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EMBRYONIC STEM CELLS

Transfer of a Human Chromosomal Vector from a Hamster Cell Line to a Mouse Embryonic Stem Cell Line

^aDipartimento di Genetica e Microbiologia "Adriano Buzzati Traverso" Università degli Studi di Pavia, Pavia, Italy;
^bIstituto di Tecnologie Biomediche del Consiglio Nazionale delle Ricerche di Segrate, Milan, Italy;
^cIstituto di Genetica Molecolare del Consiglio Nazionale delle Ricerche di Pavia, Pavia, Italy;
^d Department of Oncology Istituto di Ricerche Farmacologiche "Mario Negri" di Milano, Milan, Italy;
^e Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria Università degli Studi di Milano, Milan, Italy

Key Words. Human chromosomal vector • Mouse embryonic stem cell • Cell fusion Transchromosomic mouse embryonic stem cell line • Ploidy variation • Pluripotent gene markers

Correspondence: Luigi De Carli, Ph.D., Dipartimento di Genetica e Microbiologia, Via Ferrata 1, 27100 Pavia, Italy. Telephone: 39-0382-985554; Fax: 39-0382-528496; e-mail: decarli{at}ipvgen.unipv.it

Received on January 22, 2007; accepted for publication on June 26, 2007.

First published online in STEM CELLS EXPRESS  July 5, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Two transchromosomic mouse embryonic stem (ES) sublines (ESMClox1.5 and ESMClox2.1) containing a human minichromosome (MC) were established from a sample of hybrid colonies isolated in fusion experiments between a normal diploid mouse ES line and a Chinese hamster ovary line carrying the MC. DNA cytometric and chromosome analyses of ESMClox1.5 and ESMClox2.1 indicated a mouse chromosome complement with a heteroploid constitution in a subtetraploid range; the karyotypes showed various degrees of polysomy for different chromosomes. A single copy of the MC was found in the majority of cells in all the isolated hybrid colonies and was stably maintained in the established sublines for more than 100 cell generations either with or without the selective agent. No significant differences from the ES parental cells were observed in growth characteristics of the transchromosomic ES sublines. ESMClox1.5 cells were unable to grow in soft agar; when cultured in hanging drops, they formed embryoid bodies, and when inoculated in nude mice, they produced teratomas. They were able to express the early development markers Oct4 and Nanog, as demonstrated by reverse transcription-polymerase chain reaction assay. All these features are in common with the ES parental line. Further research using the transchromosomic ES sublines described here may allow gene expression studies on transferred human minichromosomes and could shed light on the relationships among ploidy, pluripotency, cell transformation, and tumorigenesis.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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The transgenic approach for gene localization, gene expression studies, and gene therapy is based on transfer of genetic material ranging in size from small DNA stretches to whole chromosomes. Fragmented or intact human chromosomes introduced into embryonic stem cells and segregating as autonomous elements in cell cultures or in living animals have been investigated by different authors [1–7]. The method most commonly used, although limited by its low efficiency, is the microcell-mediated chromosome transfer (MMCT), which allows the isolation of embryonic stem cell lines carrying the extra chromosome; the final goal is the generation of transchromosomic mice by injecting the chromosomally modified cells into recipient blastocysts. Using this approach, O'Doherty et al. [8] produced a chimeric mouse in which the majority of cells contained the human chromosome 21. These studies revealed the utility of transchromosomic mice for analyzing gene dosage effects on a variety of phenotypic traits and as models of disorders associated with chromosome number variation, such as Down syndrome.

Of particular interest for investigations on chromosome transfer in embryonic stem (ES) cells are human artificial chromosomes (HACs), which not only are useful for analyzing the effects of gene imbalance on embryo development and expression of tissue-specific functions but also can be exploited as potential vectors for gene therapy. Two possible sources of HACs are (a) the new formation of a functionally active chromosome from isolated minimum essential DNA sequences and (b) reduction of a pre-existing chromosome. Following this last approach, Shen et al. [2] succeeded in introducing a 4-Mb HAC derived from the long arm of the human Y chromosome into mouse embryonic stem cells, to generate transchromosomic mice. Ren et al. [9] gave the first demonstration of lineage-specific expression induction of transgenes in human mesenchymal stem cells by a HAC vector from human chromosome 21.

