Chromosome number variation in three mouse embryonic stem cell lines during culture

Abstract

Although mouse embryonic stem cell lines (mESCs) have been established since 1981, systematic studies about chromosomal changes during culture are lacking. In this study, we report the results of a cytogenetic analysis performed on three mESC lines (named UPV02, UPV06 and UPV08) cultured for a period of 3 months. At time intervals, the variation of the chromosome number together with the expression of markers of the undifferentiated status, i.e., OCT-4, SSEA-1, FOM-1 and alkaline phosphatase activity, were determined. The three mESC lines showed a progressive loss of euploid metaphases during the 3 months period of culture. Chromosome abnormalities were accumulated at the latest passages analysed. Metacentric chromosomes were the most frequent chromosome abnormality observed throughout the period of culture. Interestingly, in coincidence with, or few passages after, the drop of euploidy, the alkaline phosphatase activity was partially or totally lost, whereas the OCT-4, SSEA-1 and FOM-1 stem markers were always positive throughout the period of culture. Our results remark the necessity to perform the karyotype analysis during culture in order to develop new culture conditions to maintain the correct chromosome complement in long-term culture of mESC lines.

Established Preblastocyst- and Blastocyst-Derived ES Cell Lines Have Highly Similar Gene Expression Profiles, Despite Their Differing Requirements for Derivation Culture Conditions

“…At passage 8, karyotype analysis showed that all lines from preblastocysts had over 70% euploid chromosome count. This percentage is in normal range of reported mouse ES cell lines (Rebuzzini et al, 2008a(Rebuzzini et al, , 2008b. Teratoma study is very important assay to demonstrate pluripotent cell lines' differentiation and pluripotency.…”

Cytogenetic analysis and Dlk1-Dio3 locus epigenetic status of mouse embryonic stem cells during early passages

“…As mentioned previously, we have also observed a high level of chromosome 11 and 14 trisomies. Interestingly, we observed chromosome 1 trisomy in many cells of the ES cell lines, and its high frequency has not been previously reported (Liu et al 1997;Sugawara et al 2006;Rebuzzini et al 2008b).…”

Genome Stability in Embryonic Stem Cells

“…In other three mESC lines, named UPV02, UPV06 and UPV08, a progressive loss of euploid metaphases during culture has been observed and chromosome abnormalities, in particular metacentric chromosomes, accumulated at the latest passages analysed (passage 31, 29 and 22 for UPV02, UPV06 and UPV08, respectively). We observed that in coincidence with, or few passages after, the drop of euploidy, the alkaline phosphatase activity, one important ESC marker, was partially or totally lost (Rebuzzini et al, 2008b). Rat The rat ESCs (rESCs) are an important resource for the study of disease models, however, despite several temptatives (Brenin et al, 1997;Vassilieva et al, 2000) they have been derived only very recently (Buehr et al, 2008;Ueda et al, 2008;Zhao et al, 2010).…”

“…Mouse Unexpectedly, an accurate literature search showed that only a few papers described the genomic variation of mESCs during a long period of culture. In many mESC lines, no recurrent chromosome abnormalities, but rather random alterations have been described by some laboratories (Longo et al, 1997;Guo et al, 2005;Sugawara et al 2006;Rebuzzini et al, 2008a;Rebuzzini et al, 2008b). The most complete analysis that has been made on mESCs was performed by Sugawara and colleagues (Sugawara et al, 2006).…”

“…Generally, ESCs are derived and maintained in vitro with a co-culture protocol on a feeder layer of mitotically inactivated fibroblast cells (mouse embryonic or immortalized fibroblasts) or on defined supportive matrixes (i.e., gelatin, fibronectin or matrigel TM ). Whether using the former or the latter, genetic alterations were observed both in mESC and hESC lines (Cowan et al, 2004;Rosler et al, 2004;Mitalipova et al, 2005;Maitra et al, 2005;Guo et al, 2005;Longo et al, 2005;Imreh et al, 2006;Sugawara et al, 2006;Rebuzzini et al, 2008a;Rebuzzini et al, 2008b), suggesting that the presence or absence of a supporting cellular feeder layer can not exclude the onset of aberrations in the ESCs genome. A fundamental component of the ESC medium is the serum of animal (calf or bovine) or artificial (knockout serum replacement of defined composition) origin.…”