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Biology Direct

BioMed Central

Open Access

Research

Multipotent adult germ-line stem cells, like other pluripotent stem
cells, can be killed by cytotoxic T lymphocytes despite low
expression of major histocompatibility complex class I molecules
Ralf Dressel*†1, Kaomei Guan†2, Jessica Nolte3, Leslie Elsner1,
Sebastian Monecke1, Karim Nayernia3,4, Gerd Hasenfuss2 and
Wolfgang Engel3
Address: 1Department of Cellular and Molecular Immunology, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany,
2Department of Cardiology and Pneumology, University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany, 3Institute of Human
Genetics, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany and 4North East Stem Cell Institute and Institute of
Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK
Email: Ralf Dressel* - rdresse@gwdg.de; Kaomei Guan - kguan@med.uni-goettingen.de; Jessica Nolte - jnolte1@gwdg.de;
Leslie Elsner - lelsner@gwdg.de; Sebastian Monecke - s.monecke@med.uni-goettingen.de; Karim Nayernia - karim.nayernia@newcastle.ac.uk;
Gerd Hasenfuss - Hasenfus@med.uni-goettingen.de; Wolfgang Engel - wengel@gwdg.de
* Corresponding author †Equal contributors

Published: 28 August 2009
Biology Direct 2009, 4:31

doi:10.1186/1745-6150-4-31

Received: 19 August 2009
Accepted: 28 August 2009

This article is available from: http://www.biology-direct.com/content/4/1/31
© 2009 Dressel et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell
type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs)
present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a
source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test
whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and
iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as
previously proposed for embryonic stem cells (ESCs).
Results: We have observed that maGSCs can be maintained in prolonged culture with or without
leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in
immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic
recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility
of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major
histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on
these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However,
using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent
stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by
activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with
the SIINFEKL peptide.
Conclusion: Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for
CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow
cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent
cells might be rejected after transplantation by this mechanism if specific antigens are presented

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and if specific activated CTLs are present. Our results show that the adaptive immune system has
in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help
to develop new strategies to increase the safety of future transplantations of in vitro differentiated
cells by exploiting a selective immune response against contaminating undifferentiated cells.
Reviewers: This article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter.

Background
Pluripotent stem cells could provide the basis for restoring
tissue functions by transplantation of cellular grafts in a
broad variety of diseases. Embryonic stem cells (ESCs) are
the best characterized pluripotent stem cells. They clearly
have the capacity to self-renew and to give rise to any cell
type of the body [1]. However, the use of human ESCs is
restricted due to severe ethical concerns. Therefore, new
pluripotent cell types that are derived from adult organisms hold great promises for regenerative medicine.
Recently, it has been shown that pluripotent cells can be
obtained from spermatogonial stem cells (SSCs) of neonatal [2] and even adult mice [3-5]. Although SSCs are
known to be unipotent [6] they can undergo reprogramming during in vitro cultivation and give rise to multipotent adult germ-line stem cells (maGSCs) that can
differentiate into various cell types in vitro, and form teratomas in vivo [3,7]. The maGSCs have significant similarities to ESCs [8,9] and their differentiation capacity into
functional cardiomyocytes [10] and neuronal cells
[11,12] has been shown. Therefore, maGSCs are expected
to have a high potential for regenerative medicine [13].
The generation of pluripotent cells from adult human testis has been recently reported [14-16]. Since maGSCs can
be obtained without the need of genetic manipulation,
they might have advantages even compared to induced
pluripotent stem cells (iPSCs) that were obtained by successful reprogramming of mouse and human somatic
cells after expression of a limited number of defined transcription factors [17-19].
The possibility to generate autologous pluripotent stem
cells from adults could also reduce immunological problems that are expected to occur when allogeneic ESCs are
used for regenerative therapies [20,21]. However, in the
case of maGSC-based therapies only male patients could
theoretically benefit from fully autologous stem cells. The
complexity of maGSC generation and requirements for
quality assurance for individual stem cell lines might be a
further hurdle for the therapeutic use of individualized
maGSCs, as has been discussed in the context of iPSCs
[22,23]. Thus, although maGSCs and iPSC could theoretically allow the generation of autologous pluripotent stem
cells, they might also be of use for therapeutic purposes in
partially allogeneic settings. However, even when autologous stem cells are utilized for transplantation therapies,
cytotoxic T lymphocytes (CTLs) might recognize "oncofe-

tal" antigens [24] expressed by pluripotent stem cells or
differentiation antigens [25] as has been documented and
exploited in the context of tumor immunology. A further
concern is that syngeneic or autologous transplantations
might be associated with a higher risk of teratoma growth
than allogeneic transplantations if grafts are derived from
pluripotent cells and are contaminated with undifferentiated cells [26]. Therefore, it seems important to determine
whether new pluripotent stem cell types such as maGSCs
and iPSCs are protected against CTLs, similarly to what
has been reported for ESCs [27].
In this study, we have analyzed the teratoma growth of
maGSCs depending on the conditions used for their culture and in vitro differentiation. Furthermore, the susceptibility of three maGSC lines (maGSC Stra8 SSC5, maGSC
129/Sv, maGSC C57BL) to CTLs has been determined in
comparison to corresponding ESC lines (ESC Stra8, ESC
129/Sv R1, ESC C57BL), iPSCs, and F9 teratocarcinoma
cells.

Methods
Cell culture
The maGSC line Stra8 SSC5 (H2b) [3] was grown under
four different culture conditions that have been previously described in detail [7]. Briefly, under condition I the
cells were cultured in dishes, pre-coated with 0.1% gelatine (Sigma-Aldrich, Taufkirchen, Germany) for at least 1
h at 4°C, with basic medium, i.e. Dulbecco's modified
Eagle's medium (DMEM) supplemented with 15% fetal
calf serum (FCS, selected batches), 2 mM L-glutamine
(Invitrogen, Karlsruhe, Germany), 50 μM β-mercaptoethanol (β-ME; Promega, Mannheim, Germany), 1 × nonessential amino acids (NEAA; Invitrogen). For condition II
the cells were cultured in gelatine-coated dishes with basic
medium containing 103 units/ml leukaemia inhibitory
factor (LIF; ESGRO, Millipore, Billerica, MA, USA). Under
condition III the cells were cultured on a feeder layer of
mitomycin C-inactivated mouse embryonic fibroblasts
(MEFs) with basic medium. Cells under condition IV were
cultured on a mitomycin C-inactivated MEF feeder layer
with basic medium containing 103 units/ml LIF. The cells
were used at passages 12 to 18 (condition I), 9 to 20 (condition II), 8 to 22 (condition III), and 10 to 22 (condition
IV). The other pluripotent stem cell lines used in this
study were cultured under condition IV. These included
the lines maGSC 129/Sv (H2b) [3] (passages 24 to 37),
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maGSC C57BL (H2b) [3] (passages 24 to 31), ESC 129/Sv
MPI-II (H2b) [28] (passages 24 to 33), and ESC 129/Sv R1
(H2b) [29] (passages 19 to 38). The line ESC Stra8 SSC5
(passages 10 to 15) was derived from a Stra8-EGFP/
Rosa26 transgenic mouse [8]. A new ESC line (ESC
C57BL) (H2b) (passages 9 to 18) was generated as
described [30]. The iPSCs (passages 13 to 21) were
derived from MEFs (129/Sv × C57BL/6 F1) (H2b) and
have been described previously [31]. They were kindly
provided by Dr. Rudolf Jaenisch, The Whitehead Institute
for Biomedical Research, Cambridge, USA. F9 teratocarcinoma cells (H2b), which originated from an embryonal
testicular teratocarcinoma of a 129/Sv mouse were cultured as described [32]. The mouse cell lines YAC-1 (H2a),
RMA (H2b), and RMA-S (H2b) were maintained in
NaHCO3-buffered DMEM supplemented with 10% FCS
(Biochrom, Berlin, Germany), 2 mM L-glutamine, 1 mM
sodium pyruvate, 50 μM β-ME, 100 U/ml penicillin, and
100 μg/ml streptomycin.
In vitro differentiation of maGSCs
For differentiation, maGSC Stra8 SSC5 cells cultured
under condition III were cultivated as embryoid bodies
(EBs) in hanging drops as previously described [3].
Briefly, maGSC Stra8 SSC5 cells were cultured without
MEFs for 2 passages before cultivation as EBs. 400 cells in
20 μl of differentiation medium (Iscove's modified Dulbecco's medium supplemented with 20% FCS, 2 mM Lglutamine, 1× NEAA, and 450 μM α-monothioglycerol 3mercapto-1,2-propandiol
(MTG;
Sigma-Aldrich,
Taufkirchen, Germany)) were placed on the lids of Petri
dishes filled with phosphate-buffered saline (PBS) and
incubated in hanging drops for 2 days and in bacteriological Petri dishes for another 3 days. At day 5, single EBs
were transferred into gelatine (0.1%)-coated 6-cm tissue
culture dishes in differentiation medium for 10 further
days.
Generation of CTLs and 51Chromium release assays
To obtain peptide-specific CTLs, spleen cells from naive
OT-I mice [33] were stimulated in vitro in the presence of
1 nM SIINFEKL peptide (Ovalbumin 257-264; Bachem
Biochemica, Heidelberg, Germany) as previously
described [34]. Target cells were labeled by incubating 1 ×
106 cells in 200 μl N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES)-buffered DMEM containing
100 μl FCS and 50 μCi Na251CrO4 (Hartmann Analytic,
Braunschweig, Germany) for 1 h at 37°C and washed
three times with HEPES-buffered DMEM. Effector cells
were added to 5 × 103 51Cr-labeled target cells in triplicate
at various ratios in 200 μl HEPES-buffered DMEM/10%
FCS per well of round-bottomed microtiter plates. Spontaneous release was determined by incubation of target
cells in the absence of CTL. To allow for a peptide-specific
killing the respective wells were supplemented with 0.5

