Negligible immunogenicity of terminally
or embryonic stem cells
Ryoko Araki1,2, Masahiro Uda1, Yuko Hoki1, Misato Sunayama1, Miki Nakamura1, Shunsuke Ando1, Mayumi Sugiura1,
Hisashi Ideno1,3, Akemi Shimada3, Akira Nifuji1,3& Masumi Abe1
The advantages of using induced pluripotent stem cells (iPSCs)
instead of embryonic stem (ES) cells in regenerative medicine cen-
tre around circumventing concerns about the ethics of using ES
cells and the likelihood of immune rejection of ES-cell-derived
tissues1,2. However, partial reprogramming and genetic instabi-
lities in iPSCs3–6could elicit immune responses in transplant reci-
iPSCs are first differentiated into specific types of cells in vitro
for subsequent transplantation. Although model transplantation
experiments have been conducted using various iPSC-derived dif-
ferentiated tissues7–10and immune rejections have not been
observed, careful investigation of the immunogenicity of iPSC-
derived tissue is becoming increasingly critical, especially as this
has not been the focus of most studies done so far. A recent study
reported immunogenicity of iPSC- but not ES-cell-derived terato-
mas11and implicated several causative genes. Nevertheless, some
and ES cells, we established ten integration-free iPSC and seven
ES-cell lines using an inbred mouse strain, C57BL/6. We observed
no differences in the rate of success of transplantation when skin
cell-derived tissues. Moreover, we observed limited or no immune
responses, including T-cell infiltration, for tissues derived from
either iPSCs or ES cells, and no increase in the expression of the
immunogenicity-causing Zg16 and Hormad1 genes in regressing
skin and teratoma tissues. Our findings suggest limited immuno-
There are three major concerns about the adequacy of the design
and conclusions of a previous study that reported immunogenicity of
genicity of iPSCs was assessed by teratoma formation. Consistent with
the focus on tumour immunity rather than transplant immunity, the
causative gene identified in the regressing teratomas was tumour
related13. Second, only a single ES-cell clone was included in the com-
opmental ability of the iPSC and ES-cell lines used may have been
compromised. Given that partial genome reprogramming can elicit
immune responses and cause considerable variation among iPSC
clones, the developmental abilities of iPSC and ES-cell clones should
always be tested before such analyses. To evaluate better the immuno-
terminally differentiated cells derived from iPSCs. Our experiments
involved multiple ES-cell and iPSC lines (Supplementary Fig. 1a) that
were established from an inbred mouse strain, C57BL/6, and were
confirmed to have strong developmental capacities. We generated
iPSCs by expressing plasmid-borne Oct4 (also known as Pou5f1),
Sox2, Klf4 and Myc genes14, and then selected clones that were free
from genome-integration events (Supplementary Fig. 1b)15, as iPSCs
with integrated transgenes elicit marked immune responses11. Similar
doubling times were measured for all the cell lines (Supplementary
Fig. 1c). Germline transmission capability was confirmed for eight of
ten iPSC clones and for six of seven ES-cell clones (Supplementary
We used seven iPSC and five ES-cell lines in teratoma formation
tests to obtain more conclusive results (Supplementary Fig. 2a). The
each of the twelve clones. We observed teratomas containing three
germ layers15at a high incidence rate for both types of pluripotent
stem cells, with no statistical difference between iPSCs and ES cells
(Fig. 1a). Next, we used immunohistochemical staining to investigate
did not observe any T cells in most sections, although fewer than two
observed among the cell lines, the profiles between iPSCs and ES cells
were quite similar overall (Fig. 1b), and no statistically significant
difference between iPSC- and ES-cell-derived teratomas was detect-
able (Fig. 1c).
Pharmacology, School of Dental Medicine, Tsurumi University, Yokohama 230-8501, Japan.
2A-4F-1 2A-4F-33 2A-4F-59 2A-4F-85 2A-4F-118
Teratoma formation (%)
B6ES2-2 B6ES2-9 B6ES5-12 B6ES7-13 B6ES7-17
Figure 1 | Teratoma formation by iPSCs and ES cells. a, Time course of the
appearance of teratomas after injection. Error bars show standard error of the
mean (s.e.m.). iPSCs, n57; ES cells, n55. b, Frequency of T cells in each
each of five clones of both cell types, were stained with anti-CD3 antibody.
More than 50 frames (each 0.382mm30.507mm) were randomly chosen for
each teratoma. The numbers of T cells detected were categorized into three
groups: 0–2, 3–9 or $10 T cells per frame. c, Summary of the data shown in
b andthetotalnumberofframes investigated. Errorbars show s.e.m.(n510).
