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Tumor Progression of Culture-Adapted HumanEmbryonic Stem Cells During Long-Term Culture
Sheng Yang,1,†,‡ Ge Lin,1,† Yue-Qiu Tan,1 Di Zhou,1 Lei-Yu Deng,1 De-Hua Cheng,1 Shu-Wei Luo,1 Tian-Cheng Liu,1
Xiao-Ying Zhou,1 Zheng Sun,1 Yang Xiang,1 Tian-Ji Chen,1 Ji-Fang Wen,2 and Guang-Xiu Lu1*
1National Centerof Human StemCell Research and Engineering,Institute of Human Reproduction & StemCell Engineering,Central South University,Changsha,China2Departmentof Pathology,Basic Medical College,Central South University,Changsha,China
Human embryonic stem cells (hESCs) during long-term culture acquire chromosomal changes similar to those occurring in tu-
morigenesis. This was raised concerns about the progression from hESCs to malignant cells. This study aimed to investigate
the changes in chromosomes, cell phenotype, and genes in culture-adapted hESCs to ascertain whether tumorigenic transfor-
mation occurred. By cytogenetic analysis we found progressive karyotypic changes from simple to complex in chHES-3, one of
the hESC lines established in our laboratory, during a long-term suboptimal culture. We further compared chHES-3 cells at dif-
ferent karyotypic stages in cell surface markers, in vivo differentiation, cell cycle, apoptosis, and gene expression profiles. We
found that the karyotypically aberrant chHES-3 had higher S-phase fraction in cell cycle distributions and antiapoptosis ability.
In vivo differentiation of karyotypically normal chHES-3 resulted in relatively mature teratoma, whereas karyotypically aberrant
chHES-3 formed immature teratoma (grade III), in which more primary neural epithelium was revealed by pathological analysis.
The microarray analysis and real-time PCR results showed that some oncogenes were upregulated in karyotypically aberrant
chHES-3 cells, whereas the genes related to differentiation were downregulated, and that Wnt signal pathway was activated. In
conclusion, chHES-3 cells underwent deregulation of self-renewal and dysfunction of related genes in long-term culture adapta-
tion, leading to malignant transformation. VVC 2008 Wiley-Liss, Inc.
INTRODUCTION
Since the first human embryonic stem cell
(hESC) line was established (Thomson et al., 1998),
hESCs have held great promise for future cell- and
tissue-replacement therapy (Cowan et al., 2004).
However, concerns have been raised on the safety of
hESCs in long-term culture. Andrews et al., (2005)
reported that, after multiple passaging, hESCs
acquired chromosomal aberrations similar to those
found in embryonal carcinoma (EC) cells (Draper
et al., 2004). Several recent reports were provided
further evidence that hESCs acquire chromosomal
abnormalities when cultured in suboptimal culture
conditions, such as the presence of trypsin in passag-
ing (Cowan et al., 2004; Inzunza et al., 2004;
Zeng et al., 2004;Hoffman andCarpenter, 2005;Mita-
lipova et al., 2005; Baker et al., 2006; Imreh et al.,
2006). It has also been reported that the karyotypic
changes might lead to tumorigenesis (Enver et al.,
2005; Andrews, 2006; Baker et al., 2006). But until
now, karyotypic changes in a single subline of hESCs
has not been studied in detail. Also, the molecular
mechanisms of cellular malignant transformation of
culture-adapted hESCs remain poorly understood.
Andrews (2006) suggested that the karyotypi-
cally aberrant hESCs were likely to tend towards
the transformed phenotype of the tumor stem
cells. But until now, the relationship between kar-
yotypic changes and the malignant transformation
of hESCs has not been studied in detail. In
this study, we focused on whether karyotypic
changes of hESCs lead to cellular malignant trans-
formation or tumorigenesis, investigating cytoge-
netic changes, cellular biological characteristics,
gene expression profiles, and in vivo teratoma for-
mation of the karyotypically aberrant hESCs at
the different stages. We found that the gradual
†Present address: The 2nd Affiliated Hospital of WenZhou Medi-cal College, Wenzhou, China.Received 20 September 2007; Accepted 14 April 2008
DOI 10.1002/gcc.20574
Published online 9 May 2008 inWiley InterScience (www.interscience.wiley.com).
†Sheng Yang and Ge Lin contributed equally to this work.*Correspondence to: Guang-Xiu Lu, National Center of Human
Stem Cell Research and Engineering, Institute of Human Repro-duction and Stem Cell Engineering, Central South University,Changsha, China. E-mail: lugxdirector@yahoo.com.cn
Supported by: The Hi-Tech Research And Development ofChina, Grant number: 2006 AA02A102; The National BasicResearch Program of China, Grant number: 973 program No. 00CB51010; Bureau Science and Technology Key Project Funds ofHunan Province, Grant number: 03SSY2001; Ministry of Educationof China (Research Fund for the Doctoral Program), Grant number:20030533002; The Key Project of Chinese Ministry of Education,Grant number: 105131.
VVC 2008 Wiley-Liss, Inc.
GENES, CHROMOSOMES & CANCER 47:665–679 (2008)
karyotype changes occurred in chHES-3 cells dur-
ing long-term culture and that the aberrant chHES-
3 cells underwent malignant transformation, as
well as that the activation of Wnt signaling path-
way might be the main reason for the cellular ma-
lignant transformation and tumorigenesis.
MATERIALS ANDMETHODS
Derivation and Culturing of chHES-3
chHES-3, one of hESC lines established in our
laboratory, was isolated by immunosurgery and cul-
tured in serum-free KSR media composed of
Knock-out DMEM (Gibco BRL, Gaithersburg,
MD), supplemented with 15% serum replacement
(Gibco BRL, Gaithersburg, MD), 0.1 mM b-mer-
captoethanol (Sigma, Chemical), 1% nonessential
amino acids (Gibco-BRL, St. Louis, MO), 2 mM
L-glutamine (Gibco BRL, Grand Island), 50 U/ml
penicillin (Sigma, St Louis, MO), 50 lg/ml strepto-
mycin (Sigma, St Louis, MO), and 4 ng/ml human
recombinant basic fibroblast growth factor (Gibco
BRL, Gaithersburg, MD). The chHES-3 cells were
initially propagated on mouse embryonic fibro-
blasts (mEFs) at higher density (4–5 3 104 cells/
cm2), which were derived from inbred Kunming
White mice at 12.5 d of gestation and were inacti-
vated by mitomycin-C (10 lg/ml) (Xie et al., 2004).