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μg/ml SIINFEKL. To determine calcium-dependency of
killing 2 mM ethyleneglycol-bis(b-aminoethyl ester)N,N,N',N'-tetraacetic acid (EGTA) and 4 mM MgCl2 were
added. The microtiter plates were centrifuged for 5 min at
40 × g, incubated at 37°C for 4 h, and then centrifuged
again. Supernatant and sediment were separately taken to
determine radioactivity in each well using a Wallac
MicroBeta Trilux counter (PerkinElmer Life Sciences,
Köln, Germany). Percentage of specific lysis was calculated by subtracting percent spontaneous 51Cr release
[35].
Flow cytometry
Flow cytometry was performed on a FACScan™ flow
cytometer (BD Biosciences, Heidelberg, Germany) using
CellQuest™ data acquisition and analysis software. The
cell surface expression of major histocompatibility complex (MHC) class I molecules was analyzed using locus
and haplotype-specific antibodies (Ab) anti-H2Kb (clone
CTKb, mouse IgG2a, phycoerythrin (PE)-conjugated,
Caltag Laboratories, Hamburg, Germany), and anti-H2Db
(clone CTDb, mouse IgG2a, PE-conjugated, Caltag). The
isotype control (mouse IgG2a, PE-conjugated) was purchased from Caltag. To test whether the MHC class I
expression can be stabilized at a lower temperature, the
stem cells were incubated at 28°C for 24 h before analysis.
In order to test for the stabilization of H2Kb cell surface
expression by external peptides, the H2Kb-restricted peptide SIINFEKL or a non-binding control peptide (NGLTLKNDFSRLEG) were added for 24 h to cell cultures. The
expression analysis of pluripotency markers was done as
described previously [3] using mouse anti-SSEA-1 (clone
MC480, Developmental Studies Hybridoma Bank, IA,
USA) and mouse anti-Oct4 (clone 9E3.2, Chemicon)
mAbs.
Gene expression analysis
Total RNA was extracted from cell lines using the TRIZOL®
reagent (Invitrogen, Carlsbad, USA) according to the
manufacturer's instructions. The RNA was then treated
with RNase free DNase (RQ1, Promega, Madison, USA)
for 20 min at 37°C and purified by phenol-chloroformisoamyl alcohol (25:24:1) extraction and precipitated
with 1/10 volume 300 mM sodium acetate (pH 4.8) and
1 volume 2-propanol before washing with 70% ethanol
and solving in RNase free water. The quantity of the
extracted RNA was determined with a ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington,
USA) and the quality was analyzed using a Bioanalyzer
2100 (Agilent Techonolgies, Santa Clara, CA, USA). For
synthesis of cDNA random oligo primers (Promega, Madison, USA) were used. The reverse transcription of RNA
was performed for 60 min at 37°C with M-MLV RT
polymerase (Promega, Madison, USA) in a total volume
of 25 μl. Gene expression levels were analyzed by quanti-

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tative real time polymerase chain reaction (qRT-PCR)
assays using the following forward and reverse primers
generated according to the indicated reference sequences:
H2Kb (NM_001001892.2, 5'-CCT GGA GTG GAC TTG
GTG AC-3' and 5'-GGT GTA GAG GGG TGG ACT GG-3')
H2Db (NM_010380.3, 5'-CCC TGT GAG CTT GGG TTC
AG-3' and 5'-ACA GGG CAG TGC AGG GAT AG-3'), transporter associated with antigen processing 1 (Tap1)
(NM_001161730.1, 5'-CTG CTC TCC CTC TAC CCC TC3' and 5'-CTG AGT GGA GAG CAA GGA GTC-3'), Tap2
(NM_011530.3, 5'-GCA GAC GAC TTC ATA GGG GA-3'
and 5'-GTT GCT TCT GTC CCA CAG C-3'), β2-microglobulin (B2m) (NM_009735.3, 5'-CTC ACA CTG AAT TCA
CCC CC-3' and 5'-CAG TAG ACG GTC TTG GGC TC-3'),
and Serpinb9 (NM_011452.2, 5'-TGC AGA CAA AAC TTG
TGA AGT CCT C-3' and 5'-TGC CTG GAC ACC TCT GCT
TC-3') encoding the serine protease inhibitor 6 (SPI-6)
protein. The mRNA expression of the housekeeping gene
hypoxanthine guanine phosphoribosyl transferase 1
(Hprt1) (NM_013556.2, 5'-GTC CTG TGG CCA TCT GCC
TA-3' and 5'-GGG ACG CAG CAA CTG ACA TT-3') was
always monitored as internal control. Amplification reactions were carried out in 96-well plates in 25 μl reaction
volumes with the Power SYBR® green PCR master mix
(Applied Biosystems, Foster City, USA). The PCR reaction
plates were preheated for 2 min at 50°C and for 10 min at
95°C followed by 40 cycles of denaturation (15 s at 95°C)
and amplification (1 min at 60°C). All reactions were performed in technical triplicates using an ABI 7500 Real
Time PCR System. For the data analysis, the ABI 7500 SDS
software (Applied Biosystems) was used. The variations in
cDNA concentration in different samples were normalized to the housekeeping gene Hprt1. The cycle threshold
(ct) values obtained for the genes of interest were corrected by the ct value obtained for Hprt1 in the same sample. The relative level of transcripts was then expressed as
Δct value (ct for Hprt1 minus ct for the gene of interest).
Immunoblot
Proteins from cultured cells were isolated as described
previously [8]. The protein extracts (20 μg per lane) were
separated by sodiumdodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose (Schleicher und Schüll, Dassel, Germany). The membranes were stained with an anti-PI-9 mAb (clone JM3544, mouse IgG, MBL, Woburn, MA, USA) that has been
described to cross-react with its mouse homologue SPI-6
[27]. An anti-heat shock cognate 70 (HSC70 or HSPA8)
mAb (clone 1B5, rat IgG2a, StressGen, Biomol, Hamburg,
Germany) was used as loading control. The primary antibodies were diluted 1:2000 in PBS/0.05% Tween 20. Subsequently, blots were incubated with secondary goat antimouse IgG (115-005-003, Jackson Laboratories, Dianova,
Hamburg, Germany) and goat anti-rat IgG and IgM (112005-068, Jackson Laboratories) Abs and then with a terti-

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ary peroxidase-conjugated rabbit anti-goat IgG Ab (305035-045, Jackson Laboratories, Dianova) at dilutions of
1:5000. The substrate reaction was carried out with 0.05%
3,3'-diaminobenzidine/0.0003% H2O2 in PBS/0.05%
Tween 20.
Animal experiments
All animals were bred in the central animal facility of the
Medical Faculty of the University of Göttingen. Severe
combined immunodeficient SCID/beige mice (C.B-17/
IcrHsd-scid-bg) were kept under pathogen-free conditions. All animal experiments had been approved by the
local government. For the analysis of subcutaneous tumor
growth undifferentiated maGSC and in vitro differentiated
cells, respectively, were suspended in 100 μl PBS (2 × 106
cells/100 μl) and injected into the flank of the animals.
Tumor growth was monitored every second day by palpation and size was recorded using linear calipers. The
tumor volume was calculated by the formula V = πabc/2,
where a, b, c are the orthogonal diameters. Animals were
sacrificed at day 100, or before when a tumor volume of 1
cm3 was reached, when a weight loss of more than 10%
occurred, or when any behavioral signs of pain or suffering became evident. Autopsies were performed for all animals. Tumor tissue was immediately frozen in liquid
nitrogen or placed in phosphate-buffered 4% formalin for
16 h and then embedded in paraffin. Tissue sections (2
μm) were stained with hematoxylin and eosin (HE) for
histological examinations.
Statistics
The data were analyzed using the non-parametric Kruskal
Wallis test and the U test for subgroup analysis with the
WinSTAT software. A significance level of α = 0.05 was
used.