Here, we describe the isolation of transchromosomic mouse ES cell lines containing a HAC generated from a radiation-reduced chromosome 9-derived minichromosome (MC), which has the properties of a vector thanks to the presence of selectable markers and of the loxP sequence for site-specific recombination [10–12]. These cell lines were established from colonies isolated in fusion experiments between a human/hamster monochromosomic hybrid cell line (MClox) and a mouse ES cell line (E14). Our aim was to produce a mouse ES cell line capable of expressing human genes carried by a supernumerary, freely segregating chromosome, whose mitotic stability and distribution in the successive cell divisions could be followed over long culture periods.

To move the chromosome from the donor to the host cells, we used a simplified procedure of cell fusion-mediated chromosome transfer without separation of microcells. Using a combined pretreatment with colcemid and cytochalasin B of the MClox parental cell line, we succeeded in isolating in selective medium ES hybrid colonies retaining a single copy of the MC in the absence of hamster chromosomes. The heteroploid constitution found in all the isolated hybrid colonies and in the established cell lines would allow investigation of the effects of chromosome number variation on control of pluripotency, cell transformation, and tumorigenesis.

	MATERIALS AND METHODS

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Cell Culture
MClox is a monochromosomic human/hamster somatic hybrid originally obtained by cell fusion between hypoxanthine-quanine-phosphoribosyl-transferase–negative (HPRT^–) Chinese hamster ovary (CHO) and a lymphoblastoid line from a patient carrying an MC identified as a chromosome 9 derivative in a complex mosaic karyotype [10]. The centromeric region of MC contains the neomycin gene (neo) and five copies of the pG12 plasmid carrying a loxP site plus the puromycin resistance gene (puro) [11]. The cell line was maintained by standard culture procedures in RPMI 1640 medium (Euroclone, Pero, Italy, http://www.euroclone.net), supplemented with 10% fetal calf serum (Euroclone), and incubated at 37°C with 5% CO₂.

E14 is a mouse embryonic stem cell line from 129/Ola mice [13], kindly supplied by R. Klein (Max Plank Institute, Martinsried, Germany). The cells were grown on 100-mm Petri dishes coated with 0.1% gelatin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Euroclone) containing 15% fetal bovine serum (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and 1,000 U/ml leukemia inhibitory factor (ESGRO; Chemicon International, Temecula, CA, http://www.chemicon.com); the medium was conditioned with mouse embryonic fibroblasts (MEFs) grown for 2 days in log phase. MEFs were obtained by mincing and dissociating 13.5 days post coitum CD-1 embryos (Charles River Laboratories, Wilmington, MA, http://www.criver.com) by collagenase (0.25% in phosphate-buffered saline [PBS] plus 20% fetal calf serum); primary cultures were maintained in DMEM (Euroclone) supplemented with 10% fetal calf serum (Euroclone) and incubated at 37°C with 7% CO₂.

Cell Fusion and Colony Isolation
MClox cells arrested in mitosis and E14 unsynchronized cells were fused. MClox cells (4 x 10⁶) were inoculated in four T25 flasks (tissue culture flasks, 25 cm); colcemid was added to the cultures at a final concentration of 0.2 µg/ml when the cells reached approximately 80% confluence. After 24 hours of incubation, mitoses were mechanically removed from monolayers, incubated in growth medium containing 20 µg/ml cytochalasin B at 37°C for 4 hours with constant stirring, and then mixed with an equal amount of E14 monodispersed cells. The mixture with a total of 10⁷ cells was resuspended in 10 ml of 2.5% polyethylene glycol (PEG) (mol. wt. 1500) in DMEM and kept in this solution for 20 minutes at 4°C. After centrifugation at 160g, 1 ml of a prewarmed solution of 50% PEG was poured onto the cell pellet over 2 minutes; 10 ml of fresh DMEM was then gradually added to the cell suspension over 10 minutes. After washing with PBS, the cells were resuspended in MEF-conditioned ES medium, distributed in two 60-mm Petri dishes, and incubated for a recovery period of 48 hours at 37°C. The cells were finally plated, 10⁵ cells per 100-mm Petri dish in selective medium containing HAT (100 µM hypoxanthine, 0.4 µg/ml aminopterin, and 16 µM thymidine) and puromycin at different concentrations (0.3–10 µg/ml).

