highlighted in purple. Seven breakpoints (F8, F26-2, F31, F33, F21, G29, and 21A) showed microhomology of 2–4 base pairs. Three breakpoints (G10-1, G23, and 5A-1) had one base pair microhomology. Two samples, F1 and G19, had templated insertions at the breakpoints. The inserted sequence in G19, highlighted in green, was likely derived from part of an L1-LINE element. The deletion in 7D had short novel sequence inserted at the breakpoint.

Figure 4. CNVs arising from predetermined loci

(A) The same 275 kb deletion was identified as de novo events in F31 and F29-P12 (passage 12 of F29). The top of the panel illustrates aCGH plots delineating the deletions. In F29-P12, the 275 kb deletion is part of a larger complex rearrangement event, which includes a smaller deletion in the distal region. Below the array image are coordinates of the CNV region and an agarose gel picture of breakpoint PCR results showing the presence or absence of the breakpoint junction in the assayed samples. The results suggest that the mutations leading to the deletions in F31 and F29-P12 are both de novo events. Below the gel image is alignment of breakpoint sequence to reference sequences. Red and blue colors indicate alignment of the breakpoint sequence to the reference sequences, whereas purple indicates the interval of microhomology. (B) The same 1.4 Mb duplications were identified in both F3-P6 and F8 as independent de novo events. (C) The apparently same 699 kb duplications were identified in G5 and G30. (D) The same 337 kb deletions were identified in G23 and G10.