Chromosome Research

, Volume 16, Issue 8, pp 1075–1084

Separation and maintenance of normal cells from human embryonic stem cells with trisomy 12 mosaicism

Authors

  • Hye Won Seol
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
  • Sun Kyung Oh
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
    • Department of Obstetrics and GynecologySeoul National University College of Medicine
  • Yong Bin Park
    • Central Research InstituteSam Jin Pharm. Co. Ltd.
  • Hee Sun Kim
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
    • Department of Obstetrics and GynecologySeoul National University College of Medicine
  • Jin Ah Baek
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
  • Jin Seo
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
  • Eun Hee Kim
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
  • Seung Yup Ku
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
    • Department of Obstetrics and GynecologySeoul National University College of Medicine
  • Seok Hyun Kim
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
    • Department of Obstetrics and GynecologySeoul National University College of Medicine
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
    • Department of Obstetrics and GynecologySeoul National University College of Medicine
  • Shin Yong Moon
    • Institute of Reproductive Medicine and Population, Medical Research CenterSeoul National University
    • Department of Obstetrics and GynecologySeoul National University College of Medicine
Article

DOI: 10.1007/s10577-008-1258-y

Cite this article as:
Seol, H.W., Oh, S.K., Park, Y.B. et al. Chromosome Res (2008) 16: 1075. doi:10.1007/s10577-008-1258-y
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Abstract

Human embryonic stem cells (hESCs) are pluripotent and hold great promise as useful tools in basic scientific research and in the field of regenerative medicine. However, several studies have recently reported chromosomal abnormalities such as gains of chromosomes 12, 17 and X in hESCs. This genetic instability presents an obstacle in the application of hESCs as sources of cell therapies. We found that trisomy 12 was correlated with changes in hESC colony morphology during hESC maintenance. In this study, we investigated whether normal and trisomy 12 cells could be separated in hESC cultures displaying trisomy 12 mosaicism with two types of colony morphology using a mechanical transfer technique. Eight sublines were cultured from eight hESC colonies displaying normal or abnormal morphology. Four sublines with normal morphology had normal chromosome 12 numbers, whereas the four sublines with abnormal morphology displayed trisomy 12. These results indicate that a hESC colony with a minor degree of chromosomal mosaicism and normal morphology could proceed to a colony with normal chromosomes after prolonged cultures with mechanical transfer. Therefore, analysis of cultures for chromosomal abnormalities when changes in colony morphology are observed during culture is essential for maintaining normal hESC lines.

Keywords

chromosomal abnormalitycolony morphologyhuman embryonic stem celltrisomy 12

Abbreviations

AP

alkaline phosphatase

EC

embryonic carcinoma

FISH

fluorescence in-situ hybridization

hESC

human embryonic stem cell

IVF

in-vitro fertilization

SCID

severe combined immunodeficiency

SNUhES4

Seoul National University human Embryonic Stem cell line 4

SSEA-4

stage specific embryonic antigen-4

TGCT

testicular germ cell tumour

Tra-1-60

tumour rejection antigen-1-60

Introduction

Human embryonic stem cells (hESCs) are isolated from the inner cell mass of day 5 blastocysts procured from excess in-vitro fertilization (IVF) embryos that would otherwise be discarded (Thomson et al.1998). hESCs can be maintained in an undifferentiated state in vitro for long periods and can differentiate into all of the cell types of the human body. hESCs have the potential for use in the study of human development and in cell-based therapies for degenerative diseases or injuries (Reubinoff et al.2000, Cowan et al.2004).

hESCs appear genetically stable in prolonged culture (Rosler et al.2004, Hoffman & Carpenter 2005, Caisander et al.2006), but various chromosomal abnormalities have been reported since Draper et al. (2004) noted abnormal chromosomes of chromosomes 12 and 17 in hESCs (Buzzard et al.2004, Imreh et al.2006, Li et al.2006, Plaia et al.2006). A number of reports have indicated that bulk passaging techniques using enzymatic dissociation are accompanied by increased genetic instability, including chromosomal abnormalities seen in cancer and embryonic carcinoma (EC) cells (Draper et al.2004, Mitalipova et al.2005). hESCs with chromosomal abnormalities could easily be maintained in an undifferentiated state because of growth advantages over normal cells (Enver et al.2005, Herszfeld et al.2006). Also, hESCs with chromosomal abnormalities are often used for studying cancer development and its underlying mechanisms (Zeng et al.2004). In particular, studies of cells with trisomy for chromosome 12 could help uncover characteristics and key players in cancer development (Imreh et al.2006, Baker et al.2007, Gertow et al.2007), since this abnormality often occurs in testicular germ cell tumours (TGCT) (Summersgill et al.2001, Zafarana et al.2003) and hESCs. However, it is also critical to maintain hESCs with normal chromosome number for use in cell-based therapies.