Chromosome Analysis
Chromosome analysis was done either on slide preparations of cell suspensions or on cells grown on coverslips; for the first method, monolayer cell cultures were treated with colcemid at a final concentration of 0.1 µg/ml for 2 hours at 37°C, and mitoses were mechanically removed. After hypotonic treatment with 0.075 M KCl and fixation in methanol:acetic acid (3:1 vol/vol), the cell suspension was dropped onto a slide and air-dried. Cells grown on coverslips were treated the same way except that the colcemid concentration was 0.3 µg/ml. Chromosome counts and karyotype analyses were done on metaphases stained with a standard Q banding.

Fluorescence In Situ Hybridization
CHO genomic DNA and pMR9A plasmid, identifying a chromosome 9-specific -satellite DNA subfamily [14], were used as DNA probes. The probes were labeled using a nick-translation reagent system (Invitrogen) and Bio-16-dUTP (Roche Diagnostics S.p.A, Milano, Italy, http://www.roche-applied-science.com) according to the manufacturers' protocols. The labeled probes were resuspended in hybridization buffer (50% formamide, 10% dextran sulfate, 1x Denhart's solution, 0.1% SDS, 40 mM Na₂HPO₄, pH 6.8, 2x standard saline citrate [SSC]) containing a 10x excess of salmon sperm DNA and denatured at 80°C for 10 minutes. In situ hybridization was performed essentially as previously described [10]. In brief, slides were treated with RNase (type III) at 37°C for 1 hour and dehydrated through the ethanol series before denaturation in 70% formamide/2x SSC. Hybridization was done overnight at 42°C. Stringent washings were done in 50% formamide/2x SSC at 42°C.

For single signal detection, the slides were incubated with fluorescein isothiocyanate (FITC)-conjugated cell sorting grade avidin D (avidin DCS) (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com; Invitrogen), then with biotin-conjugated anti-avidin D antibody (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), and finally with FITC-conjugated avidin DCS. Avidin and all the antibodies were used at a final concentration of 5 µg/ml.

For double signal detection, the slides were incubated first with FITC-conjugated avidin DCS and tetramethylrhodamine B isothiocyanate (TRITC)-conjugated sheep anti-digoxigenin antibody (Roche Diagnostics), then with biotin-conjugated anti-avidin D antibody and TRITC-conjugated rabbit anti-sheep antibody (Chemicon International), and finally with FITC-conjugated avidin and TRITC-conjugated anti-rabbit antisera (Calbiochem, San Diego, CA, http://www.emdbiosciences.com). Avidin and all the antibodies were used at a final concentration of 5 µg/ml.

All the slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (0.01 µg/ml) (Sigma-Aldrich) and mounted in Tris-HCl (pH 7.5) 90% glycerol containing 2% 1,4-diazabicyclo(2.2.2)octane antifade (Sigma-Aldrich). Slides were scored under a Zeiss Axioplan fluorescent photomicroscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) equipped with a cooled CCD camera (Photometrics, Tucson, AZ, http://www.photomet.com). Images were captured with IPlab spectrum P software (BD Biosciences Bioimaging, Rockville, MD, http://www.scanalytics.com).

Indirect Immunofluorescence
Cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at 4°C; permeabilized in 0.05% Tween 20, 0.5% bovine serum albumin in PBS for 10 minutes at room temperature; and then incubated for 1 hour at 37°C with the polyclonal anti-Oct4 antibody (Santa Cruz) diluted 1:500 in PBS. The binding of the antibody was revealed with a FITC-conjugated antibody (Sigma-Aldrich), diluted 1:500. Nuclei were counterstained with 200 µg/ml DAPI. Slides were observed using an Olympus IX71 optical microscope equipped with a x60 objective (Olympus, Tokyo, http://www.olympus-global.com). Images were taken with a Cool SNAP_ES digital camera (Photometrics) using the MetaMorph software.

