Chromosome rearrangements in sublines of human embryonic stem cell lines hESM 01 and hESM 03

Due to possible proliferative effects of karyotypic reorganization of human embryonic stem cell (hESC) lines detailed genetic analysis are indicated prior to any application of hESCs. Molecular cytogenetic analysis of two different hESC sublines was performed and revealed aberrant chromosomes in both of them, i.e. in hESM01r18 (46,ХХ,-18,+mar) and hESM0309 (46,ХХ,del(4),dup(9)). This study shows that microdissection and multicolor fl uorescence in situ hybridization (mFISH) can be used to detect the chromosomal changes precisely of the derivative chromosomes that are diffi cult to identify by conventional G-banded chromosome analysis. In the present study chromosome microdissection and reverse FISH were applied using multicolor fl uorescence in situ hybridization (mFISH) for detailed characterization of the derivative chromosomes. The karyotypes of hESC lines were described as: 46,ХХ,r(18)(::p11.31→q21.2::q21.2→p11.31::) and 46,XX,del(4)(q25q31.1),dup(9) (q12q33), respectively. The potential role of the chromosomal regions involved in rearrangements for cell proliferation is discussed.


Introduction
Embryonic stem cells have attracted great interest both for their use in lab research and possible practical applications [1].However, the genetic stability of human embryonic stem cell (hESC) lines in vitro is still poorly understood and the available data remains contradictory [2,3].Undoubtedly genetic instability has an impact on the differentiation abilities of hESCs and may also lead to tumor phenotype manifestations, thus, limiting further practical applications of such cells.Thus, strict control of the chromosome content and chromosomal rearrangements in human embryonic stem cells is of importance in improving their effi ciency in research and safety in practical applications.Additionally, studies on chromosomal rearrangements in hESCs may be a helpful to obtain new insights into chromosome regions involved in pluripotency maintenance or in 'malignization' of the cells.
However, hESCs chromosome preparations frequently encounter certain diffi culties, perhaps connected with hESCs biology and chromosome organization, thus making karyotyping problematic [4].Consequently, specialized and sophisticated methods must be applied to characterize exactly chromosomal rearrangements Chromosome rearrangements in human stem cell lines and involved breakpoints.In the present molecular cytogenetic study microdissection was used to generate specifi c probes derived from rearranged chromosomes in hESCs.These probes were used for reverse multicolor fl uorescence in situ hybridization (mFISH) analysis of hESM01 and hESM03 sublines and human lymphocytes.
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 [7].

Chromosomal rearra ngements 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 [5].The patterns revealed by the staining allowed to assume that chromosome 4 contained a partial deletion and chromosome 9 a partial duplication.These chromosomes were described as del(4) and dup(9).To confi rm these results and assign precisely the breakpoints region-specifi c microdissection DNA-probes were obtained from those derivatives: (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).

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

Discussion
Since t heir isolation in 1998 [15] hESCs have attracted a tremendous interest, primarily because of their potential practical applications in regenerative medicine.However, during the last twelve years only a limited number of mechanisms involved in self-maintenance of hESCs or directed differentiation to the particular lineage have been deciphered.Even the available information concerning hESCs genetic stability in vitro is controversial [2].
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 [5].hESM01r18 and hESM0309 cells showed a higher proliferation rate and more robust cultivation conditions compared with their parental cell lines.Consequently, these cell lines could be used as a model to study the effects of some particular chromosomal regions on the pluripotency maintaining or differentiation potential, in future.
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) [16].Possibly, hESM0309 cells could be a valuable model to study causal mechanisms of genetic instability and chromosomal abnormalities.So, to look for effective remedy of its prevention.The breakpoint 18q21.2observed in hESM01r18 cells was also detected in some cases of lymphomas [17] and acute lymphoid leukemia [18].The other breakpoint in chromosome18p11.31was not seen in tumors yet.
The existence of chromosomal abnormalities in the stem cells are often associated with carcinogenesis [2].Loss of chromosome 4 is a common feature of many tumor entities summarized in Gebhart and Liehr [19].Thus, 4q25 to 4q31.1 deleted in cell line hESM0309 could be a critical region for advanced cell proliferation.For the region 9q12 to 9q33 duplicated in the same cell line, similar gains of copy numbers were observed in tumor entities like head and neck cancer, histiocytoma, esophagus-, lung-, stomach-and uterus-carcinoma [19].Chromosome 18p11.31 to 18q21.2, however, present in hESM01r18 is rather involved in loss of copy numbers in human tumors than in gain -exceptions are lung cancer and neuroblastoma [19].
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.

Figure 1 .
Figure 1.Molecular cytogenetic analysis of chromosomes of hESM0309 line.a -FISH of microdissection DNA probe del(4) (green) with metaphase chromosomes lymphocytes of a healthy donor.b -Inverted DAPI banding of lymphocytes of healthy donor.

Figure 3 .Figure 2 .
Figure 3. Molecular cytogenetic analysis of hESM0309 by FISH with microdissection probes.a -dual hybridization of microdissection probes WCPder(9), (green) and WCP9, (red) with chromosomes of normal human lymphocytes.b -hybridization of PCP9C DNA probe (green) with chromosomes of hESM03der9.c -hybridization of РСРder(9)-1 (red) and РСРder(9)-2 (green) with chromosomes of normal human lymphocytes.Profi les of signals intensity along the chromosome are shown on the right.Red line -signal from РСРder(9)-1, green line -signal from РСРder(9)-2.Ideograms and C-banding of chromosome 9 are shown to the right of a and c.

Figure 4 .
Figure 4. Analysis of hESM01r18 cells.a -FISH of telomere specifi c probe (green) with chromosomes of hESM01r18 cells.The arrow indicates ring chromosome 18.b -FISH of microdissection der(18) probe with chromosomes of hESM01r18 cells.Arrow indicates chromosome der(18), arrow head indicates normal chromosome 18.c -FISH of microdissection der(18) probe with chromosomes of healthy donor's lymphocytes.Arrows indicate sites of DNA breakage during the formation of r(18)(p11.31q21.2).Chromosomes were counterstained with DAPI. a b c