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Focusing on teratoma formation is not suitable for assessing the
immunogenic responses of relevance to regenerative medicine. Given
that teratomas are a type of tumour, it is not surprising that they elicit
immunological reactions in transplant recipients. Indeed, in a further
teratoma formation test using severe combined immunodeficiency
(SCID) mice16although, again, no difference was observed between
iPSCs and ES cells, a similar frequency but a slight increase in size
compared with wild-type mice was shown, indicating that certain
immune systems respond to the teratomas derived from these two
we did not detect any significant T-cell infiltrations in various tera-
out the possibility that substantial immune responses can be elicited
under certain situations during teratoma formation in both types of
pluripotent stem cells11,17. On the other hand, considering that iPSCs
be converted into specific types of differentiated cells before trans-
plantation, the immunogenicity of terminally differentiated cells
derived from iPSCs is critically relevant to the long-term prospects
of their use in regenerative medicine. We therefore prepared dermal
and bone marrow tissues from the highly chimaeric mice developed
from iPSCs or ES cells for subsequent transplantation experiments.
These tissues were selected on the basis of the central roles they have
in the immune system.
For each type of terminally differentiated cell, we initially set up
our assay system by using a green fluorescent protein (GFP)-labelled
iPSC clone, because it allowed us to monitor the donor cells after
transplantation, even in a single-cell manner. We used a genome-
integration-free iPSC clone, 2A-3F-EGFPtg-4, which was established
from a C57BL/6-Tg(CAG-EGFP) mouse18with Oct4, Sox2 and Klf4
genes; using Trichostatin A, we demonstrated that this iPSC line
could achieve pluripotency19. On the basis of the investigation using
the 2A-3F-EGFPtg-4 clone, we examined various non-labelled lines
of iPSCs and ES cells.
We first attempted to establish a transplantation assay system
for epidermal tissue based on a previously reported transplantation
with GFP-negative embryos to develop GFP-positive highly chimaeric
and then transplanted onto the backs of syngeneic GFP-negative
C57BL/6 mice and allogeneic Balb/c mice (Fig. 2a). All of the six
GFP-positive transplanted grafts generated could be sustained over a
10-month period. In contrast, in the control experiment, the trans-
planted grafts on the Balb/c mice remained physically attached to the
backsofrecipientmice forovera week butnotaslong as2 weeksafter
efficient way of assaying the immunogenicity of epithelial tissues.
tionaliPSC clones(2A-4F-1,-33, -60 and-100)and five ES-cellclones
(B6ES2-2, B6ES2-9, B6ES5-1, B6ES7-13 and B6ES7-15). Three mouse
strains—C57BL/6, C57BL/6-Tg(CAG-EGFP) and Balb/c—were used
asrecipients(SupplementaryFig. 4a). Becauseallofthesecelllinesare
GFP negative, each clone was aggregated with GFP-positive embryos
to ensure that the skin tissues of the chimaeric mice used for sub-
sequent transplantation tests did not contain cells other than those
forusing GFP-positive embryos forthe aggregation step was to confer
tolerance to the GFP molecules in the recipient mice used to analyse
GFP molecules can elicit immune responses21. Between 4 and 12 rep-
licate transplantations were conducted for each clone, and graft sur-
were 98.361.7% and 92.565.0% for iPSCs and ES cells, respectively
(Fig. 2c and Supplementary Fig. 4). In contrast, no long-term survival
wasnoted forgrafts derivedfromBalb/cmice. Thesedatasuggest that
iPSCs are not demonstrably more immunogenic than ES cells.
To analyse immunogenicity further, we used confocal microscopy
to investigate the invasion of the grafts by GFP-positive cells pro-
duced by the recipient C57BL/6-Tg(CAG-EGFP) mice. In the control
experiment, which involved transplantation of Balb/c tail skin, a large
number of GFP-positive cells were observed within the grafts (Sup-
plementary Fig.5a).Moreover, as expected, subsequenthistochemical
staining with anti-CD3 antibody revealed the presence of CD3-
positive cells within the GFP-positive population (Supplementary
Fig. 6a). As a result, very few GFP-positive cells were detected around
6b). However, we note the possibility that our assay system may have
missed some lymphocytes with low GFP expression levels. Never-
theless, if such T cells existed, the number that invaded the graft must
have been quite small, because flow cytometry analysis did not detect
GFP-negative T cells in the whole blood of C57BL/6-Tg(CAG-EGFP)
recipients (Supplementary Fig. 7c).