Flow Cytometric Analysis
Pellets from 5 x 10⁵ to 1 x 10⁶ cells, carefully dispersed in 100 µl of cold PBS, were fixed in 5 ml of 70% cold (–20°C) ethanol and stored at 4°C until analysis. After washing again in PBS cells were stained with 1.5 ml of propidium iodide (PI) (50 µg/ml) containing RNase (100 U/ml) and Nonidet P40 (0.05%). Samples were stored overnight at 4°C in the dark and analyzed by flow cytometry. Measurements were done with a Partec PAS II flow cytometer (Munster, Germany, http://www.partec.com) equipped with a dual excitation system (argon ion laser and HBO 100-W arc lamp). The 488 nm blue line of the laser was used to excite PI intercalated into DNA complex. A preliminary instrument alignment and control was always set up (with rat thymocytes stained with PI) to ensure the best instrumental analytical performance. Immediately before measurement, each sample was filtered through Filcons 100 (ConsulTS, Turin, Italy, http://www.consul-ts.com) to remove cell clusters. For a sample measurement, a minimum of 20,000 events were acquired. The red fluorescence emission band over 610 nm (FL3) was collected, converted, and stored as DNA distribution values by a dedicated computer integrated into the instrument.

Embryoid Bodies
A total of 400 ES E14 or ESMClox1.5 cells were inoculated in hanging drops (30 µl) on covers of 120 mm hydrophobic Petri dishes in differentiation medium (MEF-conditioned ES medium without leukemia inhibitory factor [LIF]). After 3 days of incubation, the drops were transferred to the bottom of the plates with the addition of 1 ml of medium and further incubated for 5 days. The nascent EBs were plated separately onto gelatin-coated 24-microwell plates.

Colony Formation in Soft Agar
A 3-ml layer of 2% agar (wt/vol) in MEF-conditioned medium was poured in 60-mm Petri dishes. ES E14, ESMClox1.5, or HeLa cells, used as positive control in the assay, were resuspended in 0.33% agar (wt/vol) in MEF-conditioned medium at a density of 10⁴ cells per 3 ml. Cell suspension (3 ml) was poured on the top of the base layer, allowed to solidify, and incubated at 37°C with 5% CO₂. Cells were fed twice a week with 0.3 ml of fetal calf serum (Euroclone) and observed weekly under the microscope for colony formation.

Reverse Transcription-Polymerase Chain Reaction
First-strand cDNAs were synthesized directly from ES E14, MEF, and ESMClox1.5 cells and from ES E14 EBs and ESMClox1.5 EBs using the Superscript CellsDirect cDNA Synthesis Kit (Invitrogen) according to the manufacturer's protocol. Polymerase chain reactions (PCRs) were performed using specific primer pairs (supplemental online Table 1). PCRs were performed under the following conditions: denaturing for 30s at 94°C, annealing temperature for 30s, and extension for 30s at 72°C, repeated 30 times. The PCR products were visualized by 2% agarose gel electrophoresis stained with ethidium bromide.

Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L.n.116, G.U., suppl. 40, 18 febbraio 1992, Circolare No. Eight, G.U., 14 luglio 1994) and international (EEC Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996) laws and policies.

	RESULTS

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Characterization of the Parental Cell Lines
The mouse ES cell line E14 used as the MC recipient has been adapted in our laboratory to grow in the absence of a feeder layer of MEFs: it has acquired independence from the feeder after repeated passages in MEF-conditioned medium. Under these conditions, it maintains a normal diploid karyotype; the generation time is 24 hours, and the plating efficiency is close to 10%.

The hamster cell line containing the MC (MClox) has a near-diploid/pseudodiploid chromosome constitution, with a modal chromosome number of 21. The MC is present in approximately 60% of the cells. The generation time is 12 hours, and the plating efficiency may reach 80%.

Fusion Experiments and Isolation of ESMClox Hybrid Colonies
Fusion experiments followed the procedure described in Materials and Methods. To facilitate the loss of hamster chromosomes in hybrid cells, the parental MClox cell line was pretreated with colcemid for 24 hours, and the collected mitoses were exposed to cytochalasin B for 4 hours.