The chHES-3 cells were passaged by mechanical
dissection or by collagenase IV digestion (200 U/
ml) (Sigma, St. Louis, MO) followed by mechani-
cal slicing for every 6–7 days. Thawed chHES-3
cells from early passages were cultured on either
higher-density feeders mentioned earlier or lower-
density feeders (2 3 104 cells/cm2). The chHES-3
cells were cultured on 6-well tissue culture plates
in clone-like clumps, picked out the undifferentia-
tion ES cells under inversion microscope, and were
detected by ES markers, including SSEA-4, before
any analysis. The hES cell lines used in this study
were derived from surplus embryos from IVF treat-
ment with informed consent of the patients.
Karyotype Analysis and FISH
Karyotype analysis by G-banding was performed
with about 50 metaphases for every several pas-
sages, and FISH was performed using commer-
cially available whole chromosome painting (WCP)
probes, including WCP1, WCP4, WCP6, WCP7,
and WCP8 (gift from Prof. Xinyuan Guang, The
University of Hong Kong).
CGH Analysis
CGH analysis was performed using the protocol
developed by Klein and Schmidt-Kittler (1999). In
brief, �5 3 105 karyotypically aberrant chESC-3cells were isolated and then lysed by Proteinase K.
Their genomic DNA was digested into appropriate
fragments of 300–800 bp, which were then labeled
with Spectrum Green (Vysis, USA) as probes. The
reference genomic DNA fragments (from normal
karyotypic human blood cells) was labeled with
Spectrum Red (Vysis, USA) as probes. The two
kinds of probes were co-hybridized to metaphase
slides. After hybridization in a moist chamber for
72 hr at 378C, the slides were washed and then an-
alyzed using Quips CGH karyotyping Imaging
Software (Vysis, USA). Ten to 15 metaphases were
captured and evaluated for each sample. Red/green
ratios of 1:�1.25 indicated amplified regions and
ratios of 1:�0.75 indicated deleted regions.
RT-PCR
Total RNA was isolated from cell pellets using
TRIZ reagent (Sigma, USA) and reversely tran-
scribed in a thermocycler using virus reverse tran-
scriptase and random primers, according to the
manufacturer’s protocol supplied by Fermentas
Company (Ferments, Lithuania). PCR amplifica-
tions of different genes were performed using Taq
DNA polymerase (Promega Madison, WI). The
PCR products were subjected to electrophoresis in
1.5% agarose gel containing ethidium bromide
(0.5 lg/ml) with PUC Mix marker 8 as marker.
(MBI-Fermentas, St. Leon-Roth. Nukleotide).
The primers for RT-PCR were listed in Table 1.
Real-Time RT-PCR Analysis
Total RNA was extracted from 1 3 106 hESCs
by classical method and then digested by the
DNase I (NEB, Frankfurt a.M., Germany). About
4 ll aliquots of total RNA were reversely tran-
scribed in a T-GRADIENT thermocycler (Bio-
metra, Gottingen) by using the RevertAidTM First
Strand cDNA Synthesis Kit (Fermentas, Lithua-
nia). Real-time RT-PCR was performed by using
the Lightcycler Faststart master DNA Sybr Green
I kit (Roche, Germany) with specific primer pairs
listed in Table 2. A melting curve analysis was per-
formed to determine the specificity of the ampli-
fied products. The relative gene expression level
was calculated by subtracting the cycle threshold
(Ct) value of the 28 s (control gene) from Ct valueof the target gene to generate the DCt value, andthe relative changes in mRNA expression of aber-
Genes, Chromosomes & Cancer DOI 10.1002/gcc
666 YANG ETAL.
rant hESCs versus normal hESCs were obtained
using the 2-DDCt method (Livak and Schmittgen,
2001) and 95% confidence interval was accepted.
Telomerase Activity
Telomerase activity was measured using TRA-
Peze Telomerase detection kit (Chemicon, Teme-
cula, CA) according to the manufacturer’s instruc-
tions as described (Kim et al., 1994; Weinrich et al.,
1997). In brief, after addition of 10–20 ll telomerase
assay lysis buffer (13 CHAPS), the cells were lysed
on ice. The lysate was incubated on ice for 30 min
and then centrifuged at 12,000 rpm for 20 min at
48C. The supernatant was collected and the protein
concentration was determined by standard proce-
dures (BCA protein assay). A volume of 0.33 lg pro-
tein equivalent was added to a 48 ll reaction solu-
tion consisting of TRAP buffer, dNTP Mix, TS
primer, RP primer mix, and 2 U Taq polymerase.
The PCR condition was 33 cycles of 948C for 30 sec
and 598C for 30 sec. The PCR products were deter-
mined by electrophoresis in 12.5% nondenaturing
polyacrylamide gel with silver staining.
Detection of HESC-Specific Markers
Cells were fixed in 100% ethanol, washed with
13 phosphate buffered solution (13 PBS), and
incubated with monoclonal primary antibodies
against SSEA-1, SSEA-4, TRA-1-81, and TRA-1-
60, separately. Then, the cells were stained by
using rabbit anti-mouse immunoglobulins conju-
gated to fluorescein isothiocyanate (Chemicon,
Temecula, CA). Oct-4 staining was performed
according to the manufacturer’s instructions (Santa
Cruz Biotechnology, Santa Cruz, CA). Alkaline
phosphatase activity was detected according to the
protocol of the Fast Red Substrate Pack (Invi-
trogen, Germany).