Results
Maintenance of maGSC Stra8 SSC5 cells under different
culture conditions
MaGSC lines can be grown in vitro under different cell culture conditions [3,7], of which we used four in our study.
Condition I consists of basic medium, condition II is the
basic medium plus LIF, condition III is the basic medium
plus a feeder layer of MEFs, and condition IV is the basic
medium plus LIF plus MEFs. The condition IV represents
the gold standard for ESC culture. The maGSC line SSC5
expressed the pluripotency markers stage-specific embryonic antigen 1 (SSEA-1) and Oct3/4 under all culture conditions similarly to the ESC line MPI-II (Figure 1A). This
maGSC line was generated from the Stra8-EGFP/Rosa26
transgenic mouse, which expresses the enhanced green
fluorescent protein (EGFP) under control of the Stra8 promoter [3]. In male mice the Stra8 promoter is selectively
active in spermatogonia and their immediate descendants
(preleptotene spermatocytes) [36-39] before it becomes

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silenced upon further differentiation. Therefore, the
undifferentiated maGSC Stra8 SSC5 cells are characterized
by EGFP expression [3]. We determined the proportion of
EGFP expressing cells under the four culture conditions
and after in vitro differentiation for 5 plus 10 days by flow
cytometry as exemplified in Figure 1B. Under all four culture conditions more than 75% of the maGSC Stra8 SSC5
cells expressed EGFP (Figure 1C) and the conditions did
not differ significantly (p = 0.1753, Kruskal-Wallis test).
Thus, maGSCs retain expression of pluripotency markers
even under culture conditions without either LIF or feeder
cells, at least for more than 16 passages. After spontaneous
in vitro differentiation using the hanging drop method, on
average less than 20% of the cells remained positive for
EGFP at day 10 after plating EBs at day 5 (Figure 1C).

SSEA-1+ cells [%]

120

Oct-3/4+ cells [%]

A

120

100
80
60
40
20
0

100
80
60
40
20
0

I

II

III

IV

ESC

B
II

III

IV

diff.

ESC

counts

I

EGFP

EGFP+ cells [%]

C
120
100
80
60
40
20
0

I

II

III

IV

diff.

Figure
Proportion
cells
cultured
1 of under
pluripotent
various
cells
conditions
among maGSC Stra8 SSC5
Proportion of pluripotent cells among maGSC Stra8
SSC5 cells cultured under various conditions. (A)
MaGSC Stra8 SSC5 cells were cultured in basic medium
(condition I), medium plus LIF (condition II), basic medium on
MEFs (condition III), in medium plus LIF on MEFs (condition
IV). ESCs (line ESC 129Sv MPI-II) were cultured under condition IV. The proportion of SSEA-1 and Oct-3/4-positive cells
was evaluated by flow cytometry. The means of EGFP-positive cells + standard deviation (SD) are shown (n = 3). (B)
MaGSC Stra8 SSC5 cells cultured under conditions I to IV or
cells differentiated in vitro for 5 plus 10 days (diff.) were analyzed for EGFP expression by flow cytometry. Representative histograms are shown. An EGFP-negative ESC line (ESC
129Sv MPI-II) is included as negative control. (C) The means
of EGFP-positive cells + SD are shown (n = 5).

Teratoma formation of maGSC Stra8 SSC5 cells cultured
under different culture conditions
We have previously shown that maGSC Stra8 SSC5 cells
cultured in basic medium on a feeder layer (condition III)
have the potential to form teratomas in immunodeficient
SCID/beige mice [3]. To determine whether the various
cell culture conditions have an effect on the pluripotency
of maGSCs, we compared the tumor growth of maGSC
Stra8 SSC5 cells cultured under the conditions I to IV after
subcutaneous injection into SCID/beige mice. The passage numbers of maGSC Stra8 SCC5 cells used for these
experiments was between 12 and 22. Tumors were found
in high frequency within 100 days after injection of
maGSC Stra8 SSC5 cells cultured under all conditions
(Table 1). In most cases, progressively growing tumors
occurred within the first 50 days (Figure 2). However,
some tumors remained small and stable for the whole
observation time (Figure 2); others even regressed before
the end of the experiment (Figure 2, Table 1). Usually, the
tumors were found only subcutaneously at the site of
injection. In a few cases, autopsy revealed that tumors had
grown invasively and given rise to tumors in the peritoneal cavity (Table 1). The histopathological evaluation of
tumors indicated that teratomas had formed from the
maGSC Stra8 SSC5 cells cultured under four different conditions. This is exemplified in Figures 3A-C representing a
teratoma derived from maGSC Stra8 SSC5 cells cultured
under condition I. The teratomas contained derivatives of
all three embryonic germ layers, including epithelium
with intestinal differentiation (endoderm, Figure 3A), striated muscle (Figure 3B), smooth muscle, fat, bone and
cartilage (mesoderm), and neural tissue (ectoderm, Figure
3C). Thus, the cells cultured under all four conditions
were undifferentiated, as indicated by EGFP expression
and expression of pluripotency markers SSEA-1 and Oct3/
4 (Figure 1), and also pluripotent as shown by the capability to form teratomas.

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B
1200

condition I

1000
800
600
400
200

tumor size [mm3]

tumor size [mm3]

A

0

40
20

0

condition II

1000
800
600
400
200

tumor size [mm3]

tumor size [mm3]

1200

10 20 30 40 50 60 70 80 90 100

100

condition II

80
60
40
20
0

0

0

10 20 30 40 50 60 70 80 90 100

10 20 30 40 50 60 70 80 90 100

condition III

1000
800
600
400
200
0
0

10

20

30

40

50

60

70

80

condition IV

800
600
400
200

condition III

80
60
40
20
0

90 100

1200
1000

tumor size [mm3]

100

1200

0

tumor size [mm3]

tumor size [mm3]

60

10 20 30 40 50 60 70 80 90 100

0

tumor size [mm3]

condition I

80

0
0

0

10 20 30 40 50 60 70 80 90 100

100

condition IV
80
60
40
20
0

0

0

10 20 30 40 50 60 70 80 90 100

10 20 30 40 50 60 70 80 90 100

100

differentiated

1000
800
600
400
200
0
0

10 20 30 40 50 60 70 80 90 100

days post injection

tumor size [mm3]

1200

tumor size [mm3]

100

differentiated

80
60
40
20
0
0

10 20 30 40 50 60 70 80 90 100

days post injection

Figure growth
Tumor
2
of maGSC Stra8 SSC5 cells after injection into immunodeficient SCID/beige mice
Tumor growth of maGSC Stra8 SSC5 cells after injection into immunodeficient SCID/beige mice. Undifferentiated maGSC Stra8 SSC5 cells or in vitro differentiated cells (5 + 10 days) were injected subcutaneously at day 0 into SCID/beige
mice (2 × 106 cells per mouse). The undifferentiated cells were cultured under the four conditions (I to IV) before the injection.
The tumor size was recorded every second day until day 100 using linear calipers. (A) The growth of tumors in individual mice
is shown. The mice were injected with cells from two to three independent cultures that are indicated by different colors. Animals were sacrificed after day 100 or before when ethical criteria for the stop of the experiment were reached. (B) The same
data are presented on a different scale for the tumor size in order to show small or regressing tumors.

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Table 1: Tumor formation of maGSC Stra 8 SSC5 cells in immunodeficient SCID/beige mice

Culture condition

Tumor frequency

Regression frequency

Invasive growth

I

88% (7/8)

0% (0/7)

29% (2/7)

II

80% (8/10)

0% (0/8)

13% (1/8)

III

100% (18/18)

11% (2/18)

6% (1/18)

IV

100% (7/7)

14% (1/7)

14% (1/7)

diff.

83% (11/12)

27% (3/11)

0% (0/11)

MaGSC Stra8 SSC5 cells cultured under various conditions (I to IV) and cells differentiated in vitro for 5 plus 10 days (diff.) were injected
subcutaneously into the flank of SCID/beige mice (2 × 106 cells in PBS/animal). The percentage and number of animals in which tumors were found
during autopsy or in which tumors were palpable (at least during 3 consecutive observations) at the side of injection before day 100 after injection
is indicated. In addition, the percentages and numbers of animals are given in which tumors were present but regressed before day 100 and in which
an invasive growth was observed. The differentiated cells (diff.) were obtained as described in the Materials and Methods section from maGSC
Stra8 SSC5 cells that had been cultured under condition III.