The first step in the assessment of the immunogenicity of bone
marrow tissues involved mimicking the bone marrow transplantation
treatment. Wetransplantedbonemarrowfrommicederived fromthe
GFP-positive iPSC clone 2A-3F-EGFPtg-4 into C57BL/6 wild-type
mice that had been irradiated with a lethal (9.5 gray (Gy)) dose of
X-rays (Fig. 3a). Flow cytometry analysis demonstrated the presence
of GFP-positive cells 5 months later (Fig. 3a, left, and Supplementary
Fig. 7a). We also detected B lymphocytes, T lymphocytes and granu-
tion (Fig. 3d, top) in each of the four mice that received transplants.
This indicates that both short- and long-term haematopoietic stem
cells were fixed and maintained. No major phenotypical or beha-
vioural abnormalities were observed in any of these mice more than
5 months after transplantation, although some skin ulcers were
observed in one recipient, IR-1 (Fig. 3a, left). As anticipated, bone
marrow derived from the ES-cell line B6ES5-1 also reconstituted the
bonemarrow of eachof four recipientmice afterthey received a lethal
dose of irradiation (Fig. 3b, c).
Itis noteworthythat althoughalmost all B lymphocytes,T lympho-
cytes and granulocytes were replaced with iPSC-derived cells (Fig. 3d,
top), recipient-derived cells still remained in all transplanted mice
(5 mm × 5 mm)
C57BL/6 (n = 6)
Balb/c (n = 6)
ES-cell or iPSC clone
Graft survival rate
ES cells (Balb/c)
ES cells (C57BL/6)
05 10 15 20253035 404550300
048 12 165620 24 28 32 36 40 44 48 52
(98.3 ± 1.7%)
(92.5 ± 5.0%)
Figure 2 | Skintransplantation. a, Schematic diagramof themethodforskin
transplantation using a GFP-positive iPSC clone and time course of graft
transplantation. b, Schema of the skin transplantation assay for GFP-negative
iPSCs and ES cells. c, Summary of the time course for graft survival.
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(Supplementary Fig. 7b). Importantly, the coexistence of donor- and
recipient-derivedbonemarrowcellsinthe recipientmice morethan5
months from the time of transplantation is strongly suggestive of a
limited immunogenicity of iPSC-derived bone marrow cells. This is
because substantial immune responses must occur during the fixation
and maintenance of iPSC-derived cells if the bone marrow cells
derived from iPSCs are to show even a low level of immunogenicity.
with X-ray irradiation are consistent with those of a previous study
that showed similar repopulation by iPSC- and ES-cell-derived hae-
To assess whether low levels of immunogenicity had been over-
looked, we next conducted transplantations under more severe con-
ditions. This involved transplanting iPSC-derived bone marrow cells
into wild-type C57BL/6 mice without any prior irradiation. Because
the immune activity of the bone marrow in wild-type recipient mice
must be normal, this enables better assessment of transplantation
immunity. We injected 13107bone marrow cells from chimaeric
mice developed using the iPSC line 2A-3F-EGFPtg-4 into the tail
veins of recipient wild-type mice, and analysed peripheral blood sam-
ples at 1-month intervals for 5 months. Flow cytometry and semi-
quantitative polymerase chain reaction (PCR) analysis indicated that
donor cells derived from iPSCs were sustained over 5 months even
after transplantation into non-irradiated mice (Fig. 3a, right, and
Supplementary Fig. 7a). This indicates successful engraftment of both
short-and long-termhaematopoietic stem cells. Ina comparison with
thecontrol, non-transplanted C57BL/6bonemarrowusing flow cyto-
metry, B lymphocytes, T lymphocytes and granulocytes derived from
the donor cells were detected, although the number of GFP-positive
cells was small (Fig. 3d). We also examined bone marrow cells pre-
pared 6 months after transplantation to confirm the presence of GFP-
positive lymphocytes and granulocytes (Supplementary Fig. 7d).
For the various non-labelled iPSCs and ES cells, similar bone mar-
row transplantations using non-irradiated recipient mice were per-
formed (Fig. 3b, c and Supplementary Fig. 9a). Four iPSC and three
ES-cell clones were also examined, and an assessment was done at 4
months after transplantation using genomic PCR, which enables dis-
(Supplementary Fig. 8). In addition, we prepared bone marrow from
mice of various ages (5–54 weeks). The results showed that 20 of 21
challenges involving bone marrow derived from any one of the five
iPSC lines, and 11 of 12 challenges involving bone marrow derived
from any one of the three ES-cell lines, were successful. Notably, the
of the two iPSC lines, 2A-4F-118 and 2A-4F-136, or from an ES-cell
line, B6ES7-13, were successfully engrafted (Supplementary Fig. 9a).
