DOI:http://dx.doi.org/10.7314/APJCP.2013.14.10.

5705
Teratoma Formation in Mice after Syngeneic and Allogeneic Implantation of Mouse Embryonic Stem Cells

RESEARCH ARTICLE
Teratoma Formation in Immunocompetent Mice After
Syngeneic and Allogeneic Implantation of Germline Capable
Mouse Embryonic Stem Cells
Abdullah Aldahmash1,2*, Muhammad Atteya1,3, Mona Elsafadi1, May AlNbaheen1, Husain Adel Al-Mubarak1, Radhakrishnan Vishnubalaji1, Ali AlRoalle1, Suzan Al-Harbi4, Muthurangan Manikandan1, Klaus Ingo Matthaei1,5,
Amer Mahmood1*
Abstract
Background: Embryonic stem cells (ESCs) have the potential to form teratomas when implanted into
immunodeficient mice, but data in immunocompetent mice are limited. We therefore investigated teratoma
formation after implantation of three different mouse ESC (mESC) lines into immunocompetent mice. Materials
and Methods: BALB/c mice were injected with three highly germline competent mESCs (129Sv, BALB/c and
C57BL/6) subcutaneously or under the kidney capsule. After 4 weeks, mice were euthanized and examined
histologically for teratoma development. The incidence, size and composition of teratomas were compared using
Pearson Chi-square, t-test for dependent variables, one-way analysis of variance and the nonparametric KruskalWallis analysis of variance and median test. Results: Teratomas developed from all three cell lines. The incidence
of formation was significantly higher under the kidney capsule compared to subcutaneous site and occurred in
both allogeneic and syngeneic mice. Overall, the size of teratoma was largest with the 129Sv cell line and under
the kidney capsule. Diverse embryonic stem cell-derived tissues, belonging to the three embryonic germ layers,
were encountered, reflecting the pluripotency of embryonic stem cells. Most commonly represented tissues
were nervous tissue, keratinizing stratified squamous epithelium (ectoderm), smooth muscle, striated muscle,
cartilage, bone (mesoderm), and glandular tissue in the form of gut- and respiratory-like epithelia (endoderm).
Conclusions: ESCs can form teratomas in immunocompetent mice and, therefore, removal of undifferentiated
ESC is a pre-requisite for a safe use of ESC in cell-based therapies. In addition the genetic relationship of the
origin of the cell lines to the ability to transplant plays a major role.
Keywords: Mouse embryonic stem cells - teratoma - immunocompetent - syngeneic - allogeneic
Asian Pac J Cancer Prev, 14 (10), 5705-5711

Introduction
The use of embryonic stem cells (ESCs) in the
treatment of human diseases has long been muted, because
such a treatment must not only be efficacious but must
also be shown to be completely safe (Baker et al., 2007;
Lukovic et al., 2013). Indeed, in many previous studies,
the use of injected ESCs was associated with the risk of
teratoma development (Wakitani et al., 2003; Baharvand
and Matthaei, 2004; Przyborski, 2005; Nussbaum et al.,
2007; Dressel et al., 2008; Prokhorova et al., 2009; Zhang
et al., 2012; Li et al., 2013). In a clinical setting, this is
a critical limitation of such a treatment. Therefore, much

interest among the scientific community has been on the

understanding of these processes. Most studies have used
immunodeficient recipient mice into which these cells
were implanted (Wakitani et al., 2003; Baharvand and
Matthaei, 2004; Przyborski, 2005; Nussbaum et al., 2007;
Dressel et al., 2008; Prokhorova et al., 2009; Li et al.,
2013). The resultant teratomas consisted of all three germ
layers (ectoderm, mesoderm, endoderm) representing all
tissue types in a whole individual (Prokhorova et al., 2009).
The use of immunodeficient mice appears logical since it
could be expected that immunocompetent animals would
reject these cells. However, one study (Dressel et al., 2008)
has surprisingly reported that the syngeneic implantation

Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University and King Khalid University Hospital, Riyadh,
Kingdom of Saudi Arabia, 4Biology Department, College of Science, King Abdulaziz University, Saudi Arabia, 2KMEB, Department of
Endocrinology, University of Southern Denmark, Odense, Denmark, 3Histology Department, Faculty of Medicine, Cairo University,
Egypt, 5Stem Cell and Gene Targeting Laboratory, The John Curtin School of Medical Research, The Australian National University,
Canberra ACT Australia *For correspondence: a.aldahmash@gmail.com, ammahmood@ksu.edu.sa
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Abdullah Aldahmash et al

of 129Sv ESCs into 129Sv immunocompetent mice also

gave rise to teratomas consisting of all three germ layers,
and at a high frequency. We wondered, therefore, if the
state of undifferentiation, or pluripotency, of the injected
cells may play a critical role in this process. The present
study was, therefore, designed specifically to investigate
this possibility by using three mouse ESC lines of
early passage that have been proven to have germline
transmission capacity at similar passage stages. Moreover,
we were interested in determining whether these cells
could form teratomas not only in syngeneic but also in
allogeneic immunocompetent hosts. Here we report that
ESCs can form teratomas in immunocompetent mice after
allogeneic as well as syngeneic implantation.

Materials and Methods

Ethics statement
All animal experimental procedures of this study
were approved by the Institutional Review Board (Ethical
Approval ID 11/3215/IRB), College of Medicine, King
Saud University, Riyadh, Kingdom of Saudi Arabia.
Cell culture
Three mouse ESC (mESC) lines were used in this
study: 129Sv W9.5 passage (p) 19, BALB/c p11, and
C57BL/6 p13. The cell lines were provided by the Stem
Cell and Gene Targeting Laboratory, John Curtin School
of Medical Research, the Australian National University,
Canberra ACT 0200, Australia, and were transferred to
King Saud University in liquid nitrogen. All three cell
lines have been published previously and were shown to
be germline competent at comparable passages; 129Sv
(Szab and Mann, 1994; Yang et al., 2006), BALB/c
(Royan C1) (Baharvand and Matthaei, 2004), and
C57BL/6 (Royan B1) (Baharvand and Matthaei, 2004;
Sadraei et al., 2009).
The mESCs were grown on mitomycin C-treated
embryonic feeder cells in complete ESC medium
(Dulbeccos modified Eagles medium (DMEM) (Gibco
ES cell qualified 1829, Invitrogen) supplemented with
15% ES qualified fetal bovine serum (Gibco 16141-079,
Invitrogen), 2mM Glutamine (Gibco, 25030, Invitrogen),
0.1 mM MEM nonessential amino acids (Gibco, 11140,
Invitrogen), and 0.1mM 2-mercaptoethanol (Sigma,
M7522), 50 U penicillin, and 50 U streptomycin (GIBCO
15140-148, Invitrogen), and 1,000 U/mL leukemia
inhibitory factor (LIF) (ESGRO ESG1106, Millipore) as
previously described (Matthaei, 2009) but in 5%CO2 in
air.
Mouse ESCs were grown to near confluence. Medium
was aspirated from the flask. The adhered cells were
washed twice with Ca2+ Mg2+-free phosphate-buffered
saline (PBS) (Gibco, 20012), and incubated at room
temperature for 2 minutes with PBS containing 0.5mM
ethylene glycol tetra-acetic acid (EGTA) (Sigma-Aldrich
E3889, St. Louis, USA), then for 1-2 minutes with
0.05% trypsin-ethylenediaminetetraacetic acid (EDTA)
(Gibco 25300, Invitrogen) (Matthaei, 2009). To obtain
a single cell suspension, the cells were triturated rapidly
in the trypsin solution several times through a small-bore