We have observed a variety of chromosomal abnormalities in several cultured hESC lines established in our laboratory (Kim et al.2005, Oh et al.2005a). Interestingly, in the case of trisomy 12, we found that this chromosomal anomaly was correlated with changes in hESC colony morphology. In this study, we observed morphological changes in some SNUhES4 colonies after mechanical passaging and found cells with both 47,XY,+12, and 46,XY chromosomal patterns in those colonies by cytogenetic and FISH analysis. Therefore, we investigated whether normal and trisomy 12 cells could be separated from trisomy 12 mosaic cultures with two types of colony morphology using a mechanical transfer technique, and whether this separation is helpful for maintaining a normal karyotype in hESCs.

Materials and methods

Cell culture

hESC lines used in this study (SNUhES3, SNUhES4, SNUhES11 and SNUhES16) were maintained on a feeder layer of mitomycin C-inactivated STO cells aspreviously described (Kim et al.2005, Oh et al.2005a). Briefly, undifferentiated hESCs were cultured in DMEM–F12 medium supplemented with 20% (v/v) knockout serum replacement, 1 mM l-glutamine, 4 ng/ml basic fibroblast growth factor, 1% non-essential amino acids, 50 U/ml penicillin and 50 μg/ml streptomycin and 0.1 mM 2-mercaptoethanol (all from Gibco Invitrogen, San Diego, CA, USA). All hESCs were cultured in an atmosphere of 5% CO2 in humidified air at 37°C. Culture medium was changed daily, and hESCs were passaged onto fresh STO cells once per week. hESC colonies were dissociated during serial passage by mechanical passaging with a glass pipette as previously described by Oh et al. (2005b).

Karyotyping and FISH analysis

To arrest the cell cycle at metaphase, hESCs were cultured in medium supplemented with 0.1 mg/ml colcemid (Invitrogen) for up to 4 h. Cells were harvested and fixed for conventional cytogenetic analysis.More than 20 metaphase cells were karyotyped andanalysed using chromosome image processing via ChIPS (GenDix, Seoul, Korea) and Cytovision (Genetix, New Milton, Hampshire, UK) systems. For FISH analysis, 10 hESC colonies were dissociated into single cells, fixed on glass slides, and hybridized with fluorescent probes for chromosome 12 (CEP12, SpectrumOrange; Abbott Molecular, Des Plaines, IL, USA) and chromosome X (CEPX, SpectrumGreen; Abbott Molecular). Hybridization and washing of slides were performed as manufacturer’s instructions. From 300 interphase cells, chromosome 12 number was counted using a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan) and analysed using ChIPS-FISH and Cytovision-FISH systems.

Immunostaining

Cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature. Fixed cells were washed with PBS, then 3% bovine serum albumin (BSA) was added to inhibit nonspecific binding. After antibody incubations, cells were visualized using an epifluorescence microscope (Eclipse TE2000-U, Nikon). The following primary antibodies were used for immunocytochemistry: mouse anti-Oct-3/4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SSEA-4, TRA-1-60 and TRA-1-81 antibodies (all from Millipore, Billerica, MA, USA). Alexa Fluor 488-labelled donkey anti-mouse IgG and Alexa Fluor® 594-labelled donkey anti-mouse IgG (Molecular Probes, Inc., Eugene, OR, USA) were used as secondary antibodies.

Analysis of alkaline phosphatase activity

Alkaline phosphatase activity was assayed using the Alkaline Phosphatase Detection kit (Sigma-Aldrich, St.Louis, MO, USA) according to the manufacturer’s instructions. Positively stained cells were observed using a stereo microscope (SMZ800, Nikon).