Three weeks after fusion, a total of 31 colonies, of likely clonal origin, grown in HAT medium with puromycin, were isolated and expanded in culture for determination of the DNA content and for cytological preparations. DNA flow-cytometric analysis was done on samples of 5 x 10⁵ cells from 23 colonies. Table 1 reports the data on the DNA content expressed as the mean value of the G0/G1 peak, with the corresponding coefficient of variation (CV), for 23 hybrid colonies, for ES parental cells, and for an MEF control. Whereas in the parental and MEF cells, which have a normal diploid constitution, the position of the G0/G1 peak was around 50, in the majority of the hybrid colonies (13 of 23), the peak position was between 86 and 100, corresponding to a "near-tetraploid" DNA content. In the remaining colonies, with one exception (ESMClox1.5), in addition to the main peak between 90 and 97, there was a second peak between 59 and 71, indicating a "near-triploid" DNA value. A minor peak, corresponding to a diploid DNA, value was observed in two colonies (ESMClox1.20 and ESMClox1.5), which may suggest that the cell populations contain rare normal diploid cells carrying the MC. The possibility of sorting out these cells to establish a euploid ESMClox line has been taken into consideration.

To detect the MC and the residual CHO chromosomal material, metaphases from 30 colonies were hybridized in situ with chromosome 9-specific -satellite DNA (pMR9A) and CHO genomic DNA probes, respectively. Twenty metaphase spreads for each colony were analyzed. The results are summarized in Table 1. All except two of the colonies contained the MC free or translocated on hamster residual chromosomes, the proportion of MC-positive mitoses varying from 10% to 95%. Interestingly, in 14 of 30 colonies (47%), the MC was the only chromosome transferred from the donor cell line.

One of the colonies with the highest frequency of MC-positive cells and with the typical ES morphology was expanded to establish a subline designated as ESMClox1.5. Another subline (ESMClox2.1) with the same characteristics was established from one of eight colonies isolated in an independent fusion experiment, following the same protocol as before. All the colonies showed a heteroploid mouse complement with modal chromosome numbers comparable to those in the first fusion experiment, with the majority of cells containing the MC (data not shown).

DNA and Chromosome Analyses of ESMClox1.5 and ESMClox2.1 Sublines
The DNA flow-cytometric profiles, together with the distributions of chromosome numbers of the two sublines and of the ES control line are, shown in Figure 1. The control showed a DNA distribution typical of diploid cells along the cell cycle phases. The main peak, representing G0/G1 cells, was around channel 50, whereas the corresponding G2 and mitoses were evident in channels 95–105. The DNA distribution of the sublines ESMClox1.5 and ESMClox2.1 clearly showed a shift toward double or nearly double the control values. The first G0/G1 peak was in fact located around channels 90–100, and the corresponding G2 and mitoses were between channels 180 and 200. A small subfraction of "residual" diploid population was noted in the first part of the DNA histogram of ESMClox1.5 (Fig. 1), confirming the result of the DNA analysis on the original hybrid colony (Table 1).

View larger version (20K): [in this window] [in a new window]   Figure 1. DNA cytometric profiles and chromosome number distributions (insets) of E14 line (A), ESMClox1.5 (B), and ESMClox2.1 (C) sublines. Abbreviation: ES, embryonic stem.

View larger version (49K): [in this window] [in a new window]   Figure 2. Cytogenetic analysis. Fluorescence in situ hybridization on metaphase spreads from ESMClox1.5 (A) and ESMClox2.1 (B) cells was performed with pMR9A probe labeled with digoxigenin and detected with tetramethylrhodamine B isothiocyanate (TRITC)-conjugated sheep anti-digoxigenin antibody followed by TRITC-conjugated rabbit anti-sheep antibody (red signal); the chromosomes were counterstained with 4',6-diamidino-2-phenylindole (blue). (C): Representative Q-banded karyotype of ESMClox1.5 cells.