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Asian Pacific Journal of Cancer Prevention, Vol 14, 2013

pipette. The trypsin was inactivated by the addition of

complete ESC medium. The cell suspension was plated
into a 10 cm tissue culture dish and incubated at 37C for
15-30 minutes to allow mouse embryonic feeder (MEF)
cells to attach. The ESCs were then carefully aspirated,
the cell suspension counted using a hemocytometer and
centrifuged at 1000 rpm for 5 minutes to obtain a cell
pellet for appropriate dilution.
Immunocytochemical staining
For immunostaining, cells were grown to 80-90%
confluence on 4 Permanox chamber slides. The slides were
fixed in 4% formaldehyde for 10 minutes and then washed
three times in PBS. Nonspecific immunoglobulin G
binding sites were blocked for 20 minutes with 5% bovine
serum albumin (BSA) in PBS. The cells were labeled
with primary antibodies [anti-mouse Oct3/4 (1-5 g/ml,
Abcam), anti-mouse Sox2 (3 g/ml, Abcam), and antimouse SSEA1(1/100, Abcam)], incubated for 1-2 hours,
and then stained with anti-rat or anti-rabbit FITC (Abcam)
secondary antibodies. All cells were counterstained with
DAPI for nuclei.
Teratoma formation
Forty-six BALB/c mice (a kind gift by the Stem Cell
and Gene Targeting Laboratory, John Curtin School of
Medical Research, the Australian National University,
Canberra ACT 0200, Australia) 6-10 weeks old were
used as host animals for the implantation of three mESC
lines (129Sv, BALB/c and C57BL/6). Injections were
performed under the kidney capsule in 21 mice and into
the subcutaneous tissue in 25 mice. For renal subcapsular
injection, 15106 mESCs/injection were resuspended
thoroughly in 20l cold complete ESC medium with
20mM HEPES (Gibco, 15630) in 1.5ml sterile Eppendorf
tubes. For subcutaneous injection, 15106 mESCs/injection
were resuspended in 150l cold complete ESC medium
in 1.5ml sterile Eppendorf tubes and kept on ice prior to
injection. Subcutaneous injection was performed at 2 sites
(flank and neck) in each animal.
Injection under the kidney capsule
The mice were anesthetized with Sevoflurane and the
right kidney was exposed via a right paralumbar incision.
A transverse tear was made in the ventral aspect of the
kidney capsule using microforceps. Air was then injected
using a 22G intravenous cannula through the tear to create
a pocket. The same size cannula was used to inject the
cells into the pocket. Then the tear in the renal capsule
was closed with cautery. The abdominal wall musculature
and overlying skin were closed with 6-0 vicryl sutures.
Subcutaneous injection
The mice were anesthetized with Sevoflurane. The
cells were resuspended gently using a 25G syringe needle
and then drawn into a 1ml syringe immediately before
injection. The skin at the site of injection was lifted and
the cells were injected into the subcutaneous space.
Histological analysis
Four weeks after the injection, the animals were

DOI:http://dx.doi.org/10.7314/APJCP.2013.14.10.5705
Teratoma Formation in Mice after Syngeneic and Allogeneic Implantation of Mouse Embryonic Stem Cells

euthanized and the sites of implantation were dissected

for histological examination. Harvested tissues were fixed
for 48hrs in 4% paraformaldehyde, paraffin sectioned
at 5m and stained with hematoxylin and eosin. Highresolution whole-slide digital scans of all glass slides
were then created with a ScanScope scanner (Aperio
Technologies, Inc.). The digital slide images were then
viewed and analyzed using the viewing and image analysis
tools of Aperios ImageScope software. The size of each
teratoma in terms of its cut-sectional area was measured
in mm2. The composition of each teratoma was estimated
semi-quantitatively by visual inspection of digital slide
images. Differentiated elements in the teratomas were
classified into ectodermal (skin, neural), mesodermal
(cartilage, bone, muscle), and endodermal (glandular
structures) components as a percentage of entire tissue.
The percentages of undifferentiated tissue, cysts, necrosis
and hemorrhage were also recorded. The remaining
spindle-shaped stroma was included under mesoderm.
Statistical analysis
The incidence of teratoma was compared using
Pearson Chi-square. For composition of teratoma, t-test
for dependent variables was used for within group
comparisons, while one-way analysis of variance was
used for between group comparisons. Because the size
varied widely among teratomas, it was compared between
groups using the nonparametric Kruskal-Wallis analysis
of variance and median test. Results were considered
significant when p<0.05.