In addition to the undifferentiated cells, in vitro pre-differentiated cells (5 + 10 days of differentiation culture)
were injected into 12 SCID/beige mice. In 6 cases an early
progressive tumor growth was observed, 2 tumors
remained small and stable, and 3 regressed before day 100
(Figure 2, Table 1). Thus, the in vitro pre-differentiation
protocol used here is not sufficient to deplete tumorigenic
cells from the cultures. This finding is in agreement with
the observation that a significant proportion of the in
vitro differentiated cells still expressed EGFP, indicating
the presence of undifferentiated cells (Figure 1B, C). The
histopathological analysis of these tumors showed that
the in vitro differentiated cells, similar to undifferentiated
maGSCs, formed teratomas (Figure 3D-F). The teratomas
contained derivatives of three embryonic germ layers,
including epithelium with intestinal differentiation
(endoderm, Figure 3D), striated muscle (mesoderm; Figure 3E), smooth muscle, fat, bone and cartilage (mesoderm), and neural tissue (ectoderm, Figure 3F).
The maGSC Stra8 SSC5 cells cultured under condition III
(on feeder cells) were also injected subcutaneously into
immunocompetent C57BL/6 mice. None of 9 animals
receiving 2 × 106 cells developed a teratoma within 100
days. At autopsy no residues of the injected cells were
found (data not shown). Thus, maGSCs were readily
rejected in immunocompetent allogeneic hosts.
Expression of MHC class I molecules on maGSC Stra8
SSC5 cells cultured under various conditions
Mechanisms of the immune system play an important
role for teratoma growth in immunocompetent animals.
To analyze the immunogenicity of maGSC Stra8 SSC5
cells, we determined the expression of MHC class I molecules and their susceptibility to CTL-mediated killing. A
proportion of the maGSC Stra8 SSC5 cells expressed MHC

class I molecules (Figure 4A, B). Between 35 and 60% of
the cells cultured under the four different conditions
expressed H2Kb molecules and less than 20% of the cells
expressed H2Db molecules as detected by flow cytometry.
The proportion of H2Kb expressing cells differed between
the cells cultured under the four conditions (p = 0.0127,
Kruskal-Wallis test) and was higher in cultures without
LIF (conditions I and III) compared to the respective condition with LIF suggesting an effect of the culture conditions on the immunologic phenotype of the maGSC Stra8
SSC5 cells. The results were similar (p = 0.0126, KruskalWallis test) when the analysis was restricted to the undifferentiated Stra8-EGFP expressing cells (Figure 4C). The
expression levels as determined by the mean fluorescence
intensity (MFI) on maGSC Stra8 SSC5 cells was rather low
(on average <50) and much lower than on RMA cells (on
average >350) that were used as positive control for the
staining (Figure 4A). However, the MFI for H2Kb also differed between the conditions (p = 0.0151, Kruskal-Wallis
test) and was higher in cultures without LIF. The in vitro
differentiation of SSC5 cells reduced the proportion of
H2Kb expressing cells (Figure 4A).
MaGSC Stra8 SSC5 cells cultured under different
conditions are killed by CTLs whereas in vitro
differentiated cells acquire resistance
Next we tested whether the maGSC Stra8 SSC5 cells can be
killed by CTLs. We used CTLs from TCR-transgenic OT-I
mice as effector cells, which recognize the ovalbuminderived peptide SIINFEKL in an H2Kb-restricted manner
[33]. The target cells, which do not express ovalbumin,
were pulsed with the peptide to allow for binding to H2Kb
molecules and peptide-dependent killing. RMA cells
served as positive control. The results of a representative
individual experiment are shown in Figure 4D. RMA cells
as well as maGSC Stra8 SSC5 cells were not killed when

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entiated cells (5 + 10 days of differentiation culture) were
hardly killed by CTLs.

Figure
Histology
Stra8
SCID/beige
SSC5
3 ofcells
mice
teratomas
and differentiated
formed after
cells
injection
into immunodeficient
of maGSC
Histology of teratomas formed after injection of
maGSC Stra8 SSC5 cells and differentiated cells into
immunodeficient SCID/beige mice. (A-C) Histological
analysis of a representative teratoma derived from maGSC
Stra8 SSC5 cells under condition I. (D-F) Histology of a teratoma derived from in vitro differentiated cells. The formed
teratomas contained derivatives of all three embryonic germ
layers (ectoderm, mesoderm, and endoderm). (A, D) Gut
epithelium (endoderm). (A) Cartilage at arrow (mesoderm).
(B, E) A rivulet of muscle at arrow (mesoderm). (C, F) Neural-like tissues at arrow (ectoderm). All images were
obtained from formalin-fixed and paraffin-embedded teratoma sections stained with hematoxylin and eosin.

they were not pulsed with the peptide. They were readily
killed in an effector dose-dependent manner when the
SIINFEKL peptide was present. In addition, the killing
could be blocked by EGTA indicating that the lysis of the
targets was mediated by the calcium-dependent granule
exocytosis pathway. A summary of the results of 3 experiments with maGSC Stra8 SSC5 target cells cultured under
the four conditions and differentiated cells, respectively, is
shown in Figure 4E. The lysis of the maGSC Stra8 SSC5
targets did not directly correlate with the H2Kb expression
levels since the maGSC Stra8 SSC5 cells cultured under
condition III showed the highest expression levels of
H2Kb but the lowest killing in comparison to the other
three culture conditions. Interestingly, the in vitro differ-

Further pluripotent cell lines are killed by CTLs despite
being negative for MHC class I expression in flow
cytometry
We then analyzed the killing of two further maGSC lines
of the haplotype H2b (maGSC 129/Sv and maGSC
C57BL) by CTL and compared them to ESC targets (ESC
129/Sv R1, and ESC C57BL). These cells were cultured in
the presence of LIF on feeder cells (condition IV) and
transferred two days before the experiment to gelatinecoated dishes (condition II). A summary of the results of
3 experiments is shown in Figure 5A. Similar to the
maGSC Stra8 SSC5 line, both maGSC lines (maGSC 129/
Sv and maGSC C57BL) were highly susceptible to CTL
killing. The relative lysis was between 60 and 80% compared to RMA cells. The lysis of the ESC line 129/Sv R1
was in a similar range. The ESC line C57BL was killed but
less efficiently. The relative lysis was between 40 and 50%
compared to RMA cells. The killing of all target cells was
completely peptide-dependent (data not shown). Next we
used iPSCs and F9 teratocarcinoma cells as CTL targets. A
summary of the results of 3 experiments is shown in Figure 5B. Both cell types were killed in a peptide-dependent
manner although less efficiently than RMA cells. The relative lysis was between 20 and 40% compared to RMA
cells. YAC-1 cells (H2a) which are a target for natural killer
(NK) cells were not killed, further confirming the specificity of the CTL-mediated killing and the absence of NK
cell-mediated killing.

In parallel to the killing assays, the MHC class I expression
on the target cells was determined by flow cytometry.
Interestingly, the maGSC lines maGSC 129/Sv and
maGSC C57BL, in contrast to the maGSC Stra8 SSC5 cell
line, did not express MHC class I molecules at a level
detectable by flow cytometry (Figure 5C, D). They were
very similar to the MHC class I negative ESC lines 129/Sv
R1 and C57BL (Figure 5C, D). In addition, also the iPSCs
and F9 teratocarcinoma cells were negative for MHC class
I molecules in flow cytometry (Figure 5C, D). The external
peptide pulsing of targets was apparently so efficient that
even maGSCs, ESCs, iPSC and F9 cells, on which no H2Kb
molecules could be detected by flow cytometry, were readily killed in a peptide-specific manner.
The expression of MHC class I peptides on maGSCs and
ESCs cannot be rescued by decreased temperature or
external peptides
The pluripotent stem cells could either completely fail to
express MHC class I molecules or have very few MHC
molecules at the cell surface due to a deficiency in peptide
loading, as e.g. neuronal cells that express low levels of
TAP molecules [40] or RMA-S cells that have a mutation

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A

B

MHC class I+ [%]

100

*

80

H2Kb

*

57%

1%

H2Db
I

60
40
20

1%

0

RMA

I

II

III

IV

22%

diff.

maGSC Stra8 SSC5

II

MHC class I [MFI]

500
400

isotype control

300

*

100

*

55%

4%

H2Kb

200

III

0

RMA

I

II

III

IV

diff.

C
MHC class I+ [%]

100

*

IV

H2Kb

EGFP+ cells

80

23%

2%

maGSC Stra8 SSC5

*

H2Db

60

21%

16%

40

diff.