TABLE 1. Primers Used for RT-PCR
Gene Forward primer (50-30) Reverse primer (50-30) Tm
Size ofproduct
FGFR4 GTTTCCCCTATGTGCAAGTCC GCGCTGCTGCGGTCCATGT 57 194TERF1 GCAACAGCGCAGAGGCTATTATT AGGGCTGATTCCAAGGGTGTAA 57 159TERF2 AAACGAAAGTTCAGCCCCG TCCTCCAAGACCAATCTGCTTA 55 92OCT3/4 CTTGCTGCAGAAGTGGGTGGAGGAA CTGCAGTGTGGGTTTCGGGCA 64 168REX-1 TGAAAGCCCACATCCTAACG CAAGCTATCCTCCTGCTTTGG 57 556THY1 TCTCAGGGACTTCTGCGGG GGTTGGGAAAAGCCATTTCC 57 88LEFTYA GGGAATTGGGATACCTGGAT CTAAATATGCACGGGCAAGG 57 207SOX2 AGT CTC CAA GCG ACG AAA AA GCA AGA AGC CTC TCC TTG AA 54 142NANOG ACTGTCTCTCCTCTTCCCTCCTCC GTAGAGGCTGGGGTAGGTAGGTG 64 387cripto-1 TCCTTCTACGGACGGAACTG AGAAATGCCTGAGGAAAGCA 58 139
TABLE 2. Primers Used for Real-Time RT-PCR
Gene Forward primer 50-30 Reverse primer 50-30 ImageID Ta
Nanoga CCTGTGATTTGTGGGCCTG GACAGTCTCCGTGTGAGGCAT NM_024865 60POU5F1a GTGGAGGAAGCTGACAACAA ATTCTCCAGGTTGCCTCTCA NM_002701 60Sox2a GTATCAGGAGTTGTCAAGGCAGAG TCCTAGTCTTAAAGAGGCAGCAAAC NM_003106 60BuB1 ATGTTGAGCAGGTTGTTA TATGTTAGTTAGTTGCCTCTTTC NM_004336 50TERF1 TTGCCAGTTGAGAACGATA GGGCTGATTCCAAGGGTGT NM_017489 56GSK-3b CTGTTCCGAAGTTTAGCC AAGAGGTTCTGCGGTTTA NM_002093 50b-Catenin CTGCCAAGTGGGTGGTATA GGGATGGTGGGTGTAAGAG NM_001904 54Cyclin D1b GATCAAGTGTGACCCGGACT TCCTCCTCCTCTTCCTCCTC NM_053056 56FGFR1 GGACAAGGACAAACCCAA GCGTCCGACTTCAACAT NM_000604 52CDK4 GAAACTCTGAAGCCGACCAG ACATCTCGAGGCCAGTCATC NM_053056 58V-jun ACTCGGACCTCCTCACCTCG ATGTGCCCGTTGCTGGAC NM_002228 61Tp53 CCACCATCCACTACAACTACAT AAACACGCACCTCAAAGC NM_000546 54NF1 GCTGAAAGCACCAAACG CTGCCTACTTCCTCCAT NM_000267 50AFP GGGAGCGGCTGACATTAT TGTTTCATCCACCACCAA NM_001134 53PAX6a CCAGGGCAATCGGTGGTAGT ACGGGCACTCCCGCTTATAC NM_000280 60EOMESa CGGCCTCTGTGGCTCAAA AAGGAAACATGCGCCTGC NM_005442 6028Sb GAACTTTGAAGGCCGAAGTG ATCTGAACCCGACTCCCTTT 60
aThese primers were designed as described previously (Babaie et al., 2007).bThese primers were designed as described previously (Becker et al., 2006).
Genes, Chromosomes & Cancer DOI 10.1002/gcc
667TUMOR PROGRESSION OF HUMAN EMBRYONIC STEM CELL
Teratoma Formation and Analysis
The hESCs were mechanically cut into small
pieces (�50–100 cells per piece) and about 0.5–1
3 106 cells were injected into the rear leg of 6- to
8-weeks-old NOD/SCID mice. Ten weeks later,
the mice were sacrificed and the xenografts were
removed followed by fixation in 4% paraformalde-
hyde (Sigma, St. Louis, MO) for 24 hr. After paraf-
fin embedment, the tumors were sectioned and
stained by haematoxylin and eosin (H&E). In the
experiments of the differentiation in vivo, the
SCID mice care was in accordance with guidelines
for the Care and Use of Laboratory Animals, enun-
ciated by the Ministry of Science and Technology
of the People’s Republic of China.
Cell Proliferation Analysis
For analysis of the cell cycle, 1 3 107 ES
cells were cultured for 12 hr in 20 lm EdU (5-
ethynyl-20-deoxyuridine) medium and then har-
vested and stained by The Click-iTTM EdU Alexa
Fluor1 488 Cell Proliferation Assay Kit (Invi-
trogen, USA) in accordance with the manufac-
turer’s protocol. Fluorescence data were collected
using a FACScalibur (Becton Dickinson, Mountain
View, CA).
Differentiation Experiments In Vitro
For in vitro differentiation, chHES-3 colonies
cultured on mouse embryonic feeder layer were
mechanically cut into small pieces of 50–100 cells
and continued to grow as aggregates in suspension
in ES medium without bFGF for 10 days to form
embryoid bodies. Embryoid bodies were then
transferred onto gelatin-coated 6-well plate for ad-
herent culture for 15 days in the same medium, fol-
lowed by immunocytochemistry analysis. Further-
more, the in vitro differentiation of chHES-3 cells
was examined in routine culture.
Apoptosis Analysis
To measure early apoptosis, cell apoptosis was
examined using Annexin V-FITC kit (Bender
MedSystems, Vienna, Austria). Fluorescence data
were collected using a FACScalibur (Becton Dick-
inson, Mountain View, CA) and further analyzed
using the ModFit 2.0 software (Becton Dickinson,
Mountain View, CA). The apoptotic resistance
experiments were performed by using mitomycin
C. The chHES-3 cells were cultured in 0.01 lg/ml
mitomycin C for 10 hr and then harvested to detect
cell apoptosis as mentioned earlier.