An encapsulated solid tumor was found at the

implantation site in 30% and 40% of 129Sv-implanted
mice, in 0% and 37.5% of BALB/c-implanted mice,
and in 25% and 66.7% of C57BL/6-implanted mice, for
subcutaneous and renal implantation, respectively (Table
1, Figure 2A). The overall teratoma development success
rate was 20 out of 71 injections (28.17%). The overall
incidence was highest with 129Sv (33.3%) followed by
C57BL/6 (30.43%) and lowest with BALB/c (16.67%).
For all cell lines combined, the incidence of teratoma was
significantly higher under the kidney capsule (42.86%)
than in the subcutaneous tissue (22%; p=0.0395). No
teratoma development was observed in mice that had
received the BALB/c cell line subcutaneously.
To investigate whether the site of injection plays a role
in the size of teratoma formation we analyzed the size
of teratomas based on the site of injection. The overall
tumor size appeared largest when derived from 129Sv
(66.0799.43 mm2) followed by C57BL/6 (35.0235.37
mm2) and smallest with BALB/c (5.304.77 mm2) ESCs.
However, these differences did not reach statistical
significance. Similarly the overall size appeared largest
under the kidney capsule (53.80109.84 mm2) followed
by the subcutaneous tissue (39.7829.79 mm2). The
cut-sectional area of teratomas developed by 129Sv

Results
The three mESC lines were cultured as undifferentiated
ESCs in the presence of MEFs and LIF. Colonies from
all cell lines exhibited positive staining for Oct3/4,
Sox2, and SSEA1 (Figure 1). We then analyzed the
tumor formation ability of ESCs (129Sv, BALB/c and
C57BL/6) by syngeneic and allogeneic implantation
into immunocompetent BALB/c mice. We found that
the incidence of teratoma formation after syngeneic
implantation of BALB/c ESCs into immunocompetent
BALB/c mice was 16.67% (Table 1). Unexpectedly,
however, we observed that when we implanted 129Sv
and C57BL/6 ESCs into immunocompetent BALB/c mice
(allogeneic implantation) teratomas were also formed in
33.3% and 30.43%, respectively (Table 1).

Figure 1. Characterization of the Three Different

mESC Lines. Upper panel shows live cell image and colony
formation of the three mESC lines grown under standard ESC
conditions. Lower 3 panels show immunofluorescence cell
staining of pluripotent colony cells with positivity for Oct4, Sox2
and SSEA1. Nuclear stain DAPI was used as a counter staining

Table 1. Incidence, Mean Size and Percentages of Components of Teratomas Formed After Subcutaneous and
Renal Subcapsular Injection of Three mESC Lines
Cell Line Site

Incidence

Size (mm2) Ectoderm Mesoderm Endoderm

Undiff.

H/N Cysts

129Sv SC 30.00% (6/20) 35.4023.99 40.33%17.91 36.33%21.69 9.33%5.61 4.83%2.93 2.17%3.92 5.33%10.03

Renal 40.00% (4/10) 112.09154.89 53.5%34.58 7.75%4.50 8.00%4.00 14.5%20.60 3.75%1.50 12.5%9.57

Total 33.30% (10/30) 66.0799.43 45.6%24.96 24.9%22.05 8.8%4.83 8.7%13.08 2.8%3.16 8.2%10.01
BALB/c
SC
0.00% (0/10)


Renal 37.50% (3/8)
5.304.77
83.33%15.28 5.0%5.00 3.33%2.89 5.67%5.13 1.67%1.53 1.0%1.73

Total 16.67% (3/18) 5.304.77 83.33%15.28 5.0%5.00 3.33%2.89 5.67%5.13 1.67%1.53 1.0%1.73
C57BL/6 SC 25.00% (5/20) 45.0437.88 73.60%6.12 14.40%7.54 6.20%2.77 3.00%1.00 1.60%1.53 1.2%2.17