20
0

I

II
III
maGSC Stra8 SSC5

IV
EGFP

RMA + P
maGSC Stra8 SSC5 II + P
maGSC Stra8 SSC5 III + P
RMA
maGSC Stra8 SSC5 II
maGSC Stra8 SSC5 III
RMA + P + EGTA
maGSC Stra8 SSC5 II + P + EGTA
maGSC Stra8 SSC5 III + P + EGTA

specific lysis [%]

70
60
50
40
30
20
10
0

RMA
maGSC Stra8 SSC5 I
maGSC Stra8 SSC5 II
maGSC Stra8 SSC5 III
maGSC Stra8 SSC5 IV
differentiated

120
100

relative lysis [%]

D

EGFP

E

80
60
40
20
0

10:1

5:1

2.5:1

1.25:1

0.63:1

effector : target ratio

0.31:1

0.16:1

10:1

5:1

2.5:1

1.25:1

0.63:1

0.31:1

0.16:1

effector : target ratio

Figure 4Stra8 SSC5 cells can be killed by CTLs despite low expression of MHC class I molecules
MaGSC
MaGSC Stra8 SSC5 cells can be killed by CTLs despite low expression of MHC class I molecules. (A) MaGSC
Stra8 SSC5 cells cultured under conditions I, II, III, and IV, in vitro differentiated maGSC Stra8 SSC5 cells (diff.), and RMA cells
were analyzed for MHC class I expression (H2Kb and H2Db) by flow cytometry. The mean percentages of positive cells + SD
(staining with MHC class I mAbs minus isotype control) and the mean fluorescence intensities (MFI) + SD are given as determined in 5 experiments. Significant differences between culture conditions that differed in the presence of LIF are indicated (*
p < 0.05, U test). (B) Individual dot plots from these experiments are shown. The left panel shows staining with an isotype control and the right panel with an anti-H2Kb mAb. The percentages of EGFP and H2Kb (or isotype control) double positive cells
are indicated in the upper right quads. (C) The evaluation of the proportion of MHC class I-positive cells in the experiments
was restricted to the EGFP-positive maGSC Stra8 SSC5 cell population. Significant differences between culture conditions that
differed in the presence of LIF are indicated (* p = 0.05, U test). (D) A representative experiment is shown in which the susceptibility of maGSC Stra8 SSC5 cells cultured under conditions II and III to CTLs was determined. RMA cells served as highly
CTL susceptible controls. The target cells were pulsed with the SIINFEKL peptide P (0.5 μg/ml) and exposed to CTLs derived
from TCR-transgenic OT-I mice. The mean of specific lysis plus SD at different effector:target ratios (10:1 to 0.16:1) in the
presence or absence of the SIINFEKL peptide measured in a 51Cr release assay is shown. To confirm granule exocytosis
dependency of killing EGTA was added to the test. (E) The means of relative lysis ± SD of the various target cell lines by OT-I
CTLs are shown as determined in three independent experiments. The mean percentage of specific lysis of RMA cells (serving
as positive control) at the highest effector to target ratio (10:1) was adjusted to 100% in each test and the relative lysis of the
various target cells at different effector to target ratios was calculated.
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in the Tap2 gene [41,42]. We analyzed the expression of
H2K, H2D, Tap1, Tap2, and β2-microglobulin (B2m) at
the mRNA level by qRT-PCR (Figure 6). The pluripotent
stem cell lines expressed H2K and B2m mRNA although at
lower levels than RMA and RMA-S cells. The H2D mRNA
was less expressed in the stem cell lines. The expression of
Tap1 and Tap2 was indeed low in most stem cell lines.
Only maGSC Stra8 SSC cells that expressed H2Kb molecules at the cell surface (Figure 4A, B) had similar Tap1
mRNA levels as RMA cells. F9 teratocarcinoma cells were
the only pluripotent cell type that expressed all these
mRNAs at rather high levels. Interestingly, iPSCs
expressed much less of these mRNAs than MEFs that were
used for their generation.
The mRNA expression data are in accordance with the
hypothesis that the peptide loading might be impaired in
the pluripotent stem cell lines. Thus, we tested the expression of MHC class I molecules at decreased temperature
and in the presence of external peptides. Incubation at
28°C for 24 h stabilized unloaded MHC class I molecules
on RMA-S cells (Figures 7A, B), whereas a subsequent culture at normal temperature (2 h 37°C) decreased the cell
surface expression again. A culture for 24 h at 37°C in the
presence of the SIINFEKL peptide (0.5 μg/ml) did not
increase the H2Kb expression as compared to untreated
cells or cells that were incubated with a peptide that cannot bind to H2Kb (Figures 7A, B). The combination of culture at 28°C and presence of the SIINFEKL peptide further
augmented the H2Kb but not H2Db expression and the
peptide-loaded H2Kb molecules were found to be more
stable when the cells were transferred afterwards for 2 h to
37°C (Figures 7A, B). However, on the stem cell lines
maGSC 129Sv and ESC 129Sv/MPI-II that were tested in
parallel, MHC class I molecules were not similarly stabilized by these treatments. Thus, a low expression of TAP
molecules is unlikely to be the main reason for the low
MHC class I cell surface expression on these cell lines.
MaGSCs as well as in vitro differentiated cells do not
express the serine protease inhibitor 6 that can protect
target cells against CTLs
It has been reported that mouse ESCs express SPI-6 that
confers protection against cellular cytotoxicity mediated
by the granule exocytosis pathway [27,43]. We analyzed
the expression of SPI-6 in maGSC Stra8 SSC5 cells cultured under the four conditions (I to IV) described above.
The protein was expressed in activated CTLs from OT-I
mice in which it contributes to the self protection against
granzymes [44]. However, we did not detect the SPI-6 protein in the maGSCs (Figure 8A). In contrast to undifferentiated maGSCs, cells differentiated in vitro from maGSCs
were rather resistant to lysis by CTLs (see Figure 4E).
Therefore, we analyzed the expression of SPI-6 in cells differentiated from maGSC Stra8 SSC5 cells and included

http://www.biology-direct.com/content/4/1/31

two ESC lines for comparison. The protein was neither
detected in the in vitro differentiated cells nor in the ESC
lines (Figure 8B). Serpinb9 that encodes SPI-6 was also
hardly expressed in these cell lines at the mRNA level (Figure 8C). Similarly, we did not detect SPI-6 expression in
any of the other pluripotent cell lines analyzed in this
study (data not shown). Thus, expression of SPI-6 is
apparently not responsible for the relative resistance of
the in vitro differentiated maGSCs cells to CTLs that we
observed.

Discussion
One advantage of maGSCs over ESCs is that they could
theoretically be a source of autologous pluripotent stem
cells for individualized medicine. One criterion for assessing the pluripotency of stem cells is the capacity to give
rise to teratomas in immunodeficient mice [45]. Therefore, the potential of maGSCs and their differentiation
products to form teratomas has to be analyzed very carefully before any therapeutic application can be envisaged.
In syngeneic or immunocompromised recipients, the risk
of teratoma growth is inherent to transplantation therapies that are based on pluripotent cells. In immunocompetent allogeneic mice, transplanted ESCs do not usually
form teratomas [26], unless very high numbers of pluripotent cells are injected [46]. We have compared the capacity
of the maGSC line Stra8 SSC5 to give rise to teratomas
depending on the culture conditions used to propagate
them, and after differentiation. Cells cultured under four
different conditions that varied in the presence of LIF and
MEFs as feeder cells did not differ with respect to teratoma
growth. This result was consistent with the presence of a
similar proportion of undifferentiated cells under the four
culture conditions as demonstrated by expression of
pluripotency markers SSEA-1 and Oct-3/4, as well as EGFP
expression driven by the Stra8 promoter in undifferentiated cells. Thus, the pluripotency of maGSCs was preserved under a variety of culture conditions for over 16
passages including those conditions that do not require
feeder cells and might, therefore, have advantages in view
of future therapeutic applications. Furthermore, cells differentiated in vitro (5 + 10 days of differentiation culture)
without lineage selection still contained about 20% undifferentiated cells and gave rise to teratomas at high frequency in immunodeficient recipients, suggesting that
lineage selection is necessary before the differentiated
cells can be used for therapeutic application [47,48].
The risk of teratoma growth after transplantation of
pluripotent cells does not only depend on the pluripotency of the injected cells but also on their immunogenicity and their susceptibility to cytotoxic effector
mechanisms of the immune system because maGSC Stra8
SSC5 cells did not form teratomas in allogeneic immunocompetent mice. Therefore, we have started to character-

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A
120

RMA
iPSC
F9
YAC-1

120

100

relative lysis [%]

relative lysis [%]

