Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells

Abstract

Reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variations (CNVs)1-4. To explore this issue, we performed a whole-genome and transcriptome analysis of 20 human iPSC lines derived from primary skin fibroblasts of 7 individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two CNVs not apparent in the fibroblasts from which the iPSC was derived. Using qPCR, PCR, and digital droplet PCR (ddPCR), we show that at least 50% of those CNVs are present as low frequency somatic genomic variants in parental fibroblasts (i.e. the fibroblasts from which each corresponding hiPSC line is derived) and are manifested in iPSC colonies due to the colonies’ clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSC, since most of line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations

“…The size, nature and frequencies of these segmental CNVs are comparable to the abnormalities recently reported in fibroblasts 6 , differentiated hiPSC and neurons studied using aCGH and single-cell sequencing 19 . Remarkably, we only found full chromosome aneuploidy in two hESCs and none in the somatic cells: one hESC contained a trisomy 20 and one was triploid.…”

“…As these CNVs are present at very low frequencies in the culture, they also could not be detected using the straight-forward analysis of pooled DNA, as used by most studies for high-resolution screening of cell lines. To get around this, Abyzov et al 6 derived hiPSC lines from skin biopsies to clonally amplify the genetic content of single fibroblasts and described low-frequency kilo-base scale CNVs unique to each subclone. This is indicative for low-grade mosaicism in the original fibroblasts; however, the CNVs described by Abyzov et al 6 are considerably smaller than the CNVs described in this study.…”

“…To get around this, Abyzov et al 6 derived hiPSC lines from skin biopsies to clonally amplify the genetic content of single fibroblasts and described low-frequency kilo-base scale CNVs unique to each subclone. This is indicative for low-grade mosaicism in the original fibroblasts; however, the CNVs described by Abyzov et al 6 are considerably smaller than the CNVs described in this study. A very recent publication reports genetic mosaicism of megabase-scale CNVs in differentiated hiPSC, cultured fibroblasts and neurons using various high-resolution single-cell genomics 19 .…”

“…These studies have shown that the grade of mosaicism varies from tissue to tissue and the percentage of aneuploid cells increases with age, but provide limited insight on the occurrence of small genetic imbalances [2][3][4][5] . More recently, the group of Abyzov et al 6 made use of the clonal nature of humaninduced pluripotent stem cells (hiPSCs) to show that human skin fibroblasts are highly mosaic, with up to 30% of cells carrying copy number variants (CNVs). In the field of human pluripotent stem cell research, a large number of studies have shown that human embryonic stem cell (hESC) cultures are frequently taken over by chromosomally abnormal cells 7,8 , most probably because of a selective advantage caused by the chromosomal aberration 9,10 .…”

“…In recent years, many groups have performed high-resolution screening of somatic cell populations 1,6,11,12 as well as hiPSC and hESC 7,8 and have reported de novo CNVs ranging from 10 kb to several Mb. It is important to bear in mind that these studies are based on the analysis of DNA from extractions from large numbers of cells, which only allows detection of CNVs if they are present in at least 5-10% of the cells 11,12 .…”