Microarrary and Data Analysis
The normal chHES-3 cells (P30) and aberrant
chHES-3 cells with a simple duplication karyotype
(SIMP) of passage 72 (P72) and complex karyotype
(COMP) (P182) were collected for gene expression
profiles. Total RNA was extracted from normal and
karyotypically aberrant chHES-3 cells using the
RNeasy kit (Qiagen, Chatsworth, CA). One micro-
gram of total RNAwas primed with 100 ng of Oligo
dT-T7 primer and reverse transcribed with Super-
script II (Invitrogen, USA). A second strand was
synthesized and the double strand cDNAwas puri-
fied with DNA Clean and Concentrator (Zymo
Research, Orange, CA). The in vitro transcription
reactions were performed with T7 RNA polymer-
ase. The amplified RNA (aRNA) in the first round
amplification was purified with the RNeasy Mini
Kit (Qiagen, Valencia, CA). The second round
amplification was performed similarly to the first
round, but with 100 ng of aRNA and 500 ng ran-
dom hexamers. The ENZO BioArray HighYield
RNA Transcript Labeling Kit (Affymetrix, USA)
was used to incorporate biotin-labeled nucleotides
in the second round dscDNA, after which RNA
was purified using RNeasy. Fragmentation was
completed using a standard protocol. Prior to
hybridization on Gene Chip array, a test3 array of
housekeeping controls was analyzed to determine
sample suitability for Gene Chip arrays. Hybri-
dized arrays were subsequently scanned for data
analysis. A detailed RNA amplification protocol is
available upon request. The hybridization mixture
was heated at 998C for 5 min and then at 458C for
5 min, followed by centrifugation at 13,000g for
5 min. Gene chips were prehybridized in 200 ml of
13 hybridization buffer for 10 min at 458C with
mixing at 60 rpm in the hybridization oven. The
prehybridization buffer was replaced with 200 ml
hybridization mixture and incubated for 16 hr at
458C, mixed at 60 rpm. The hybridization mixture
was removed and stored at 2708C. Each chip was
filled with 250 ml of nonstringent wash buffer (63SSPE, 0.01% Tween-20). The chips were scanned
with an Affymetrix Scanner 3000 (Affymetrix), and
the gene expression signal was collected using
Affymetrix GCOS V1.1.1 software. The distribu-
tion of upregulated and downregulated genes on
each chromosome was analyzed by the Dchip2004
software. Hierarchical cluster analysis was per-
formed using the AVADIS software.
Genes, Chromosomes & Cancer DOI 10.1002/gcc
668 YANG ETAL.
RESULTS
Characterization of the chHES-3 Cell Line
The chHES-3 cell line used in these studies was
established in our laboratory, and was cultured on
feeder cells at higher density than normal ES cells
(see Material and Methods). The cell immunohis-
tochemistry always showed that the chHES-3 cells
are positive for SSEA-3, SSEA-4, TRA-1-60, TRA-
1-81, and oct-4, and negative for SSEA-1 and that
the cells had alkaline phosphatase activity and
telomerase. The results of RT-PCR indicated that
the cells expressed special markers, such as Oct-4,
Nanog, Sox-2, TERF1, TERF2, REX, FGF4,
Cripto, Thy1, and LEFTYA. The chHES-3 cells
presented a normal karyotype and could form tera-
toma after injection into SCID mice, which con-
tained many kinds of cells derived from three
germ layers (see later). All the above mentioned
results confirmed that the chHES-3 cells had basic
features of the normal HES cells. The chHES-3
cell lines kept these basic features of hESCs even-
though their karyotype and characteristics of tera-
toma changed in later passages during long-term
culture (Figs. 1A–1C).
Gradually Karyotypic Changes of the chHES-3 Cell
Line During Long-Term Culture
To monitor the genetic stability of hESCs, we
routinely examined karyotype of hESCs every 5–
10 passages, and found that the karyotype of
chHES-3 cell line displayed a slowly progressive
changes. The G-banding results showed that the
chHES-3 cell line retained a normal karyotype up
to P27 (Fig. 2A); at P34, all analyzed 50 meta-
phases had the same karyotype of 46,XX,dup(1)
(p32p36) (Fig. 2B); from P44 to P99, more chromo-
somal abnormalities accumulated and showed a
karyotype of 46,XX,dup(1)(p32p36), t(1;6;4)(q25;
q23;p16),ins(4;1)(p16;q21q25), t(7;8) (q32;q13)
(Fig. 2C); at P114, a mosaic karyotype was
observed, including the chromosomal abnormal-
ities mentioned above and three kinds of addi-
tional chromosomal abnormalities (Figs. 2D–2F);
from P142 to P153, the cells only demonstrated the
Figure 1. Identical morphology of normal chHES-3, simple duplication chHES-3 (SIMP), and karyotypicallycomplex chHES-3 (COMP) colonies. (A) All colonies stained positive for AKP, SSEA-4, OCT-4, TRA-1-60, andTRA-1-81 and negative for SSEA-1. (B) RT-PCR detection of pluripotency-related genes showed that thesegenes were all expressed in normal, SIMP and COMP chHES-3 cells. (C) The telomerase activity was presentin normal chHES-3 cells (Lane 1), SIMP chHES-3 cells (Lane 2), and COMP chHES-3 cells (Lane 3).
Genes, Chromosomes & Cancer DOI 10.1002/gcc
669TUMOR PROGRESSION OF HUMAN EMBRYONIC STEM CELL
karyotype 47,XX1X,dup(1)(p32p36),t(1;6;4) (q25;
q23;p16), ins(4;1)(p16;q21q25),t(7;8)(q22;q22) (Fig.