Renal 66.70% (2/3)
9.972.60
80.00%1.41 4.5%2.12 3.5%0.71 10.00%0.00 1.00%0.00 1.00%1.41

Total 30.43% (7/23) 35.0235.37 75.43%5.91
11.57%7.87 5.43%2.64 5.00%3.51 1.43%1.72 1.14%1.86100.0
100.0
Overall
28.17% (20/71) 46.0974.82 61.7%24.71 17.25%17.86 6.8%4.34 6.95%9.54 2.15%2.52 4.65%7.88
6.3
10.1
20.3
*SC, subcutaneous; undiff., undifferentiated tissue; H/N, hemorrhage and necrosis

Asian
14, 2013
75.0 Pacific Journal of Cancer Prevention, Vol 25.0
56.3

46.8

6.
61

5707 75.0
30.0

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Abdullah Aldahmash et al

Figure 2. Development of Teratomas by Three Different

mESC Lines in BALB/c Mice. A) Average teratoma
development at the different sites as a percentage; and B) Average
teratoma size at the different sites (mm2). SC, subcutaneous;
*Significant difference (p<0.05)

Figure 4. Germ Layer Composition of Teratomas

Derived from the Different Cell Lines. A) to C) comparison
between sites for the 129Sv, C57BL/6 and BALB/c, respectively;
and D) Overall comparison of germ layer composition of all
teratomas formed, **Significant difference from meso- and
endoderm; *Significant difference from endoderm (p<0.05)

Figure 5. Germ Layer Composition of Teratomas

Derived from the Different Cell Lines. Comparison of
cell lines in subcutaneous A) and renal subcapsular B) teratomas

Figure 3. Histological Analysis of Teratomas. A) Gross

anatomy and overview of teratoma cut-section; B) Histological

analysis of teratomas identifying the presence of derivatives of
all three germ layers: neural tissue and keratinizing epithelium
(ectoderm), cartilage, striated muscle and trabecular bone
(mesoderm) and respiratory-like, gut-like and mucinous
glandular epithelium (endoderm); and C) Other tissue elements:
undifferentiated tissue with malignant nuclear criteria including
pleomorphism, hyperchromatism, frequent mitotic figures
(arrowheads) and tumor giant cells with bizarre nuclei (white
arrows), necrotic tissue and cysts lined by cuboidal epithelium
(black arrow). Scale bars represent 100 m

cell line measured an average of 35.4023.99 mm2 and

112.09154.89 mm2 in the subcutaneous tissue and under
kidney capsule, respectively. No teratoma development
was observed in the subcutaneous tissue of mice that
received the BALB/c cell line, while those that developed
under the kidney capsule were only small and measured
an average of 5.34.77 mm2 (Figure 2B). The teratomas
that developed from the C57BL/6 cell line measured an
average of 45.0437.88 mm2 in the subcutaneous tissue
and 9.972.60 mm2 under the kidney capsule (Table 1,
Figure 2B). The largest sizes were observed with the

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Asian Pacific Journal of Cancer Prevention, Vol 14, 2013

129Sv cell line injected under the kidney capsule and with
the C57BL/6 cell line in the subcutaneous tissue (Figure
2B). Notably, the larger the teratoma had grown, the more
tissue types could be identified histologically. We also
found that the incidence and size of teratoma formation
are site-dependent; injection under the kidney capsule
having the highest success rate and producing the largest
teratoma sizes (Figure 3A).
Diverse ESC-derived tissues, belonging to the three
embryonic germ layers, were encountered, reflecting the
pluripotency of the ESCs. Most commonly present tissues
were nervous tissue, keratinizing stratified squamous
epithelium (ectoderm), smooth muscle, striated muscle,
cartilage, bone (mesoderm), and glandular tissue in the
form of gut- and respiratory-like epithelia (endoderm), in
that order (Figure 3B). The overall average composition of
teratomas (n=20) was toward significantly more ectoderm
(61.70%24.71) than mesoderm (17.25%17.85,
p=0.0001) or endoderm (6.80%4.34, p=0.0000) and
significantly more mesoderm than endoderm (p=0.0085)
(Table 1, Figure 4D). Teratomas that developed from
the 129Sv cell line in the subcutaneous tissue showed
significantly more mesoderm than teratomas that
developed under the kidney capsule (p=0.017) (Figure
4A). However, C57BL/6 and BALB/c did not show any
significant difference in germ layer composition between
injection sites (Figure 4B-C). Teratomas that developed

