Embryonic stem cells have attracted great interest both for their use in lab research and possible practical applications [
However, hESCs chromosome preparations frequently encounter certain difficulties, perhaps connected with hESCs biology and chromosome organization, thus making karyotyping problematic [
Materials and methods
Cell lines: Sublines hESM01r18 (46,ХХ,-18,+mar) and hESM0309 (46,ХХ,del(4),dup(9)) were derived from hESM01 and hESM03 parental cell lines, respectively, as described previously [
Metaphase chromosome preparations: Chromosome samples were prepared according to standard procedures. In brief, PBL metaphase and prometaphase chromosomes for FISH analysis were performed as described by Henegariu [
DNA probe preparation: Chromosome microdissection was performed according to previously described methods [
A telomere-specific biotin-11-dUTP labeled (TTAGGG)n DNA probe was generated by PCR as described by Ijdo [
Fluorescence in situ hybridization (FISH): The chromosomal location of the dissected chromosome fragments was assessed by reverse painting as previously described [
Chromosomes were counterstained with 1 µg/ml 4’,6-diamidino-2-phenylindole (DAPI) and analyzed using an AXIOPlan2 Imaging (Carl Zeiss, Jena, Germany), equipped with CCD camera, filter sets, and ISIS5 image-processing package of MetaSystems GmbH. Chromosomes and chromosomal regions were identified by inverted DAPI-banding using human an International Systems for Human Cytogenetic Nomenclature [
Chromosomal rearrangements in hESM0309 cell line
Using DAPI staining, it was shown previously that in all cells of hESM0309 cell line chromosomes 4 and 9 were rearranged [
(i) The breakpoints involved in chromosome 4 deletion formation were characterized as 4q25 and q31.1 (Fig. 1). Based on the data abnormal chromosome 4 was described as del(4)(q25q31.1).
(ii) Another abnormal chromosome found in hESM0309 cells was previously described as dup(9)(pter→q3::q12→qter) by DAPI banding . C-banding revealed two C-positive regions confirming the location of one of the breakpoints in a C-positive region 9q12 (Fig. 2a). To describe chromosome dup(9) more precisely the set of DNA probes were generated by microdissection from this chromosome and normal chromosome 9 as shown schematically in Fig. 2b. E.g. DNA probes WCP9 and WCPder(9) (Fig. 2b) completely painted chromosome 9 and dup(9), however, the intensity of the FISH staining of these chromosomes was different (Fig. 3). Further studies summarized in Fig. 3 revealed a dup(9)(pter→q33::q12→qter) in hESM0309.
Thus, the karyotype of hESM0309 cells was described 46,XX,del(4)(q25q31.1),dup(9)(q12q33).
Chromosomal rearrangements in hESM01r18 cells
A previously performed hESM01r18 karyotype analysis allowed to hypothesize that the marker, most likely an abnormal ring chromosome was a derivative of normal chromosome 18 and hESM01r18 cells were described as 46,ХХ,-18,+?r(18) [
Thus r(18) contain two copies of the indicated region and the karyotype of hESM01r18 cells was 46,ХХ,r(18)(::p11.31→q21.2::q21.2→p11.31::).
Since their isolation in 1998 [
Karyotyping of hESM01r18 and hESM0309 cell lines demonstrated that chromosomes r(18) and dup(9), respectively, were present in all cells from given cell lines [
The breakpoints in chromosome 9 revealed in hESM0309 cell line coincides with the breakpoints quite often observed during loss of heterozygosity in transitional cell carcinoma of urinary tract tumor (9q12, 9q22.3, 9q33-34) [
The existence of chromosomal abnormalities in the stem cells are often associated with carcinogenesis [
Overall, this study shows that hESCs should be (i) molecular cytogenetically characterized in detail and (ii) that such studies may be extremely helpful in understanding tumor initiation and progression, as well.
This work was supported by Russian Federal contract 02.512.11.2060.
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