2G); from P188 to P197, the karyotype further
changed in which a novel marker chromosome and
disomy X appeared but the trisomy X disappeared
(Fig. 2H). FISH analysis (Figs. 3A–3D) further con-
firmed some of the changes in the karyotypes. Fur-
thermore, CGH analysis of chHES-3 cells at P96
and at P187 both showed only gain of 1p32-p36
(Figs. 4A and 4B) although routine G-banding
revealed more complex chromosome rearrange-
ment. All those results suggest that chromosomal
abnormalities in hESCs can be passed on to the
next generation and accumulate.
To clarify whether high-density feeder cells
have a detrimental effect on karyotypic stability,
feeder cells at different density were used to cul-
ture chHES-3 cells. When karyotypically normal
chHES-3 cells of early passages (P19) were thawed
and cultured on lower density feeder layer of 2.5 3104 cells/cm2, they maintained a normal karyotype
up to P74; when cultured on feeder cells at higher
density of 4.0–5.0 3 104 cells/cm2, they also
acquired the aberrant karyotype with the simple
duplication in chromosome 1 (triplet experiments),
which when were transferred back onto lower den-
sity feeder layer, only the duplication karyotype
were observed without further changes up to P114.
Figure 2. G-banding of chHES-3 cells at different passages. (A) Nor-mal karyotype of 46,XX at passage 27. (B) Abnormal karyotype of46,XX,dup (1)(p32p36) at passage 34. (C) Abnormal karyotype withcomplex chromosome rearrangement involving chromosomes 1, 4, and6, and a reciprocal translocation between chromosomes 7 and 8 wasfound at passage 44–99, seen as 46,XX, dup(1)(p32p36),t(1;6;4)(q25;q23;p16),ins(4;1)(p16;q21q25), t(7;8)(q32;q13), and was also foundin 2 of 19 metaphases at P114. (D) One of 19 metaphases at P114 was46,XX,dup(1)(p32p36),t(1;6;4)(q25;q23;p16),ins(4;1)(p16;q21q25),
t(7;8)(q32;q13),1mar(2?). (E) One of 19 metaphases at passage 114was 46,XX,dup(1)(p32p36),t(1;6;4)(q25;q23;p16),ins(4;1)(p16;q21q25),t(7;8)(q32;q13),1mar(2?),1mar(6?),1mar(12?). (F) Eleven of 19 meta-phases at passage 114 were 47,XX1X,dup(1)(p32p36),t(1;6;4)(q25;q23;p16),ins(4;1)(p16;q21q25),t(7;8)(q22;q22). (G) There existedonly one karyotype described in (f) at passages 142–153. (H) Thekaryotype at passages 188–197 became 46,XX,dup(1)(p32p36),t(1;6;4)(q25;q23;p16),ins(4;1)(p16;q21q25),t(7;8)(q22;22),1mar(15?).
Genes, Chromosomes & Cancer DOI 10.1002/gcc
670 YANG ETAL.
All the above mentioned results suggest that
higher-density feeder layers might have an effect
on chromosomal changes.
Lower Differentiation of Karyotypically Aberrant
chHES-3 Cells In Vivo
Previous reports have shown that karyotypic
changes give rise to a malignant phenotype of
hESCs in vivo (Andrews et al., 2005). To deter-
mine the tumorigenicity of aberrant chHES-3, we
compared the teratomas derived from chHES-3
cells with a complex karyotype at P96 (COMP)
with those derived from chHES-3 cells with a nor-
mal karyotype at P17. In xenografts from COMP
chHES-3 cells, there were tissues from three germ
layers, predominantly immature mesenchymal tis-
sues, and primitive neural tissues with neural tube-
like rosette structure (Fig. 5A) in which there were
more cellular layers with apoptosis bodies and nu-
clear division metaphases (Fig. 5B). In every sec-
tion there were more than three low power fields
(403) containing the primitive neural tissues.
According to the diagnostic criteria defined by the
World Health Organization (Mikuz, 2002), the
tumors were classified as immature teratoma of
grade III. And in the teratoma, nested regions with
Oct4-positive cells were also observed (Fig. 5C).
Compared to the teratoma derived from the
COMP chHES-3 cells, the teratoma derived from
the normal chHES-3 presented higher degree of
differentiation with few immature neural tissues
(Fig. 5D) and less cellular layers in the immature
neural tissue (Fig. 5E), as well as Oct4-negative
(Fig. 5F). Similar to in vivo, the abnormal chHES-3
Figure 3. FISH analysis of chHES-3 cells at different passage. FISHanalysis at passage 34 using WCP1 (red ) and WCP4 (green) showed anadditive red signal on one chromosome 1 (red), while the other chro-mosome 1 is normal, confirming that the additive fragment originatedfrom chromosome 1 (i). FISH analysis at passage 65 using WCP1 (red)and WCP6 (green) (j), WCP1 (red) and WCP4 (green) (k), and WCP7(red) and WCP8 (green) (l), respectively. In (j), there is a red signal(Chr 1) on der(6) ( green), and a red signal (Chr 1) and a green signal
(Chr 6) on der(4); In (k), there is a green signal (Chr 4) on der(1) (red),a red signal (Chr 1) on der (6), and a green signal (Chr 6) and a red sig-nal (Chr 1) on der(4); In (l), there is a red signal (Chr 7) on der(8)(green) and a green signal (Chr 8) on der(7) (red). These results con-firm that there are complex rearrangements involving chromosomes 1,4, and 6, an insertional between chromosome 1 and 4, and a reciprocaltranslocation between chromosome 7 and 8.
Genes, Chromosomes & Cancer DOI 10.1002/gcc
671TUMOR PROGRESSION OF HUMAN EMBRYONIC STEM CELL
cells make more rosettes in vitro (data not shown).
Furthermore, the aberrant chHES-3 cells formed
embryoid bodies in vitro and had capacity of differ-
entiation to three germ layers after 3–4 weeks of
differentiation (Fig. 6).
