Abstract
The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC.
Highlights
The generation of induced pluripotent stem cells from adult somatic cells is one of the most remarkable discoveries in recent decades
As direct measure of replication stress (RS), we observed that replication fork speed, measured by single molecule DNA combing analysis, is lower in cells expressing OSKM than in green fluorescent protein (GFP)-expressing cells (Supplementary Fig. 3f)
Fork symmetry was not altered in OSKM-expressing fibroblasts when compared with GFP control cells
Summary
The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. Recent reports have shown evidence of DNA damage and genomic instability in iPSC2–8, raising concerns on their potential biomedical use. The source of genomic instability on iPSC remains unresolved, several evidence suggest that it could be linked to replication stress (RS), a type of DNA damage occurring at stalled replication forks and limited by the ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) kinases[9]. We hypothesized that similar to oncogene-induced RS; an analogous reprogramming-induced RS could drive genomic instability in iPSC Supporting this view, we and others have recently demonstrated that iPSC contain genomic structural variations such as copy number variants (CNV) that were highly enriched in fragile sites[3,7,8], a hallmark of RS. If RS were to significantly contribute to the genomic rearrangements found in iPSC, we reasoned that strategies directed to lowering reprogramming-induced RS could offer a strategy to reduce genomic instability on iPSC
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