DOI:http://dx.doi.org/10.7314/APJCP.2013.14.10.5705
Teratoma Formation in Mice after Syngeneic and Allogeneic Implantation of Mouse Embryonic Stem Cells

from the C57BL/6 cell line in the subcutaneous site

contained significantly more ectoderm than teratomas
that developed from the 129Sv cell line at the same
site (p=0.0017) (Table 1, Figure 5A). Teratomas that
developed under the kidney capsule did not show any
significant difference in germ layer composition between
the three cell lines; however they all showed a high
tendency towards ectoderm (Figure 5B).
Clusters of undifferentiated cells with malignant nuclear
criteria (including pleomorphism, hyperchromatism
and frequent mitotic figures) were common in most
of the tumors (Figure 3C). The overall percentage of
undifferentiated cells was higher with 129Sv (8.7%13.08)
than BALB/c (5.67%5.13) or C57BL/6 (5%3.51) with
no statistically significant differences (Table 1). Site
comparison showed that, overall, renal subcapsular
implantation was associated with a significantly higher
percentage of undifferentiated cells than subcutaneous
implantation.

Discussion
The use of stem cell therapy for the treatment of
human diseases would dramatically improve our ability
to alleviate debilitating conditions. The potential of using
ESCs for this purpose has been muted since these cells
have the ability to differentiate into every cell type in
an entire individual. This would suggest that all organs
could be treated once methods had been developed to
direct the ESCs to differentiate into specific target tissues
thereby eliminating the problem of limited donor tissue for
treatments. However, concerns have been raised over the
safety of this promising therapeutic approach, including
whether the transplanted ESCs have the potential to form
tumors after engraftment because of their unlimited selfrenewal and high differentiation potential (Arnhold et al.,
2004; Amariglio et al., 2009; Lukovic et al., 2013).
A number of studies (Thomson et al., 1998; Wakitani
et al., 2003; Stojkovic et al., 2005; Nussbaum et al.,
2007; Dressel et al., 2008; Prokhorova et al., 2009;
Zhang et al., 2012; Li et al., 2013; Lukovic et al., 2013)
have shown the potential of ESCs to form teratomas by
injecting undifferentiated ESCs into mice. Most studies
used immunodeficient hosts such as severe combined
immunodeficient (SCID) mice (Thomson et al., 1998;
Wakitani et al., 2003; Stojkovic et al., 2005; Zhang et
al., 2012). Dressel et al. (2008) surprisingly showed that
teratoma development could also occur after injection of
ESCs into syngeneic immunocompetent mice. This made
us consider whether the state of differentiation of the ESCs
could influence the outcome. The question, therefore, is
whether the more undifferentiated ESCs are, the more
they are prone to form teratomas.
We have for many years been using mouse ESCs from
3 different mouse strains to generate genetically modified
mice. This process is completely reliant on the ability of
the ESCs to remain totally pluripotent and integrate into
the germline of chimera after injection into blastocysts;
thereby producing ESC-derived offspring after mating
with wild-type mice. Our cell lines had all been proven
to be fully undifferentiated and germline competent using