Higher Ratio of S Phase and Lower Ratio of
Apoptosis in Karyotypically Aberrant chHES-3 Cells
We systematically compared normal chHES-3,
SIMP chHES-3, and COMP chHES-3 cells in cell
cycle distribution and cell apoptosis ratios. The
chHES-3 cells were detected by ES markers
including the SSEA-4 before analysis. In the popu-
lation of ES cells, 84–96% cells are SSEA-4 posi-
tive. The percentage of cells in S-phase gradually
increased from 33.1% 6 1.27% (normal chHES-3
cells) to 58.2% 6 0.71% (SIMP chHES-3 cells) and
to 72.7% 6 1.5% (COMP chHES-3cells Fig. 7A),
while the apoptosis ratio sharply decreased from
10.3% 6 0.4% (normal chHES-3 cells) to 4.0% 60.9% (SIMP chHES-3 cells) and to 1.0% 6 0.3%
(COMP chHES-3 cells Fig. 7B). To detect the
capacity of apoptosis resistance, normal chHES-3,
SIMP chHES-3, and COMP chHES-3 cells were
Figure 4. CGH analysis of COMP chHES-3 cells at passage 96 (m) and P187 (n). An obvious gain (green)of 1p32?p36 was found.
Genes, Chromosomes & Cancer DOI 10.1002/gcc
672 YANG ETAL.
cultured in 0.01 lm mitomycin C, the apoptosis
ratios were 13.3% 6 0.89%, 4.83% 6 0.93%, and
1.06% 6 0.12% respectively. Furthermore, during
routine culture, the ratios of single clone-formation
were 0.165% 6 0.01%, 0.18% 6 0.03%, and 0.96%
6 0.11%, respectively. All these results indicate
that compared to that of normal chHES-3 cells, the
aberrant chHES-3 cells exhibited gradually
increased capacities of proliferation and antiapop-
tosis with increased complexity of karyotype.
Gene Expression Profile Analysis
The SIMP (P53) and COMP (P182) chHES-3
cells were selected as karyotypically aberrant sam-
ples to detect gene expression profiles, with the
normal chHES-3 (p30) cells being used as control.
A simple present/absent analysis of gene expres-
sion in the samples is shown as Venn diagrams
(Fig. 8). In total, 12,045, 11,001, and 11,375 genes
were expressed in the normal chHES-3 cells, SIMP
cells, and COMP cells, respectively, showing that
the numbers of expressed genes were clearly dif-
ferent among them. More genes were expressed in
the normal chHES-3 cells when compared with
those in two aberrant chHES-3 samples. Of them,
965, 310, and 346 genes were uniquely expressed
in normal chHES-3 cells, SIMP chHES-3 cells, and
COMP chHES-3 cells, respectively.
Upregulation of Pluripotency-Related Genes and
Downregulation of Differentiation-Related Genes
The expression of all 10 genes related to pluripo-
tency and self-renewal mentioned in a previous
study (Mitalipova et al., 2005), including POU5F1,
Figure 5. Histopathological and immunohistological analyses oftumors. (A–C) Teratoma derived from COMP chHES-3 cells, showing alarge area of primitive nerve tissue containing many neural tube-likestructre (arrows) (A), neural tube-like structure with multilayer of cells(B) and nested region of Oct4-positive cells (C). (D–F) Teratoma
derived from normal chHES-3 cells, showing well differentiated tissues(D), neural tube-like structure with only a few layers of cells (E) and noOct4-positive cells (F). Insert shows the DAPI staining for cell nuclei.(triplet experiments).
Figure 6. Differentiation analysis of hESC-derived embryoid bodies in vivo. (A) Embryoid body derivedfrom aberrant karyotypic HES cells. (B) Expression of genes related to the three germ layers in differentcells from embryonic body. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Genes, Chromosomes & Cancer DOI 10.1002/gcc
673TUMOR PROGRESSION OF HUMAN EMBRYONIC STEM CELL
Nanog, LDB2, GABRB3, FGF4, LEFTY2, LDB2,
DNMT3B, BUB1, and CD9, were gradually upre-
gulated with increased complexity of chromosomal
aberration, while the expression of differentiation-
related genes such as FN1, MSI2, NEDD1, SEPT2,
NEFH, NEDD4, PAX6, OTX2, MCFD2, and
SOX3 in 21 ectoderm development-related genes,
EOMES, FLT1, HLA-C, MYL4, PECAM1, T,
THBS1, THBS2, and VCAM1 in 28 mesoderm de-
velopment-related genes, and AFP, CER1, FOXA2,
GATA4, GATA6, and HNF4A in 7 endoderm devel-
opment-related genes (Mitalipova et al., 2005) had a
tendency to decrease with increased karyotypic
complexity, whereas the expression level was
unchanged for the rest of them (Table.3). Hierarchi-
cal cluster analysis of pluripotency-related genes
showed that normal chHES-3 cells and SIMP
chHES-3 cells are more closely related to each other
than to the COMP chHES-3 cells (Fig. 9A).
Downregulation of Tumor-Suppression Genes and
Upregulation of Oncogenes
Thirteen tumor suppressor genes, including
PTPRG, PTCH, SMAD4, PTEN, RERE,
RPL10A, TIMP1, CDH1, APC, TP53, BRCA1,
MSH2, and NME1, were expressed in all three
samples. Of these, SMAD4, RERE, CDH1, APC,
TP53, and BRCA1 exhibited a gradually decreased
expression with increased karyotypic complexity.
Hierarchical cluster analysis demonstrated that
normal and SIMP cells were closely related to each
other but distinct from COMP cells (Fig. 9B).
Analysis of 10 oncogenes, including FGFR1,
MDM2, BCL2, LMO2, ERBB2, TPM3, NTRK1,
MET, CDK4, and LMO1, in SIMP, COMP chHES-3
cells, and normal chHES-3 cells showed no significant
difference in their expression levels (data not shown).