View larger version (62K): [in this window] [in a new window]   Figure 3. Phase contrast photographs of cultured cells and expression of differentiation markers. (A): Colonies of E14 cells. (B): Monolayer of MClox cells. (C): Colonies of ESMClox1.5 cells. (D): ESMClox1.5 embryoid body (EB). Scale bar = 10 µm (A–C) and 300 µm (D). (E): Reverse transcription-polymerase chain reaction analysis on germ layer markers. Lane 1, E14 cells; lane 2, E14 EBs; lane 3, ESMClox1.5 EBs.

View larger version (30K): [in this window] [in a new window]   Figure 4. Analysis of pluripotent markers. (A): Oct4 and Nanog expression analysis by RT-PCR; β-actin, positive control. (B): Immunofluorescence analysis. Cells were stained with an anti-Oct4 antibody (yellow signal; 1, 3, and 5) and counterstained with 4',6-diamidino-2-phenylindole (2, 4, and 6). MEF cells were negative to the antibody (1); E14 (2) and ESMClox1.5 (3) cells were positive. Abbreviations: ES, embryonic stem; MEF, mouse embryonic fibroblast.

View larger version (138K): [in this window] [in a new window]   Figure 5. Histology of teratomas developed in nude mice after injection of ESMClox1.5 cells. The mesenchymal component consists of abundant fibro-adipose tissue embedding portions of well-differentiated cartilaginous tissue (*) peripherally associated with immature haphazardly arranged striated muscular fibers (arrowhead). H&E staining; magnification, x100.

Propagation of the ESMClox1.5 Subline
After the first passages in culture, the ESMClox1.5 subline was cryoconserved in liquid nitrogen. Cell samples were thawed at intervals of 3–4 months. The overall period of culture was 6 months, with 50–60 passages resulting in 100–120 cell population doublings. The growth characteristics remained constant, and no changes were seen in the morphology of cells and colonies. The distribution of chromosome numbers was the same as in the subline at the first passages, with a modal class of 65. The MC was found in up to 95% of cells at passage 40, either with or without the selective agent.

A derivative of ESMClox1.5 was established in vitro from dissociated fragments of teratomas produced in mice. The chromosome number distribution and the growth characteristics of this newly isolated subline from teratoma tissue were the same. The MC was retained in 80% of the cells, as indicated by FISH analysis with the pMR9A probe (Fig. 6).

View larger version (21K): [in this window] [in a new window]   Figure 6. Fluorescence in situ hybridization (FISH) analysis of metaphase spreads prepared from cells of the ESMClox1.5 subline derived from teratoma fragments. FISH was performed as indicated in Figure 2.

	DISCUSSION

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In view of its pluripotency, the recipient cell line may constitute a suitable host for the expression of exogenous genes transferred into the human minichromosome, used as a tool for genetic analysis or for physiological studies.

Since the fusion method that we used did not involve the separation of microcells from the donor cells arrested in mitosis, these donor cells should have contained an intact chromosome complement. We were then faced with the problem of directional chromosome segregation in mouse/hamster hybrids. In this regard, the literature is conflicting, although it indicates a tendency to loss of mouse chromosomes [16–18]. In our experiments, the construction of a hybrid containing only the minichromosome would have required the opposite dynamics, leading to the loss of hamster chromosomes. This reverse effect is indeed what we obtained, presumably because of the combined treatment of the MClox cell line with a spindle formation inhibitor and a microfilament disrupting agent coupled to a potent selective system, which proved highly effective in inducing the loss of hamster chromosomes. The process took place in one or a few steps, presumably before the formation of the hybrid colonies, between three and four cell divisions after fusion. This suggests a mechanism of massive loss of hamster chromosomes, possibly through unbalanced distribution of chromosomes in multipolar mitoses, with the persistence of the MC carrying the selective marker.

The human MC was identified in the majority of cells of all the isolated hybrid colonies. In the sublines maintained in culture, it was detected in more than 90% of the cells in the periodic controls performed every 10 passages, in either the presence or absence of the selective agent. This points to a high mitotic stability of the MC, probably thanks to a normal centromeric function and regular interactions with the spindle apparatus. No structural alteration could be detected by cytogenetic analysis. These data agree with the report by Kazuki et al. [19] on a human chromosome 21 fragment transferred in mice. On the other hand, Shen et al. [2] found that a minichromosome derived from the human chromosome Y was unstable in mouse ES cells but could be stabilized by exchanges of mouse centromeric sequences. These contrasting findings might be explained by structural differences between individual chromosomes and/or between methods used to incorporate chromosomes into the host cell.

