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Karyotype analysis of the euploid cell population of a mouse embryonic stem cell line revealed a high incidence of chromosome abnormalities that varied during culture

Dipartimento di Biologia Animale, Laboratorio di Biologia dello Sviluppo, Università degli Studi di Pavia, Pavia, Italy.
Cytogenetic and Genome Research (Impact Factor: 1.91). 02/2008; 121(1):18-24. DOI: 10.1159/000124377
Source: PubMed

ABSTRACT It is common knowledge that mouse embryonic stem cell (mESC) lines accumulate chromosomal changes during culture. Despite the wide use of mESCs as a model of early mammalian development and cell differentiation, there is a lack of systematic studies aimed at characterizing their karyological changes during culture. We cultured an mESC line, derived in our laboratory, for a period of 3 months investigating its chromosome complement at different times. About 60% of the metaphases analysed were euploid throughout the culture period but, from passage 13, only 50% of the euploid metaphases had a proper chromosome complement. The remaining 50% showed chromosome abnormalities, mainly gain or loss of entire chromosomes, both within the same passage and among different passages analysed. The very heterogeneous spectrum of abnormalities indicates a high frequency of chromosome mutations that arise continuously during culture. The heterogeneity of the aberrant chromosome constitution of 2n = 40 metaphases, observed at different passages of culture, might be due either to their elimination or to a shift towards the hypoeu- or hypereuploid population of those metaphases that accumulate further chromosome abnormalities. The stability of the frequency of eu-, hypoeu- and hypereuploid populations during culture might, however, be due to the elimination of those cells that carry a high mutational burden. Based on our results, we suggest that karyotype analysis of the euploid cell population of mESC lines is necessary when such lines are used in the production of chimeric mice, for their contribution to the germ line, or when they are differentiated into specific cell types.

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    • "The presence of chromosomal abnormalities in mouse and human ESCs started to be systematically described in the mid 2000s, and most of them appear in a recurrent manner. Trisomy 8 and trisomy 11 are frequent in mESCs9111213 whereas trisomy 12, 17 (which is partially syntenic with mouse chromosome 1114) and amplifications of 20 p are typical of hESCs1516171819202122. All these changes have been reported to confer a growth advantage1516171819212223 at the expense of cell differentiation16181923, as a result of the unbalanced dosage of key genes involved in self-renewal, differentiation or cell-cycle regulation. "
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    ABSTRACT: Embryonic Stem Cells (ESCs) are expected to show a stable euploid karyotype, but in the last decade (sub)chromosomal aberrations have been systematically described in these cell lines when maintained in vitro. Culture conditions and long-term culture have been traditionally proposed as possible factors involved in the acquisition of chromosomal abnormalities. Thus, we analyzed the chromosome constitution, the undifferentiated state and the functional pluripotency of three different mouse ESCs grown under the same culture conditions. Two cell lines were unstable from early passages, whereas the third one retained its chromosome integrity after long-term culture despite using enzymatic methods for cell disaggregation. Trisomy 8 and 11 were clonally selected in both unstable cell lines, which also showed a higher growth rate than our normal cell line and suffered morphological changes in colony shape with increasing passage number. Regardless of the length of culture or the chromosome instability, all cell lines preserved their differentiation potential. These results confirm that double trisomy 8 and 11 confers a growth advantage to the abnormal cells, but not at the expense of cell differentiation. The presence of chromosome instability, widely related to tumor development and cancer disease, highlights the risk of using pluripotent cells in regenerative medicine.
    Scientific Reports 06/2014; 4:5324. DOI:10.1038/srep05324 · 5.58 Impact Factor
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    • "Interestingly, in the progeny of irradiated cells the distribution of the number of chromosomes among cells was not altered. Karyotypic alterations in mESCs have already observed in previous studies and have been attributed to culture conditions and passage number [45]. Since both, numerical and structural abnormalities may affect gene expression [46] [47], and subsequently change the properties of a cell population, the outcome of toxicity tests or differentiation assays using mESCs might be affected underlining the need for the improvement of culture conditions for mESCs. "
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    ABSTRACT: The risk of radiation exposure during embryonic development is still a major problem in radiotoxicology. In this study we investigated the response of the murine embryonic stem cell (mESC) line D3 to two radiation qualities: sparsely ionizing X-rays and densely ionizing carbon ions. We analyzed clonogenic cell survival, proliferation, induction of chromosome aberrations as well as the capability of cells to differentiate to beating cardiomyocytes up to 3 days after exposure. Our results show that, for all endpoints investigated, carbon ions are more effective than X-rays at the same radiation dose. Additionally, in long term studies (≥8 days post-irradiation) chromosomal damage and the pluripotency state were investigated. These studies reveal that pluripotency markers are present in the progeny of cells surviving the exposure to both radiation types. However, only in the progeny of X-ray exposed cells the aberration frequency was comparable to that of the control population, while the progeny of carbon ion irradiated cells harboured significantly more aberrations than the control, generally translocations. We conclude that cells surviving the radiation exposure maintain pluripotency but may carry stable chromosomal rearrangements after densely ionizing radiation.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/2014; DOI:10.1016/j.mrgentox.2013.12.004 · 4.44 Impact Factor
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    • "Generally, cells in vitro for numerous passages acquire chromosomal changes. The proportion of euploid cells starts to decrease abruptly after passage 15, and only 20 to 30% of cells remain euploid by passage 25 [5,11]. The efficiency of germ line transmission declines as the ES cell passage number increases in number, at least partially due to the presence of aneuploidy in the cell population [5,12]. "
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    ABSTRACT: Background Obtaining a germ cell line is one of the most important steps in developing a transgenic or knockout mouse with a targeted mutated gene of interest. A common problem with this technology is that embryonic stem (ES) cells often lack, or are extremely inefficient at, germ line transmission. Results To determine whether chromosomal anomalies are correlated with inefficient ES cell germ line transmission, we examined 97 constructed ES cell lines using conventional cytogenetic analysis, and fluorescence in situ hybridization (FISH). Chromosomal abnormalities occurred in 44 (45%) out of the 97 specimens analyzed: 31 specimens had trisomy 8 or mosaic trisomy 8, eight specimens had partial trisomy 8 resulting from unbalanced translocations, and five specimens had other chromosomal anomalies. Conclusions Our data suggest that chromosomal analysis is an important tool for improving the yield and quality of gene targeting experiments.
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