However, the karyotype and CGH analysis showed
that duplication of 1p32–1p36 was the only dosage
variation of the genome in SIMP chHES-3 cells,
which might be an important event in malignant
transformation and genomic instability. Therefore,
we further investigated the expression of oncogenes
in this region, including HKR3, DJ1, FGR, LCK,
MPL, BLYM, MYCL1, BLF, TAL1, and JUN by
using semiquantitative PCR and real-time PCR. Of
these, the expression levels of HKR3 and LCK in
SIMP chHES-3 cells were higher than in the normal
chHES-3 cells, while the expression levels of the
other genes showed no difference (Fig. 10).
Activation of Wnt Signaling Pathway
Previous studies have shown that important
genes related to the Wnt signaling pathway are
involved in self-renewal of hESCs; and tumorigen-
esis exhibit significantly differential expression in
stem cells, cancer stem cells, and cancer (Reya
et al., 2001; Ying et al., 2003; Sato et al., 2004). In
this study, the microarray results showed that of all
analyzed signaling pathways, the intracellular Wnt
Figure 7. Analyses of cell cycle distribution and cellular apoptosis.(A) The percentage of S-phase cells of normal chHES-3 cells, simpleduplication (SIMP) chHES-3 cells, and complex karyotype (COMP)chHES-3 cells (three independent experiments) (mean 6 s.d., n 5 3; *P< 0.05; **P < 0.05 by ANOVA). (B) The percentage of apoptotic cellsin normal, SIMP and COMP chHES-3 cells (mean 6 s.d., n 5 3; *P <0.05; **P < 0.05 by ANOVA).
Figure 8. The number of expressed genes in three types of cells.Normal chHES-3 cells, simple duplication chHES-3 cells, and complexkaryotypic chHES-3 cells are denoted by Normal, SIMP, and COMP,respectively. [Color figure can be viewed in the online issue, which isavailable at www. interscience.wiley.com.]
Genes, Chromosomes & Cancer DOI 10.1002/gcc
674 YANG ETAL.
signaling pathway was preferentially activated with
increased karyotypic complexity. Of all differen-
tially expressed genes related to the Wnt signaling
pathway, the inhibitor GSK3b and Wnt-antagonists
SFRP1, SFRP2, and FRZB were gradually down-
regulated with increased karyotypic complexity,
while b-catenin and its downstream oncogenes Jun
and cyclin D1, and other genes, such as E-cadherin
and the Wnt receptor Fzd2, were gradually upregu-
lated (Table 3). However, the Wnt genes were not
expressed differentially. These findings were fur-
ther supported by real-time PCR analysis (Figs.
11A–11F). These results suggested that activated
Wnt signaling pathway might be a main reason for
malignant transformation of chHES-3 cells, similar
to the results observed in other tumorigenesis in
tumors (Gat et al., 1998; Moon et al., 2002).
Figure 9. Hierarchical cluster analyses. (A) pluripotency-related genes and (B) tumor-suppressiongenes. The normal chHES-20 cells (normal) and the simple duplication (SIMP) chHES-3 cells are moreclosely related to each other than to the complex karyotype (COMP) chHES-3 cells.
Figure 10. Real-time RT-PCR analysis of LCK and HKR3 demon-strated a 2.4-(LCK) and a 1.3-(HKR3) fold expression increase in thesimple duplication (SIMP) chHES-3 cells compared with the normalchHES-3 cells, respectively (n 5 3).
Genes, Chromosomes & Cancer DOI 10.1002/gcc
675TUMOR PROGRESSION OF HUMAN EMBRYONIC STEM CELL
Verification of Microarray Results by Real-Time
RT-PCR
To verify the results of the gene chips, we con-
firmed the expression changes of some important
genes by real time RT-PCR with primers listed in
Table 2. The results are shown in Figure 12 and
summarized in Table 4.
DICUSSION
More and more evidence has shown that the
karyotype of hES cells change during long-term
culture. The typical changes are additional copies
of chromosomes 12, 17, 1, and X, similar to germ
cell tumors, such as malignant EC (Sandberg et al.,
1999; Andrews, 2002;). In this study, the karyotypic
changes started with a duplication of 1p32-36. Fur-
thermore, this study is the first detailed report on
the karyotypic change process, exhibiting progres-
sive chromosomal aberrations during long-term
culture; from an early simple duplication to a late
complex karyotypes similar to those in tumors
through gradual accumulation of new abnormal-
ities. Draper (2004) indicated that karyotypic
changes of chromosomes 12, 17, and X confer hES
cells with a tumorigenic capacity (Baker et al.,
2006). Our results show that karyotypic changes of
chromosome 1 might be related to tumorigenesis,
not least because there are many oncogenes and
tumor suppression genes on chromosome 1. Karyo-
typic change affected the function of ES cells. At
the early stage of karyotypic changes, LCK,
HKR3, two oncogenes mapping to the duplication
region of 1p32-36, were upregulated. Furthermore,
genes important for proliferation in the canonical
Wnt pathway (Wnt4) and survival pathways
(EGF), or cell apoptosis (FAF1, TP73, TNFRSF8,
TNFRSF9, TNFRSF14, CASP9, and JUN), or
cell cycle regulation (CDKN2C, CDC42,
CDC2L1, DDEFL1) map to 1p32-36. This might
be one reason for chromosomal duplication and the
high cell proliferation.