Moreover, semi-quantitative PCR showed donor-origin cells in B-
lymphocyte, T-lymphocyte and granulocyte fractions prepared from
mice developed using the non-labelled pluripotent stem cell lines 2A-
4F-60 and B6ES5-1 (Supplementary Fig. 9b). These data indicate that
iPSC-derived haematopoietic stem cells can be engrafted and sus-
tained in recipients even under conditions of normal immunocompe-
tence. Although seven-to-twelve months have now passed since the
The Zg16 and Hormad1 genes have been causally linked to the
immunogenicity of iPSC-derived teratomas, and both genes are
regression in both transplanted skin grafts and teratomas in these
experiments, although no statistically significant difference was appa-
rent between iPSC- and ES-cell-derived tissues (Supplementary Figs
10 and 11). Investigation of the expression of Zg16 and Hormad1
indicated no expression of Zg16 in non-shrinking or shrinking grafts
in dermal tissues, and a low level of expression of these genes in
teratomas. A low level (less than 100-fold lower expression than
that seen in the testis) of Hormad1 expression was also observed in
both transplanted grafts and teratomas. However, neither gene was
expressed at an elevated level in the shrinking tissues. The expression
profiles were quite similar between iPSC and ES-cell derivatives
(Fig. 4a, b). Thus, the expression profiles of both genes were similar
(1 × 107 cells)
ES-cell or iPSC clone Bone marrow
(1 × 107 cells)
2 3 1 2 1 2 1 2 3 4
2-2 5-1 5-1*
33 60 118 136
1 2 1 2 3 4
2-2 5-1 7-13
Donor: 7–8 weeks
0 1 2 3 4 5
0 1 2 3 4 5
GFP-positive cells (%)
Recipient Graft survival rate
iPSCs (1 line) with IR 4/4 (100%)
ES cells (1 line) with IR 4/4 (100%)
iPSCs (5 lines) w/o IR 20/21 (95.2%)
ES cells (3 lines) w/o IR 11/12 (91.7%)
r-1 + Mac-1
B lymphocytes T lymphocytes Myeloid cells
Gr-1 + Mac-1
Gr-1 + Mac-1
GFP-positive cells (%)
Donor: >16 weeks
1 2 3 4 1 2 1 2 3 4
16.0 16.016.0 3.913.913.91
Figure 3 | Bone marrow transplantation. a, Schematic diagram of the bone
marrow transplantation experiments using 2A-3F-EGFPtg-4 iPSCs and the
survival of donor bone marrow cells. Time-course analysis of the survival of
by flow cytometry over 5 months (Supplementary Fig. 7a). IR-1, -2, and so on,
non-labelled ES cells, and of PCR assessment of the engraftment of donor-
derived bone marrow cells. The existence of donor-derived bone marrow cells
over 4 months was investigated by PCR using genomic DNA prepared from
peripheral blood cells harvested at 4 months after transplantation. Asterisk
indicates the use of irradiated recipients. c, Summary of the incidence of graft
survival. d, Long-term reconstitution of bone marrow by 2A-3F-EGFPtg-4
iPSC-derived bone marrow cells. Peripheral blood cells at 4 months after
transplantation were analysed using flow cytometry. The results of irradiation
recipient IR-4 (top) and recipient without (w/o) irradiation IR-1 (middle),
which are described in a, are shown as a representative case. The C57BL/6
mouse is shown as a control (bottom).
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in both non-regressing and regressing teratomas, and we could not
teratomas derived from the iPSC clone 2A-4F-33.
Finally, we examined whether Zg16 and Hormad1 are expressed
at higher levels in the various differentiated tissues derived from
iPSCs relative to differentiated cells derived from ES cells. Ten tissue
types were prepared from the adult mice generated by tetraploid
complementation22–24with the iPSC line 2A-4F-33 and the ES-cell
lines B6ES2-9 and B6ES5-1 for semi-quantitative expression analyses
of the Zg16 and Hormad1 genes. The expression profiles of Zg16 and
Hormad1 were indistinguishable between the three tissue panels
derived from 2A-4F-33, B6ES2-9 and B6ES5-1 (Fig. 4c).