As far as the origin of the subtetraploid chromosome set in the hybrids containing the MC is concerned, it may be supposed that the initial hybrid cell was derived from a polykaryocyte containing two nuclei of the parental ES and one nucleus of the CHO line. Alternatively, the initial fusion product might have been a heterodikaryon with one hamster nucleus and one mouse nucleus; then, the mouse chromosome set would have been doubled by endoreduplication, which is occasionally observed in the cell lines used in our experiments. Successive losses and gains of individual chromosomes, following the massive segregation of hamster chromosomes, would have generated a hybrid with the chromosome distribution observed. Whatever the mechanism of origin of the heteroploidy in the ES cells carrying the MC, cell fusion is likely to be the causative event. Notably, the same chromosome pattern was observed in the vast majority of colonies isolated in two independent experiments. Reports in the literature on the dynamics of chromosome complements in mouse ES cell lines point to a marked stability of the karyotype, although karyotypic abnormalities are not infrequent and prolonged cell culturing may affect the diploid chromosome constitution [20, 21]. In contrast, mouse cell cultures from a variety of somatic tissues, after a period of crisis, gradually turn into stable cell lines with a heteroploid chromosome constitution associated with karyotype instability [22, 23]. A similar chromosomal pattern is exhibited by our ESMClox sublines. As ascertained by the flow cytometric analysis, cells with a diploid DNA content are present in the hybrid cell population but tend to be eliminated because the hyperdiploid cells are at a selective advantage. The probability of recovering diploid cells by sorting and establishing a euploid cell line would greatly increase if the isolation were made a few generations after fusion, when the cell population displays a wide spectrum of chromosome sets. The ploidy variation and the incorporation of the MC in the ESMClox1.5 subline apparently had no consequences on the pluripotent phenotype or on the ability to differentiate in vitro as shown by RT-PCR analyses and by immunofluorescence studies on Oct4 marker. This finding appears unusual for a cell line that has undergone heteroploid transformation, an event generally associated with the loss of many of the differentiated properties that distinguish the tissue from which the cells were isolated [24]. Apparently, ES cells do not follow these dynamics. Similar conclusions can be drawn for the ability to produce teratomas, which was retained in the ESMClox1.5 subline. An evidence against occurrence of cell transformation is also provided by the lack of an anchorage-independent growth. In this connection, it is worth mentioning that transfection with a dominant-negative allele of the MKK4 gene, a putative suppressor gene implicated in several transforming functions, makes ES cells, normally unable to grow in soft agar, anchorage-independent and more tumorigenic [25].

There is no doubt that the observed heteroploid constitution of our transchromosomic mouse embryonic stem cell lines changes the prospects of in vivo experimentation originally aimed at generating mice exploitable as models for human chromosomal pathology; however, the ESMClox hybrid cells hold promise for use in in vivo studies on the effects of chromosome number variation on early embryogenesis, cell transformation, and tumor development. In this regard, it is worth mentioning that Pralong et al. [26] demonstrated that tetraploid mouse ES cells produced by fusion are capable of integrating into inner cell mass of diploid preimplantation blastocysts.

Moreover, the established mouse ES cell lines containing a human chromosomal vector may provide an in vitro model system suitable for analyzing the expression and regulation of cell lineage-specific genes. These genes may either be already present in the MC or be inserted by gene targeting through homologous recombination, with a view to possible applications in stem cell-mediated gene therapy.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank Prof. Silvia Garagna and Dr. Paola Rebuzzini (University of Pavia) for providing the anti-Oct4 antibody and for help in the immunofluorescence experiments. The work was supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (Cofinanziamento 2004), from the University of Pavia (FAR), and from Eurostells (STELLAR). M.P. is a fellow of the Fondazione A. Buzzati Traverso.

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