Results

We established and cultured several hESC lines (Kim et al.2005, Oh et al.2005a) and observed normal karyotypes as well as a variety of chromosomal abnormalities in cultured hESC lines. Particularly in the case of the SNUhES3 hESC line, the morphology of colonies with trisomy 12 differed from that of colonies with a normal karyotype. SNUhES3 colonies showed two types of morphology: normal colonies appeared thin and bright and were homogeneous in the centre of the colony (Figure 1A) and colonies with trisomy 12 appeared thick, dark and heterogeneous (Figure 1B). With this criterion, we could separate normal and trisomy 12 karyotype cells in SNUhES11 and SNUhES16 as well as in SNUhES3 (Figure 2). We observed similar morphological changes in cultures of the SNUhES4 hESC line, with mixed populations of colonies with normal and abnormal morphology in a single culture dish (Figure 3A). Enlarged images of colonies with normal morphology and abnormal colony morphology are shown in Figure 3B and C, respectively. Using cytogenetic and FISH analyses, we observed chromosomal mosaicism (e.g., 47,XY,+12/46,XY) in hESCs with these two types of colony morphology (Figure 3D). Cells containing two or three red signals (chromosome 12) were simultaneously observed in cultures at passage 21 + 6. Cells with two red signals were confirmed as 46,XY (normal chromosome number) and cells with three red signals were confirmed as 47,XY,+12 by karyotyping (Figure 3E–H). We also observed normal karyotypes and 47,XXY cells in SNUhES4 cultures after freezing and thawing (Figure 3I). However, at passage 7 + 39, all of the cells in SNUhES4 cultures possessed the 47,XXY chromosomal abnormality. Because we did not monitor hESC colony morphology over time, we could not determine the exact timing of changes from normal karyotypes to abnormal karyotypes.
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Figure 1

Two types of colony morphology according to karyotype were observed in hESCs cultured on mitomycin C-inactivated STO feeder cells by mechanical passaging using a stereo microscope. (A) Colony morphology with normal karyotype (SNUhES3, day 6, passage 97). This colony type was thin, bright and homogeneous. (B) Colony morphology with trisomy 12 (SNUhES3, day 6, passage 97). This colony type was thick, dark and heterogeneous.

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Figure 2

Separation of normal and trisomy 12 cells using mechanical passaging in hESCs with morphological difference of colonies. Normal morphology colony (A) with 46,XY normal karyotype (G) of SNUhES3 (day 7, passage 95), (B, H) SNUhES11 (day 5, passage 26) and (C, I) SNUhES16 (day 6, passage 72). Abnormal morphology colony (D) with 47,XY,+12 abnormal karyotype (J) of SNUhES3 (day 7, passage 95), (E, K) SNUhES11 (day 5, passage 26) and (F, L) SNUhES16 (day 6, passage 72). Colony morphologies of SNUhES3 and SNUhES11 were imaged by stereomicroscopy, those of SNUhES16 were imaged by phase-contrast microscopy.

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Figure 3

Colony morphology, chromosome analysis and karyotyping pedigree after freezing and thawing of SNUhES4 cells. (A) Two types of colony morphology (at passage 21 + 6) were mixed in a culture dish. One type had normal morphology (thin and bright). The other type had abnormal morphology (thick with a dark centre). In the dish, the ratio of the two types of colony (normal:abnormal) was 1:2. (B) Normal colony enlarged (solid lined black circle). (C) Abnormal colony enlarged (dotted black circle). (D) Two or three red signals (chromosome 12) were observed in cells of colonies at passage 21 + 6. (E) Cells with two red signals were confirmed as having a normal 46,XY karyotype. (F) Cells with three red signals were confirmed as 47,XY,+12 (D, E, F, magnification: ×1000). (G) 46,XY normal karyotype. (H) 47,XY,+12 abnormal karyotype. (I) Upon initial derivation, SNUhES4 showed diploid karyotypes; chromosome composition up to passage 100 is shown. After repeated freezing and thawing, normal and abnormal karyotypes such as 47,XXY or trisomy 12 were observed. pm+n: m indicates freezing and thawing passage. n indicates cultured passage after thawing.