Previous studies indicated that the karyotypic
changes in the hESCs during long-term culture in
vitro were adaptive changes to the suboptimal cul-
ture and the adaptive karyotypic changes conferred
hESCs with more power of self-renewal (Buzzard
et al., 2004; Baker et al., 2006). In present studies,
the aberrant karyotypic chHES-3 cells exhibited
the characteristics similar to normal ES cells, such
as the special cellular markers, alkaline phospha-
tase activity, and telomerase activity, whereas the
results from the apoptosis analysis, distribution of
cell cycle, and in vitro differentiation demonstrated
TABLE 3. The Expression Changes of Wnt SignallingPathway-Related Genes in Microarrary
Gene title
Changea
SIMPb COMPc
Glycogen synthasekinase 3b
NCd D
Catenin (cadherin-associated protein),b 1, 88 kDa
De I
Casein kinase 1, epsilon If IWingless-type MMTVintegration sitefamily, member 6
NC NC
Frizzled homolog 6 D NCWingless-type MMTVintegration site family,member 1
D D
Wingless-type MMTVintegration sitefamily, member 4
D D
Frizzled homolog 3 (Drosophila) D DFrizzled homolog 7 (Drosophila) D DFrizzled homolog 2 (Drosophila) I IFrizzled homolog 6 (Drosophila) D DFrizzled homolog 1 (Drosophila) NC DFrizzled homolog 5 (Drosophila) NC DFrizzled homolog 2 (Drosophila) D DFrizzled homolog 3 (Drosophila) D DFrizzled homolog 10 (Drosophila) D DFrizzled homolog 5 (Drosophila) NC DCyclin D2 D DProtein kinase C, iota NC NCPlatelet-activating factoracetylhydrolase, isoform Ib,a subunit 45 kDa
D D
Platelet-activating factoracetylhydrolase,isoform Ib,b subunit 30 kDa
D D
Protein kinase D1 NC DLow density lipoproteinreceptor-related protein 6
NC I
Ras homolog gene family,member A
NC D
Adenomatosis polyposis coli NC NCras-related C3 botulinumtoxin substrate 1
I NC
v-jun sarcoma virus 17oncogene homolog (avian)
I I
Secreted frizzled-relatedprotein 1
D D
Secreted frizzled-relatedprotein 2
D D
Frizzled-related protein D DJUN I I
aChange, the change tendency of abnormal hESCs compared to normal
hESCs.bSIMP, chHES3 cells with a simple duplication karyotype.cCOMP, chHES3 cells with a complex karyotype.dD, decrease.eI, increase.fNC, no change.
Genes, Chromosomes & Cancer DOI 10.1002/gcc
676 YANG ETAL.
that their power of antiapoptosis and proliferation
gradually increased with the increased karyotypic
complexity. In a previous study, karyotypic changes
in mouse ES cells correlated with a reduced ability
to colonize the germ line in the chimeric mice,
implying that karyotypic changes decreased the
capacity of cellular differentiation (Longo et al.,
1997). Recent studies have also shown that adapt-
ive karyotypic changes confer hESCs with more
power of self-renewal at expense of cellular death
and differentiation capacity (Keith, 2004). In our
experiments of in vivo differentiation, the imma-
ture neural tissues dominantly existed in the tera-
toma derived from the COMP chHES-3 cells and
were more than that derived from karyotypically
normal chHES-3 cells, indicating that the karyo-
typic changes might lead to the restricted differen-
tiation power to the neural tissues, and further
illustrating that the karyotypically aberrant
chHES-3 cells had stronger power of self-renewal
and proliferation at expense of cellular death and
differentiation capacity. Consistent with this, the
results of gene chips and real-time RT-PCR indi-
cated that the expression of genes related to self-
renewal, such as POU5F1, Nanog, LDB2, GABRB3,
FGF4, LEFTY2, LDB2, DNMT3B, BUB1, and
CD9, increased in karyotypically aberrant chHES-3
cells, wheras the expression of genes related to dif-
ferentiation of the three germ layers decreased.
From the tissue characteristics in teratoma derived
from the karyotypically aberrant chHES-3 cells we
propose that the chHES-3 cells are gradually trans-
formed to malignant cells due to the dysregulated
stem cell self-renewal and proliferation/survival,
with the accumulated karyotypic changes.
More evidence shows that the Wnt signaling
pathway, which normally controls stem cell fate, is
activated in tumorigenesis (McWhirter et al., 1999;
Figure 11. Relative expression level of genes in Wnt signal pathwayin karyotypically aberrant HES cells versus normal HES cells. (A) mRNAlevels of FED2, (B) E-cadherin, (C) FRZB, (D) SFRP1, and (E) SFRP2were detected by real-time RT-PCR of normal chHES-3 cells (n 5 3),
SIMP chHES-3 cells (n 5 3), and COMP chHES-3 cells (n 5 3). The rela-tive expression levels were determined by normalizing the DCt valuesof aberrant HES cells against the average DCt values of normal HES cellsfor the specified genes.
Genes, Chromosomes & Cancer DOI 10.1002/gcc
677TUMOR PROGRESSION OF HUMAN EMBRYONIC STEM CELL
Derksen et al., 2004). In this study, the results of
the microarray and real-time RT-PCR analysis
demonstrate that in all investigated signaling path-
ways the intracellular Wnt signaling pathway was
preferentially activated with increased karyotypic
complexity, while no differentiated expression of
Wnt genes was detected in karyotypically normal
and aberrant hESCs. We conclude that the Wnt
signaling pathway might be activated by Wnt pro-
teins from the feeder cells because our previous
proteomics results on feeder cells showed that the
Kunming-White mouse feeder cells produced
Wnt-3 protein (Xie et al., 2004). We further investi-
gated the expression of Wnt family genes in the
feeder cells and found that 13 of 19 Wnt family
genes were expressed, including WNT1 etc (data
not shown) and that the expression amount of most
expressed genes were positively correlated to the
density of feeder cells, including Wnt2 etc (data
not shown). We propose that excessive production
of Wnt proteins from high-density feeder cells
might be one of the main reasons for cellular tumor
progression in chHES-3 cells. Whether Wnt pro-
teins or other factors are involved in this process
remaine to be determined.
In conclusion, chHES-3 cells undergo deregula-
tion of self-renewal and dysfunction of related
genes in long-term culture adaptation, leading to
malignant transformation; the activation of
Wnt signaling pathway might be involved in this
process.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Alan Troun-
son for helpful suggestions and kindly revising
manuscript, Dr. Hsiao Chang Chan for helpful
comments and suggestions and Dr. Liang Hu for
kindly carrying out the CGH analysis.
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679TUMOR PROGRESSION OF HUMAN EMBRYONIC STEM CELL
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