this technique (Campbell et al., 2002; Baharvand and

Matthaei, 2004; Yang et al., 2006; Meyer et al., 2010). We
therefore decided to test the ability of all 3 mouse ESC
lines to form teratomas in immunocompetent mice, both
in syngeneic as well as allogeneic combinations.
The present study shows that our ESCs had the
capability to form teratomas after injection into syngeneic
as well as allogeneic immunocompetent mice (Table 1).
The incidence of teratomas was higher after implantation
of ESCs from the 129Sv strain when compared to ESCs
from the C57BL/6 and BALB/c strains. This might be
expected since the 129Sv strain is most commonly used
due to its high germline competence and stability during
continued cell culture and expansion. Interestingly, in this
study, syngeneic implantation of the BALB/c cells into
BALB/c mice failed to produce teratomas in subcutaneous
tissue while those that developed under the kidney capsule
were only small. Previous studies reported teratomas
developing after syngeneic implantation (129Sv ESCs
into 129Sv mice (Arnhold et al., 2004; Swijnenburg et
al., 2005; Dressel et al., 2008) or C57BL/6 ESCs into
C57BL/6 mice (Nussbaum et al., 2007), but syngeneic
implantation of BALB/c ESCs into BALB/c mice has not
been described previously. In previous studies (Nussbaum
et al., 2007; Xie et al., 2007) allogeneic ESCs also caused
teratomas. However, in these previous studies, allogeneic
teratomas initially detected were gradually rejected by an
increasing inflammatory immune response. These findings
suggested that immunogenicity of ESCs increases on their
differentiation, i.e. once ESCs reach a more differentiated
state, they can be more effectively recognized and rejected
by the recipient immune system. In the present study, the
reason why we did not encounter such immune rejection
after allogeneic implantation is, at this stage, not known.
Histologic analysis of the teratomas showed that
the most commonly present tissues were nervous
tissue, keratinizing stratified squamous epithelium
(ectoderm), smooth muscle, striated muscle, cartilage,
bone (mesoderm), and glandular tissue in the form of
gut- and respiratory-like epithelia (endoderm), in that
order. Hence diverse ESC-derived tissues, belonging
to the three embryonic germ layers, were encountered,
thereby reflecting the pluripotency of the ESCs (Thomson
et al., 1998; Oosterhuis and Looijenga, 2005; Mahmood
et al., 2010; Bustamante-Marn et al., 2013; Pal et al.,
2013). Importantly, this suggests that the differentiation
potential of the 3 mouse ESC lines used is independent
of microenvironmental factors that may be encountered
at the injection site. These results are in agreement
with previous studies that implanted a heterogeneous
differentiated ESC population into brain (Riess et al.,
2007), heart (Xie et al., 2007) and retina (Cui et al.,
2013) and found that any remaining undifferentiated cells
formed teratomas. Hence, our data also support that care
should be taken that ESCs are fully differentiated before
their use for cell-based therapy (Prokhorova et al., 2009),
since any remaining undifferentiated cells, if irresponsive
to microenvironmental differentiation signals, can form
teratomas. Moreover, even after thorough selection of a
highly puried population of ESC-derived neural (Arnhold
et al., 2004; Lukovic et al., 2013) and islet (Fujikawa et al.,

