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. 2008 Sep;58(1):17-23.
doi: 10.1007/s10616-008-9164-x. Epub 2008 Sep 30.

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

Paola Rebuzzini  1 , Tui Neri, Maurizio Zuccotti, Carlo Alberto Redi, Silvia Garagna
Affiliations

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

Paola Rebuzzini et al. Cytotechnology. 2008 Sep.

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.

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Figures

Fig. 1
Fig. 1
Reverted images of DAPI banded karyotypes of a 2n = 40 (a) and a 2n = 77 (b) metaphase from UPV02 at passage 41. Chromosome abnormalities observed during the period of culture (black arrows): metacentric chromosomes (c), fragments (d), gaps (e) and spots (f)
Fig. 2
Fig. 2
In (a), frequency of euploid metaphases in UPV02, UPV06 and UPV08 mESC lines. In (b), (c) and (d), frequency of chromosome number distribution of UPV02, UPV06 and UPV08 mESC lines, respectively, during the period of culture
Fig. 3
Fig. 3
The three mESC lines expressed OCT-4 protein (b), SSEA-1 (d) and FOM-1 (f) antigens throughout the period of culture. a, c, e, DAPI counterstaining. Cells from the UPV06 cell line, at passage 10, have been shown as an example. At early passages, the three mESC lines formed round-shaped colonies and had high alkaline phosphatase activity (g, colonies of UPV06 at passage 23 are shown as a representative example of all three mESC lines, black arrowhead). At passage 34, the alkaline phosphatase activity is maintained in some colonies (black arrowhead) whereas is lost at the edge of most of the others (white arrowhead) in UPV02 cell line (h); at passage 32 it is either present at the edge of some colonies (white arrowhead) or it has strongly decreased in others (black arrow) (i) in UPV06 cell line; at passage 28, it is completely lost in UPV08 cell line (j). Dark bar: 200 μm; white bar: 50 μm
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