Embryonic stem cells and somatic cells differ in mutation frequency and type

Abstract

Pluripotent embryonic stem (ES) cells have been used to produce genetically modified mice as experimental models of human genetic diseases. Increasingly, human ES cells are being considered for their potential in the treatment of injury and disease. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differs from that in embryonic somatic cells. The mutation frequency in ES cells is significantly lower than that in mouse embryonic fibroblasts, which is similar to that in adult cells in vivo. The distribution of spontaneous mutagenic events is remarkably different between the two cell types. Although loss of the functional allele is the predominant mutation type in both cases, representing about 80% of all events, mitotic recombination accounted for all loss of heterozygosity events detected in somatic cells. In contrast, mitotic recombination in ES cells appeared to be suppressed and chromosome loss͞reduplication, leading to uniparental disomy (UPD), represented more than half of the loss of heterozygosity events. Extended culture of ES cells led to accumulation of cells with adenine phosphoribosyltransferase deficiency and UPD. Because UPD leads to reduction to homozygosity at multiple recessive disease loci, including tumor suppressor loci, in the affected chromosome, the increased risk of tumor formation after stem cell therapy should be viewed with concern.T he frequency and types of spontaneous mutation that occur in pluripotent stem cells may have an impact on their future clinical uses. Embryonic stem (ES) cells are intrinsically different from somatic cells in that they retain the capacity to differentiate into multiple cell types (1, 2). Maintenance of genomic stability in these cells is essential for their potential to serve as donor cells for tissue transplants. Here we have examined spontaneous and induced mutagenic events in ES cells. We have taken advantage of a recently described murine model that utilizes mice heterozygous at the selectable marker encoding the purine salvage enzyme adenine phosphoribosyltransferase (APRT) to examine this issue (3-5).The Aprt gene, resident on mouse chromosome 8, is an endogenous marker that supports selection for cells that are APRT deficient and, conversely, for cells that have APRT activity (6). The model uses cells derived from 129 SvEv ϫ C3H͞HeJ mice or embryos in which the strain 129 SvEv Aprt allele has been interrupted by a targeted neo insertion (7). These cells are therefore heterozygous at Aprt, with the neo insertion allowing for rapid discrimination between the targeted and untargeted alleles. Polymorphic microsatellites that span the length of chromosome 8 can be used to confirm loss of heterozygosity (LOH) resulting from either mitotic recombination, or multilocus deletion. Centromeres in mouse strains 129 SvEv and C3H͞HeJ are different sizes. Hence, the parental origin of copies of chromosome 8 can be determined, providing visual determination of uniparental disomy (UPD), defined as the presence of homolo...

Mouse embryonic stem cells are hypersensitive to apoptosis triggered by the DNA damage O6-methylguanine due to high E2F1 regulated mismatch repair

“…This has indeed been found to be the case. 29 What regulates MMR in ES cells? As outlined in Figure 7b, MSH2 gene expression is governed by the transcription factor E2F1, 21 which is regulated by Rb.…”

The decatenation checkpoint

“…The gain and loss of whole chromosomes and chromosome fragments are expected, while point mutations and mitotic recombination events are not. An analysis of loss-of-heterozygosity (LOH) mutations found that in mouse embryonic stem (ES) cells, the majority of LOH mutations were attributable to nondisjunction, whereas in mouse embryonic fibroblasts, LOH owing to nondisjunction was not observed (Cervantes et al, 2002). The LOH events in ES cells are likely to arise from the decatenation checkpoint deficiency in those cells.…”

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

“…A similar system in mice, based on a heterozygous mutation in the (autosomal) Aprt gene, also allows mutation analysis in vivo by screening for loss of the remaining wild-type allele in somatic cells[47]. These assays, however, can only be performed on cells that can actively proliferate in culture.…”