B

RMA
maGSC 129/Sv
maGSC C57BL
ESC 129/Sv R1
ESC C57BL

80
60
40
20

100
80
60
40
20

0

0
10:1

5:1

2.5:1

1.25:1

0.63:1

0.31:1

0.16:1

20:1

10:1

effector : target ratio

5:1

2.5:1

1.25:1

0.63:1

0.31:1

effector : target ratio

C
iPSC

ESC 129/Sv R1

maGSC 129/Sv

isotype control
H2Kb

counts

H2Db

ESC C57BL

maGSC C57BL

RMA

F9

MHC class I

300

20

150
100
50

maGSC 129/Sv

F9

iPSC

ESC C57BL

ESC 129/Sv R1

maGSC C57BL

maGSC 129/Sv

0

RMA

0

200

F9

40

H2Kb
H2Db

iPSC

60

250

ESC C57BL

H2Db

ESC 129/Sv R1

H2Kb

80

maGSC C57BL

MHC class I [MFI]

MHC class I+ [%]

100

RMA

D

Figure 5 cells can be killed by CTLs despite very low expression of MHC class I molecules
Pluripotent
Pluripotent cells can be killed by CTLs despite very low expression of MHC class I molecules. (A) The means of
relative lysis ± SD of maGSC and ESC lines by CTLs are shown as determined in three independent experiments. The target
cells were pulsed with the SIINFEKL peptide (0.5 μg/ml) and exposed to CTLs derived from TCR-transgenic OT-I mice. For
normalization between experiments, the mean percentage of specific lysis of RMA cells (serving as positive control) at the highest effector to target ratio (10:1) was adjusted to 100% in each test and the relative lysis of the various target cells at different
effector to target ratios was calculated. (B) The means of relative lysis ± SD of iPSCs and F9 teratocarcinoma cells by CTLs are
shown as determined in three independent experiments. The target cells were pulsed with the SIINFEKL peptide (0.5 μg/ml)
and exposed to CTLs derived from TCR-transgenic OT-I mice. The relative lysis was calculated as described above. YAC-1
cells (H2a) served as a negative control. (C) The target cells were tested in each experiment for MHC class I expression (H2Kb
and H2Db) by flow cytometry. Representative individual histograms are shown. (D) The mean percentages of positive cells +
SD (staining with MHC class I mAbs minus isotype control) and the MFI + SD are given for the three experiments.

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ize the immunological features of maGSCs and analyzed
their susceptibility to killing by CTLs. CTLs recognize peptides presented by MHC class I molecules, which are
highly polymorphic in the population. Alloreactive CTLs
recognize MHC class I molecules either by direct or indirect allorecognition [20,21]. The most important immunological barrier for transplantation of pluripotent stem
cell-derived allografts is presumably represented by MHC
class I molecules [20,21], but minor histocompatibility
antigens have also been shown to contribute to the rejection of ESC-derived grafts by CTL [49]. Interestingly, MHC
class I molecules are not detectable by flow cytometry on
mouse ESCs [26,27,50-54]. This is in contrast to human
ESCs [55-57]. However, the expression of MHC class I
molecules has been reported to increase during in vitro
[55] or in vivo differentiation of mouse ESCs [54,58].
MHC class I molecules are inducible by interferon-γ in
mouse ESCs [51,54] or in differentiated cells [27]. Thus,
ESCs and their differentiation products might become targets for CTLs after transplantation [49,53,54,59].
Whilst the mouse maGSC line Stra8 SSC5 was found to
express comparatively low levels of MHC class I molecules
by flow cytometry, the other maGSC lines (maGSC 129/
Sv and maGSC C57BL), ESCs, and iPSCs that were used
for comparison were negative for these molecules, resembling the phenotype known for mouse ESCs and also F9
teratocarcinoma cells [60]. At the mRNA level H2K and
B2m transcripts were detected in all pluripotent stem cell
lines, although less abundantly than in RMA cells or
MEFs. A low expression of Tap1 and Tap2 mRNA in most
pluripotent stem cell lines suggested that a failure of peptide loading could explain the lack of MHC class I molecules at the plasma membrane. However, a decrease of
temperature to 28°C or incubation with an H2Kbrestricted peptide did not stabilize the MHC class I molecules on maGSC 129Sv or ECS 129Sv MPI-II cells. A previous report showed a marked increase of expression of
H2Dd molecules on mouse embryonic cell lines (including one ESC line) that were transfected with an H2Ddexpression construct and incubated with H2Dd-binding
peptides [61]. Thus, the endogenous H2K and B2m transcription in the stem cell lines analyzed here might still be
insufficient to produce enough MHC class I molecules
that can be detected by flow cytometry after stabilization
by external peptides and decreased temperature.
However, all pluripotent cell lines analyzed here were
killed by CTLs derived from TCR-transgenic OT-I mice
that recognize the peptide SIINFEKL in an H2Kb-restricted
manner [33]. This finding is in accordance with studies
showing that extremely low numbers of ligands on target
cells are sufficient for CTL recognition and killing [62-64].
In addition, it has been reported previously that the MHC
class I expression on mouse ESCs is sufficient for recogni-

http://www.biology-direct.com/content/4/1/31

tion by the SIINFEKL-specific T cell hybridoma B3Z
[27,65]. We now show that the MHC class I expression
level on ESCs, maGSCs, and iPSCs is functionally relevant
since it is also sufficient for peptide-specific CTL killing,
although the presence of the peptide did not markedly
increase the H2Kb molecules at the cell surface of maGSCs
and ESCs. The iPSCs appeared to be less susceptible to
CTLs than the ESC and maGSC lines. Whether this is characteristic for iPSCs in general or only for the iPSC line analyzed here has to be determined. Interestingly, in vitro
differentiation of maGSC Stra8 SSC5 cells appeared to
reduce the sensitivity to CTLs because the in vitro differentiated target cells became much less sensitive to killing
compared to their undifferentiated counterparts. The
experiments using CTLs from TCR-transgenic mice demonstrated clearly that pluripotent stem cell lines can be
killed by this mechanism. However, it has to be further
clarified whether the antigen processing machinery of
pluripotent stem cells is capable of generating peptide
loaded MHC class I molecules for recognition by activated
CTLs. Furthermore, it remains to be determined whether
alloreactive CTLs that recognize their targets directly can
kill the pluripotent stem cells with similar efficacy.
The observation that killing of the maGSCs, ESCs, iPSCs,
and F9 cells could be inhibited by EGTA strongly suggests
that the granule exocytosis pathway which involves perforin and granzymes [66,67] was used for killing.
Recently, it has been reported that the mouse ESC line
CGR8 is resistant to antigen-specific CTLs due to the
expression of SPI-6 [27], which is an endogenous inhibitor of granzyme B [43]. We therefore looked for SPI-6 by
western blot in the maGSC and ESC lines but could not
detect expression of this protein in any of these cells, even
after in vitro differentiation of maGSC Stra8 SSC5 cells.
Their relative resistance to CTL cytotoxicity must therefore
be caused by another mechanism. Our results are in
accordance with recent reports by us [26] and others [65]
showing that several ESC lines can be readily killed by NK
cells. Although the mechanisms of target cell recognitions
are different, NK cells and CTLs share the granule exocytosis pathway of target cell killing [66,67]. Thus, individual
ESC lines might differ with respect to SPI-6 expression and
resistance to CTL and possibly NK cells.
Several studies have suggested that mouse or human ESCs
are immune-privileged. They may suppress immune
responses by contact dependent [51,57] and contact independent mechanisms [46] or induce apoptosis in T cells
[51]. It has also been reported that ESCs are resistant to
CTL and NK cell-mediated cytotoxicity [27,46,51]. Here
we have demonstrated that maGSC, ESC, and iPSC lines
can be killed by activated peptide-specific CTLs despite
levels of MHC class I expression that were below the
detection level of flow cytometry. Therefore, it has to be

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6

assumed that these pluripotent cells can become targets
for CTLs after transplantation.

H2K

' ct

4

Conclusion

2

MaGSCs represent a new pluripotent cell type derived
from adult mouse testis. They retain pluripotency under
several cell culture conditions and form teratomas in
immunodeficient mice. No expression of MHC class I
molecules could be detected by flow cytometry in most
maGSC lines and the iPSCs analyzed here, a phenotype
shared with ESC lines and F9 teratocarcinoma cells. However, in immunocompetent recipients the immune
response could suppress growth of the maGSCs lines as
teratomas, and all analyzed pluripotent cells including
maGSCs, ESCs, iPSCs, and F9 teratocarcinoma cells could
be killed efficiently by activated CTLs. Thus, maGSCs,
ESCs, and iPSCs can be recognized as targets for CTLs and
they might be rejected by this mechanism after transplantation in immunocompetent recipients. However, the
capacity of the immune system to kill pluripotent and teratoma forming stem cells could also be used to develop a
new strategy to increase the safety of transplantation of in
vitro differentiated cells. Indeed, it might be possible to
selectively induce an adaptive immune response in recipients against antigens expressed selectively in pluripotent
stem cells in order to kill any teratoma forming cells that
could not be depleted before grafting. Further studies are
required to validate or disprove this hypothesis.