To verify that colonies were completely composed of either normal or abnormal cells in trisomy 12 mosaic cultures, eight hESC colonies were selected by their morphological characteristics and cultured. Four colonies with normal (a, b, c, d) and four colonies with abnormal (e, f, g, h) morphology were isolated, and chromosome 12 number was analysed at every passage by FISH (Figure 4). After colony isolation, the features of each colony were retained (Figure 5A, B). Two red signals for chromosome 12 were detected in normal colonies (a, b, c, d), but three red signals (trisomy 12) were detected in morphologically abnormal colonies (e, f, g, h) (Figure 5C). Regardless of morphology or karyotype, all of the colonies expressed undifferentiated hESC markers such as alkaline phosphatase activity, Oct-4, SSEA-4, Tra-1-60 and Tra-1-81 (Figure 5D). In sublines with normal colony morphology (a, b, c, d), a lower percentage of chromosome 12 abnormalities were present at passage 21 + 9. After passage 21 + 12, each of these colonies possessed cells with normal chromosome 12 numbers. Likewise, sublines with abnormal morphology (e, f, g, h) displayed complete trisomy 12 after passage 21 + 12, although these sublines included a low percentage of cells with normal chromosome 12 numbers at passage 21 + 9 (Figure 6).
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Figure 4

Strategy for separation of normal hESCs from trisomy 12 mosaic cultures. Detection: Two types of colony morphology, normal and abnormal, were observed in a culture dish at six passages after thawing (passage 21 + 6) after previous freezing at passage 21 in SNUhES4 cells. Selection: Four colonies (a, b, c, d) with normal morphology and four colonies (e, f, g, h) with abnormal morphology were selected by mechanical transfer. Propagation: One colony was split into a four-well culture dish and expanded for several passages. Thus, eight colonies were expanded into eight sublines. Separation: Cells with normal or trisomy 12 karyotypes were separated from cultures with trisomy 12 mosaicism, maintained by mechanical transfer and assayed by FISH analysis.

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Figure 5

Colony morphology, detection of chromosome 12 number and expression of undifferentiated cell markers in eight sublines of SNUhES4. (A) Normal colony morphology at passage 21 + 14. (B) Abnormal colony morphology at passage 21 + 14. (C) Two red signals corresponding to chromosome 12 centromeres (CEP 12, SpectrumOrange) were detected in sublines a, b, c, d and three red signals were detected in e, f, g, h. One green signal (chromosome X) in each interphase cell was detected in colonies with normal and abnormal morphology (magnification ×1000). (D) Expression of undifferentiated markers for hESCs was detected in SNUhES4 with normal and abnormal colony morphology.

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Figure 6

Separation of normal or trisomy 12 cells from hESCs with trisomy 12 mosaicism. After eight sublines (a–h) were separated according to colony morphology from the SNUhES4 line containing trisomy 12 mosaicism on passage 21 + 7, these sublines were independently expanded by mechanical transfer. FISH analysis was performed from passage 21 + 9 to passage 21 + 15 to detect trisomy 12. After passage 21 + 12, the sublines consisted entirely of normal or trisomy 12 cells.

Discussion

hESCs are derived from the inner cell mass at the blastocyst stage of embryogenesis and represent undifferentiated embryonic cells. Interest in stem cell research has increased because of their potential use as a source for cell replacement therapies in degenerative diseases and injury (Lerou & Daley, 2005, Peura et al.2007).

It is important not only to understand the mechanisms of differentiation into various cell types but also to be able to stably culture hESCs with normal karyotypes. Recently, chromosomal abnormalities have been reported in several hESC lines (Buzzard et al.2004, Draper et al.2004, Mitalipova et al.2005, Imreh et al.2006, Plaia et al.2006). Interestingly, several reports identified expansion methods as potential causes of chromosomal abnormalities (Caisander et al.2006, Baker et al.2007). Mitalipova et al. (2005) reported increased chromosomal abnormality when hESCs are expanded using bulk passaging methods with collagenase IV and trypsin or cell dissociation buffer compared with when they are passaged using mechanical transfer methods. In addition, Grandela & Wolvetang (2007) mentioned that hESCs maintained by mechanical passaging methods have not been reported to develop genetic abnormalities during culture. They suggested that the mechanical passaging method is superior to enzymatic passaging for propagation of hESCs in culture.

However, after freezing and thawing, we detected cells with the chromosomal abnormalities +8, +12, i(Xq) and XXY in eight different SNUhES cell lines cultured on STO feeder cells by mechanical passaging. These abnormalities were observed both in early and late passage hESCs. But chromosome abnormalities such as 47,XY,+8 and 46,Y,i(X)(q10) and 47,XXY were sporadic events. Thus we could not establish the correlation of morphological change with these abnormalities. Although we detected chromosomal abnormalities in hESCs cultured by mechanical passaging, we also suggest that mechanical transfer is more reliable for maintaining normal karyotypes in hESC cultures, because mechanical transfer allows for the selection of normal or abnormal colonies from mixed cultures. Using mechanical passaging, we separated normal and trisomy 12 karyotype cells in SNUhES11 and SNUhES16 as well as SNUhES3 and SNUhES4 according to their morphological differences, and also maintained normal hESCs in those hESC lines.