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Abdullah Aldahmash et al

2005) precursor cells, there remains a high risk of teratoma

formation after engraftment. However, transplantation
of FACS purified ESC-derived neural precursors
expressing the neural precursor marker Sox1 into mouse
brains (Fukuda et al., 2006) and targeting hESC-specific
antiapoptotic factor(s) (i.e., survivin or Bcl10) represent
an efficient strategy to selectively eliminate pluripotent
cells with teratoma potential (Lee et al., 2013).
We found, in accordance with Cooke et al. (2006),
that the incidence and size of teratoma formation are
site-dependent; injection under the kidney capsule
having the highest success rate and producing the largest
teratoma sizes. Because of its rich vascular bed, the renal
subcapsular space of mice is a suitable environment
for growing and studying solid tumors (Press et al.,
2008). This makes renal subcapsular implantation better
to subcutaneous implantation for teratoma induction
experiments, in terms of success rate and size of teratomas
produced.
In this study, clusters of undifferentiated cells
exhibiting malignant nuclear criteria (including
pleomorphism, hyperchromatism and frequent mitotic
figures) were common in most of the teratomas. These
can be considered as malignant teratomas based on the
presence of tissues of the three germ cell layers and the
presence of both anaplastic foci and immature tissues
(Lpez and Mrcia, 2008). The overall percentage
of undifferentiated cells (malignant-looking clusters)
was higher with 129Sv than BALB/c or C57BL/6
ESCs. Renal subcapsular implantation was associated
with a higher percentage of undifferentiated cells than
subcutaneous implantation. This agrees with Cooke
et al. (2006) who reported that subcutaneous implants
were significantly slower growing and formed tumors
composed of differentiated tissues, in contrast to cells
grafted into the liver which rapidly produced large tumors
containing predominantly immature cells. It is still largely
unknown why ESCs, which lack overt chromosomal
abnormalities, are tumorigenic. One hypothesis is based
on the identification of the canonical WNT signaling
as a critical determinant for the tumorigenicity and
therapeutic function of ESC-derived retinal progenitors.
The function of WNT signaling is primarily mediated by
TCF7, which directly induces expression of Sox2 and
Nestin (Cui et al., 2013). Also, similarity between ESCs
and cancer cells has been proposed. The embryonic stem
cell factor Sox2 is reported to be expressed in a variety
of early stage postmenopausal breast carcinomas and
metastatic lymph nodes (Lengerke et al., 2011). The study
of Wong et al. (2008) demonstrated that embryonic stem
cells and multiple types of human cancer cells share a
genetic expression pattern that is repressed in normal
differentiated cells. Recent discoveries also indicated that
testicular carcinoma in situ (CIS) cells have a striking
phenotypic and genotypic similarity to ESCs (Almstrup
et al., 2006). The similarities between ESCs and cancer
cells should be further investigated.
In the present study, implantation of mESCs
caused teratomas in both syngeneic and allogeneic
immunocompetent mice. It could be argued that our data
are not consistent with a previously published report

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(Dressel et al., 2008) in which allogeneic transplants of

mESC did not result in teratomas in immunocompetent
hosts. However, the state of pluripotency of the ESCs
used in that study (Dressel et al., 2008) was not clearly
described, in contrast to our ESCs with proven germline
competence. In addition, because of their early stage of
development, ESCs are considered immune privileged,
i.e, unrecognizable by the recipient immune defenses
(Magliocca et al., 2006; Ichiryu and Fairchild, 2013).
Moreover, it has been reported that mESCs are resistant
to antigen-specific cytotoxic T lymphocyte (CTL) due to
the expression of serpin-6, an endogenous inhibitor of
granzyme B (Abdullah et al., 2007). Dressel et al. (2008)
stated that the MPI-II cells they used were susceptible to
granule exocytosis-mediated killing by rat NK cells and
also by peptide-specific mouse CTL. So their cells may
not have been protected against cellular cytotoxicity and
hence their allogeneic transplants into immunocompetent
mice were rejected soon after injection. In contrast, our
cells were not rejected after allogeneic implantation
in immunocompetent mice since they may have been
immune privileged and thereby escaped cytotoxic immune
destruction and formed teratomas. Nevertheless, such
argument warrants that the susceptibility of different ES
cell lines to the cytotoxic immune response be further
investigated.
In conclusion, we have confirmed that ESCs can form
teratomas in immunocompetent mice. Furthermore, we
showed that germline competent ESC can form teratomas
not only in syngeneic but also in allogeneic hosts. It is
clear, therefore, that in a clinical setting, it must be ensured
that no undifferentiated cells remain which could form
teratomas. Without this assurance, the use of ESCs should
not be considered as a therapeutic option for patients with
degenerative disease.

Acknowledgements
This work was supported by a grant (No. 10-BIO130402) and (10-BIO1308-02) from the National Plan for
Sciences and Technology Program, King Saud University
and a grant from the International Scientific Twinning
Program, King Saud University. Our thanks must also go
to Professor Moustapha Kassem.

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