0
-2

H2D

4

' ct

2
0
-2
-4
-6

Tap1
2

' ct

0
-2
-4
-6

Tap2

2

' ct

0
-2
-4

Abbreviations

-6
8

B2m

' ct

6
4
2
0
-2

RMA-S

RMA

iPSC

MEF

F9

ESC C57BL
ESC 129/Sv MPI-II

ESC 129/Sv R1

maGSC Stra8
SSC5

maGSC C57BL

II III IV

maGSC 129/Sv

I

and
Quantitative
Figure
B2m6expression
real timeinPCR
stemanalysis
cell lines
of H2K, H2D, Tap1, Tap2,
Quantitative real time PCR analysis of H2K, H2D,
Tap1, Tap2, and B2m expression in stem cell lines. The
transcripts of H2K, H2D, Tap1, Tap2, and B2m genes were
analyzed in triplicates by qRT-PCR in the indicated cell lines
in comparison to the housekeeping gene Hprt1. The maGSC
Stra8 SSC5 cells had been cultured under conditions I, II, III,
and IV before mRNA preparation. Means and SDs of the Δct
values (Hprt minus gene of interest) are shown.

Ab: antibody; B2m: β2-microglobulin; β-ME: β-mercaptoethanol; ct: cycle threshold; CTL; cytotoxic T lymphocyte; EB: embryoid bodies; EGFP: enhanced green
fluorescent protein; EGTA: ethyleneglycol-bis(b-aminoethyl ester)-N,N,N',N'-tetraacetic acid; ESC: embryonic
stem cell; DMEM: Dulbecco's modified Eagle's medium;
FCS: fetal calf serum; HE: hematoxylin and eosin; HEPES:
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid;
HPRT1: hypoxanthine guanine phosphoribosyl transferase 1; HSC70: heat shock cognate 70; iPSC: induced
pluripotent stem cell; LIF: leukemia inhibitory factor;
mAb: monoclonal antibody; maGSC: multipotent adult
germ-line stem cell; MEF: mouse embryonic fibroblast;
MFI: mean fluorescence intensity; MHC: major histocompatibility complex; NEAA: nonessential amino acids; NK:
natural killer; PBS: phosphate-buffered saline; qRT-PCR:
quantitative real time polymerase chain reaction; SDSPAGE: sodiumdodecyl sulphate-polyacrylamide gel electrophoresis; SPI-6: serine protease inhibitor 6; SSC: spermatogonial stem cell; SSEA-1: stage-specific embryonic
antigen 1; TAP: transporter associated with antigen
processing; TCR: T cell receptor

Competing interests
The authors declare that they have no competing interests.

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Biology Direct 2009, 4:31

http://www.biology-direct.com/content/4/1/31

A

RMA-S

24h 37°C
24h 28°C
100

101

102

103

104 100

101

102

103

104 100

101

102

103

104

24h 28°C + 2h 37°C
24h 37°C + SIINFEKL
24h 37°C + con. pep.
24h 28°C + SIINFEKL + 2h 37°C

maGSC
129/Sv

24h 28°C + con. pep. + 2h 37°C

100

101

102

103

104 100

101

102

H2K

104 100

RMA-S

101

102

103

104

RMA-S
30

80
70

MHC class I [MFI]

MHC class I+ [%]

B

103

b

60
50
40
30
20
10
0

25
20
15
10
5
0

H2Kb

H2Kb

H2Db

H2Db

24h 37°C
24h 28°C

10

80

maGSC 129/Sv

MHC class I [MFI]

MHC class I+ [%]

70
60
50
40
30
20
10
0

8

maGSC 129/Sv

24h 37°C + SIINFEKL
6

24h 37°C + con. pep.
4

24h 28°C + SIINFEKL
24h 28°C + con. pep.

2

24h 28°C + SIINFEKL + 2h 37°C

0

H2Kb

24h 28°C + 2h 37°C

H2Db

H2Kb

H2Db
24h 28°C + con. pep. + 2h 37°C

10

70

ESC 129/Sv MPI-II

ESC 129/Sv MPI-II

MHC class I [MFI]

MHC class I+ [%]

80

60
50
40
30
20
10
0

8
6
4
2
0

H2Kb

H2Db

H2Kb

H2Db

Figure
Analysis 7of MHC class I expression on stem cell lines at low temperature and in the presence of peptides
Analysis of MHC class I expression on stem cell lines at low temperature and in the presence of peptides.
MaGSC 129/Sv and ESC 129/Sv MPI-II cells and as positive control RMA-S cells were cultured for 24 h at 37°C or at 28°C, in
the presence of the H2Kb-binding peptide SIINFEKL (0.5 μg/ml) or a non-binding control peptide (con. pep.) NGLTLKNDFSRLEG. The culture at 28°C was either followed by a further incubation at 37°C for 2 h or not before flow cytometric analysis of
H2Kb and H2Db cell surface expression. (A) An individual experiment with RMA-S and maGSC 129/Sv cell is shown by histogram overlays. The different experimental conditions are indicated by the colors of the lines. The H2Kb staining is indicated by
full lines and staining with the isotype control by dotted lines. (B) The mean percentages of three independent experiments for
H2Kb and H2Db positive cells + SD (staining with MHC class I mAbs minus isotype control) and the MFI + SD are given.

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CTL (OT-I)

Biology Direct 2009, 4:31

A

http://www.biology-direct.com/content/4/1/31

Authors' contributions
maGSC Stra8 SSC5
I

II

III

RD, KG, and WE designed the experiments; RD, KG, JN,
SM, and LE performed experiments; KG, GH, KN, and WE
provided study material; RD wrote the manuscript, all
authors discussed the data and commented on the manuscript draft; all authors read and approved the final manuscript.

IV

SPI-6
HSC70

Reviewers' report 1
Dr. Bhagirath Singh, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada

ESC 129/Sv R1

ESC 129/Sv MPI-II

CTL (OT-I)

B

maGSC differentiated

Reviewers' comments

In most part this manuscript deals with the immunogenicity of multipotent adult germ-line stem cells (maGSCs) that are derived from spermatogonial stem cells
(SSCs) present in adult testis. The authors use these cells
as targets for a peptide specific cytotoxic T lymphocytes
(CTL) restricted to a major histocompatibility complex
(MHC) class I plus OVA (Ovalbumin 257-264) peptide,
SIINFEKL.

SPI-6

II

III

IV

CTL (OT-I)

I

ESC 129/Sv R1

maGSC Stra8 SSC5

ESC 129/Sv MPI-II

C

maGSC differentiated

HSC70

0
-1

' ct

-2
-3
-4
-5
-6

Figure 8of SPI-6 expression in stem cell lines
Analysis
Analysis of SPI-6 expression in stem cell lines. (A) Protein extracts of maGSC Stra8 SSC5 cells cultured in basic
medium (condition I), medium plus LIF (condition II), basic
medium on MEFs (condition III), or in medium plus LIF on
MEFs (condition IV) were separated by SDS-PAGE and the
blot was probed with an anti-SPI-6 Ab and with an antiHSC70 mAb as loading control. A protein extract of activated CTLs from OT-I mice served as positive control for
SPI-6 detection. (B) The expression of SPI-6 was similarly
probed in differentiated cells derived from maGSC Stra8
SSC5 cells and in two ESC lines for comparison. (C) Expression of Serpinb9 gene that encodes SPI-6, was analyzed in
triplicates by qRT-PCR in the same cell lines in comparison
to the housekeeping gene Hprt1. The maGSC Stra8 SSC5
cells had been cultured under conditions I, II, III, and IV
before mRNA preparation. Means and SDs of the Δct values
(Hprt minus gene of interest) are shown.

1) Based upon the data presented the authors conclude
that these cells can become targets for CTL even if the
expression level of MHC class I molecules is below the
detection limit by using flow cytometry. This is not surprising because little as 100 molecules of MHC-peptide
complex are sufficient for CTL killing of target cells. Therefore, pluripotent cells might be rejected after transplantation by this mechanism and provides the hope that
transplantations of in vitro differentiated cells may be
possible and the undifferentiated cells may be killed by
their immunogenicity. However, immunologically this
approach does not provide any particular benefit as both
differentiated cells and undifferentiated cells would be
killed in allogeneic conditions. In syngeneic situation it is
unclear if there is an advantage to the approach. Authors
have to demonstrate this in vivo.
Authors' response: We indeed agree that it is not surprising that
pluripotent stem cells can be killed by CTL. However, contrary
data have been reported in the literature [27]. Therefore, it was
important to demonstrate that pluripotent stem cells are in general not protected against the CTL-mediated cytotoxicity. It
remains to be analyzed whether alloreactive CTL that often
have lower affinity T cell receptors will similarly kill those targets. Nonetheless, antigens that are specifically expressed in
pluripotent teratoma forming stem cells might be relevant targets for CTL and this might be helpful to avoid teratomas after
transplantation. However, we completely agree that this is presently a pure speculation that has to be validated or disproved by
in vivo experiments in subsequent studies.