Baker et al. (2007) showed that chromosomal abnormality in hESCs could result from stress during adaptation of the cells to their environments in prolonged culture. They also found a chromosomally homogeneous staining region in chromosome 17 in hESC cultures, a genetic change that is associated with oncogenesis in vivo. Other studies have reported that chromosomal abnormalities in hESCs can occur via the influences of culture medium (Pyle et al.2006), feeder cell type (Stojkovic et al.2005) or freezing–thawing periods (Iwarsson et al.1999) as the cells adapt to their environment.

Draper et al. (2004) and Baker et al. (2007) mentioned chromosomes 12, 17, and X as chromosomes that are commonly abnormal in hESCs. Abnormalities such as trisomy 12 and aberrant chromosome X number were also reported in feeder-free cultures of hESCs using defined media (Ludwig et al.2006). In our laboratory, we observed trisomy 12 and abnormalities in chromosome X, but no abnormalities of chromosome 17 in hESCs cultured on STO cells by passaging using mechanical or enzymatic methods. Therefore, trisomy 12 and XXY in hESCs might occur under suboptimal culture conditions regardless of feeder cell type or passaging technique.

In this study, we observed morphologically abnormal colonies in the SNUhES4 line during prolonged culture. Chromosomal mosaicism (47,XY,+12/46,XY) was confirmed in these cells by conventional cytogenetic and FISH analyses. Eight colonies that were clearly distinguishable by morphology were isolated: four colonies with normal morphology (Figure 5a, b, c, d) and four colonies with abnormal morphology (Figure 5e, f, g, h). When these colonies were first isolated, they allpossessed slight chromosomal mosaicism with normal and trisomy 12. After seven passages, hESC colonies with initial chromosomal mosaicism (normal and trisomy 12) were completely separable on the basis of colony morphology: two red signals for chromosome 12 were detected in normal colonies, while three red signals (trisomy 12) were detected in colonies with abnormal morphology.

Baker et al. (2007) found that during long-term culture of hESCs with chromosomal mosaicism (normal and trisomy 12), hESC karyotypes became entirely abnormal via the progressively increasing frequency of trisomy 12. In this case, completely abnormal cultures only appeared more than 20 passages after chromosomal mosaicism was first detected in long-term cultured hESCs, owing to random selection of hESCs by the enzymatic passaging method. On the other hand, we selected hESCs mechanically based on differences in colony morphology. Because of this, we derived completely normal or trisomy 12 sublines within seven passages, which is much quicker than the results reported by Baker et al. (2007).

Several studies have reported that hESCs with chromosomal abnormalities express markers for undifferentiated hESCs, much like karyotypically normal hESCs, and had extensive pluripotency as assessed by teratoma formation after injection into SCID mice (Andrew et al.2005, Enver et al.2005, Gertow et al.2007). We also found that undifferentiated markers were expressed equally in karyotypically normal and abnormal hESCs.

Karyotypically abnormal hESCs are used for many purposes including drug screening, in-vitro toxicology, and the development of new defined media because they are easier to maintain and expand than normal hESCs (Herszfeld et al.2006, Ludwig et al.2006). However, abnormal hESCs are unsuitable as a source for cell-based therapies. Differences in cell growth and control mechanisms could result in problems if differentiated cells derived from abnormal hESCs were transplanted into damaged tissue (Grandela & Wolvetang, 2007). It is therefore important to detect chromosomal abnormalities and to maintain normal karyotypes in hESC culture for cell replacement therapy applications.

We have reported a method to verify chromosomal abnormality via colony morphology during mechanically passaged hESC cultures. In this study we also confirmed that complete trisomy 12 develops in colonies with chromosomal mosaicism within seven passages. Trisomy 12 might underlie morphological changes due to the overexpression of proliferative genes (e.g. nanog). In conclusion, when changes in colony morphology or cell state are recognized in hESC cultures, chromosomal analyses should be carried out immediately to maintain normal karyotypes.

Acknowledgements

This research was supported by a grant (SC1150) from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, Republic of Korea.

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© Springer Science+Business Media B.V. 2008