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Biology Direct 2009, 4:31

2) I agree with the conclusion that suggests that pluripotent cells are not protected against CTL, even if MHC class
I molecules are not detectable by flow cytometry. However, authors should explore this further by RT-PCR analysis for the results to be valid. Similarly the SPI-6 data (Fig.
6) should be confirmed by RT-PCR.
Authors' response: As suggested, we have analyzed the MHC
class I (see new Figure 6) and SPI-6 expression (see Figure 8)
by quantitative real time PCR.
3) Finally, it is very important that authors provide a wellbalanced analysis to the differences where their results differ from previously published information about the
immune-privileged nature of mouse or human ESC as
quote in the last paragraph of their Discussion section.
Authors' response: We have altered the discussion in order to
clarify these issues.
4) Overall the manuscript is well written and the question
posed, methods used are well defined although not new.
Additional data is needed to validate the study. The discussion and conclusions need to be refined as outlined
above. The title and abstract are well constructed but conclusions need to be modified to identify the shortcomings
of the approach and the usefulness of the findings.
Authors' response: We tried also to further clarify the advantages but also the limitations of our present approach in the discussion.
Comment to the revised manuscript
The authors have considerably revised and updated the
manuscript based on my comments. Additional experiments have been done and added as new figures. Authors
have discussed these results in the text and in my view, the
revised manuscript provides a more evidence for the
observations and conclusions.

Authors' response: We would like to thank Dr. Singh for his
thoughtful review of our manuscript and the helpful suggestions
for improvement.
Reviewers' report 2
Dr. Etienne Joly, Equipe de Neuro-Immuno-Génétique Moléculaire, IPBS, UMR CNRS 5089, Toulouse, France

1) Have you considered the possibility that all these
pluripotent cells express very low levels of TAP, and have
thus very few properly folded MHC molecules on their
surface, but probably quite a few empty, peptide-receptive
ones. On this subject, I refer you to Bikoff et al. [61] and
to my own paper on neuronal cells [40].

http://www.biology-direct.com/content/4/1/31

Authors' response: We have analyzed now the expression of
Tap1 and Tap2 at the mRNA level (see the new Figure 6) and
found rather low levels in most of the pluripotent stem cell lines.
H2K and B2m transcripts were more abundant suggesting that
indeed a failure of peptide loading could be responsible for the
low MHC class I cell surface expression levels. However, culture
at 28°C or in the presence of an H2Kb-binding peptide did not
markedly increase the H2Kb cell surface expression (see the new
Figure 7). Thus, it is unlikely that a low TAP expression alone
is the reason for the low MHC class I expression on maGSCs
and ESCs.
2) Considering this previous point, and the fact that the
effectors in graft rejection would be alloreactive CTL
rather than peptide specific ones, I think that you may
have found quite a different picture if you had used antiH2b alloreactive CTLs obtained either after an in vitro
mixed lymphocyte reaction, or after priming non-H2b
mice with H2b splenocytes. Indeed, whereas OT-I cells
have very high affinity TCRs that can kill upon recognition
of just a handful of peptide-MHC complexes, the TCRs of
alloreactive CTLs would be much more likely to be of low
affinity, and would thus not have been triggered by the
low levels found at the surface of the various stem cells
used in your study.
Authors' response: We agree that using alloreactive CTL might
give different results. Therefore, it will be of interest to determine whether the pluripotent cells used in this study can induce
an alloreactive immune response and whether they are good
targets for alloreactive CTL. However, we think that this question is beyond the scope of the present manuscript. In the
present study we wanted to clarify whether the pluripotent cells
can in principle be killed by CTL since it has been proposed previously that ESCs are resistant to cytotoxicity mediated by CTL
[27]. Therefore, we used a well-controlled and very potent system of peptide-specific CTL derived from T cell receptor-transgenic mice instead of less well-defined alloreactive CTL. Using
this system we could clearly demonstrate that the targets were
killed and that the killing was peptide-dependent. We clarified
the advantages but also the possible limitations of our present
experimental approach in the discussion.
3) The last issue is that of the presentation of the FACS
data. I find that histograms showing percentages of positive cells or even means of fluorescence are really much
less informative than the actual plots. If they all look similar, then you should at least provide one example for each
case. And if they are not so similar (for example different
shapes), then you should definitely show the plots, and
not the percentage of positive cells.
Authors' response: We agree and we have now included representative FACS plots for most of the data in addition to the his-

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Biology Direct 2009, 4:31

tograms that summarize several experiments and that are
therefore more representative than individual plots.
We would like to thank Dr. Joly for his important and very helpful scientific and editorial suggestions for our manuscript.
Reviewers' report 3
Dr. Lutz Walter, Department of Primate Genetics, German
Primate Center - Leibniz Institute for Primate Research, Göttingen, Germany

This manuscript reports on pluripotent stem cells and
their susceptibility to killing by CTL. Pluripotent stem
cells include ESCs, iPSCs, and maGSCs. The latter type
was examined in detail in this paper. MaGSCs are derived
from spermatogonial stem cells after reprogramming via
in vitro cultivation. These maGSCs harbour enormous
potential in regenerative medicine, as they are able to differentiate into a variety of cell types. Additionally, in an
autologous transplantation of differentiated maGSCs no
immunologic barrier is expected to occur. Yet, a known
problem is formation of teratoma after transplantation of
pluripotent cells.
The authors compared teratoma growth in immunodeficient SCID/beige mice after in vitro differentiation in various culture conditions. Differences in teratoma
formation were not observed. In contrast, immunocompetent mice were able to reject maGSC and did not
develop teratoma, suggesting cytotoxic immune cells
mediated killing. These cells usually recognise MHC class
I molecules. Therefore, the authors tested expression levels of MHC class I on the surface of different maGSC cell
lines, which turned out to be either very low or not detectable, similar to what is known for ESCs. Interestingly, all
maGSC lines could be killed by CTL, despite their low or
undetectable expression level of MHC class I molecules.
As the authors used well-defined CTL from TCR-transgenic OT-I mice, they could demonstrate the peptidedependency of CTL killing and could exclude NK cellmediated killing. Furthermore, it was found that maGSCs
do not express the serine-protease inhibitor 6, which is
known to confer protection against the granule exocytosis
effector mechanism of cytotoxicity, providing a potential
explanation for the observed CTL killing and the lack of
CTL inhibition, which can usually be observed for ESCs.

http://www.biology-direct.com/content/4/1/31

cells and RMA. It was concluded that the level for maGSC
was low. While I have no doubt that this is true, the question arises whether RMA cells are an adequate control. I
guess this cell line was used as a control for antibody binding. Would it be possible to compare the level with spermatogonia or spermatogonial cell lines? At the bottom of
page 15, last sentence, the authors should add "and
absence of NK-mediated killing."
Authors' response: We would like to thank Dr. Walter for his
encouraging review of our work. We have added the information on the MHC haplotype of the studied cell lines in the methods section. The RMA cells were indeed used as positive control
for MHC class I expression and antibody binding but also for
killing by the CTL. Recently, we have analyzed two newly generated cell lines that represent spermatogonial stem cells
(SSCs). Similarly to most maGSCs, they did not express H2K
or H2D molecules at the cell surface when analyzed by flow
cytometry. However, these cell lines are so far not fully characterized. Therefore, we decided not to present these data in this
manuscript. The suggested addition on page 15 has been done.

Acknowledgements
The authors would like to thank Dr. Rudolf Jaenisch, The Whitehead Institute for Biomedical Research, Cambridge, USA, for the iPSCs used in this
study. The technical assistance of A. Cierpka, B. Kaltwasser, J. Ulrich, and
L. Piontek, is gratefully acknowledged. This work was supported by grants
MRTN-CT-2004-512253 (TRANS-NET) from the European Union and
01GN0819 from the German Federal Ministry for Research and Technology (BMBF) to RD, EN 84/22-1 and SSP 1356 from the Deutsche Forschungsgemeinschaft (DFG) to WE, 01GN0601 from BMBF to GH,
01GN0822 from BMBF to KG and GH, and KFO 155 (Clinical Researcher
Group 155) from the DFG to KG, WE and GH.

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