An important finding of this study is that the immunogenicity of
iPSC-derived tissues is indistinguishable from that of ES-cell-derived
tissues. Although all of the assays we performed demonstrated that
the terminally differentiated cells developed from iPSCs elicit limited
immunogenicity, given the limitations of the experimental approaches
at our disposal. Whether the immunogenicity of iPSCs is higher than
a previous study11. We have successfully established seven ES-cell lines
derived from C57BL/6 mice and confirmed germline transmission for
six of these (Supplementary Table 1). Our results using these ES cells
ES-cell-derived tissues were indistinguishable when scored using all
three immunogenicity assays tested—teratoma formation, skin trans-
plantation and bone marrow transplantation. None of our data show
meaningful differences between iPSCs and ES cells.
Our data may indicate that the differences between specific clones
of iPSCs and ES cells are more crucial than the difference between
iPSCs and ES cells per se. Indeed, we observed a variation among
iPSC clones in another experiment involving three iPSC clones from
which chimaeric mice exhibited low chimaerism and no germline
transmission (Supplementary Fig. 12). We performed teratoma for-
mation tests in each case followed by T-cell infiltration analysis.
Intriguingly, several lines exhibited significant T-cell infiltration.
Furthermore, considerable variations in T-cell infiltration were
observed among teratomas generated even from identical cell lines,
although the frequency was similar not only to that among the three
iPSC clones examined, but also to that of the lines of which fully
developmental abilitywasconfirmed. Our datamay thus alsoindicate
that the teratomas generatedfrom the iPSCs for which developmental
ability is not complete are prone to eliciting immune responses, and
that this issue is crucial for the medical use of iPSCs. Because testing
such as chimaera formation is not possible for human iPSCs, these
findings underscore the need for careful discussion and longitu-
dinal investigations of interclonal differences in iPSC and ES-cell
lines25,26, and the need to develop culture conditions that will achieve
a pluripotent state27–29. This is in addition to broader generalizations
regarding the immunogenicities of the two cell types.
In this study, we used terminally differentiated cells of adult mice,
which had beengeneratedfrom either iPSCs or ES cells as donor cells,
as our aim was to determine whether iPSC-derived differentiated cells
whether iPSC-derived differentiatedcells canbe used as donor cells in
the same way as ES cells when the complete in vitro differentiation of
these stem cells became possible. In this regard it must be noted that
ourstudy does notdirectly contribute to resolving the possibleimmu-
nogenicity of in vitro differentiated cells. Further investigations are
needed to assess this issue and hence the actual clinical use of these
tration when we used in vitro derived cells, cardiomyocytes, as donors
(Supplementary Fig. 13).
We have demonstrated that the limited immunogenicity of iPSC-
derived differentiated cells is indistinguishable from that of com-
parable ES-cell-derived cells. However, the possibility remains that
even terminally differentiated cells derived from iPSCs might elicit
some immune responses. For example, the genetic aberrations that
have been demonstrated recently in iPSCs could account for their
immunogenicity5,6. However, considering the advantages of iPSCs
compared with ES cells, such as the ease of direct establishment from
patients without the same complex ethical considerations, the discus-
sion surrounding the relative merits of using autologous iPSCs and
resolved by a direct comparison between autologous iPSCs and allolo-
C57BL/6J Jms Slc and C57BL/6-Tg(CAG-EGFP)18mice were used for iPSC and
ES-cell generation. iPSCs were generated with pCX-OKS-2A and pCX-c-Myc
Transplanted dermal cellsTransplanted dermal cells
2A-4F-33 B6ES2-9 B6ES5-12A-4F-33 B6ES2-9 B6ES5-1
Shrinking Shrinking Shrinking Shrinking
≈ ≈ ≈
iPSCs ES cells
iPSCs ES cells
Relative expression level
Figure 4 | Expression of Zg16 and Hormad1 genes in various tissues
generated from iPSCs and ES cells. a, Expression of the Zg16 and Hormad1
genes in grafted skin 2 months after transplantation. b, Expression of the Zg16
and Hormad1 genes in the teratomas at 4 or 5 weeks after injection
(Supplementary Fig. 11b). c, Expression of the Zg16 and Hormad1 genes in
various tissues of iPSC tetraploid complementation chimaeric mice. Ten types
of tissues were prepared from adult, 2–8-month old, tetraploid-
complementation chimaeric mice derived from iPSC clone 2A-4F-33 or from
the ES-cell clones B6ES2-9 and B6ES5-1, and then subjected to semi-
quantitative RNA analysis. The intestines and the testes of C57BL/6 wild-type
mice were used as a control for Zg16 and Hormad1 gene expression,
respectively. Error bars show standard deviation (n52).
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plasmids (Addgene) as described previously14,15. We performed PCR analysis to
confirm the absence of plasmid integration14and established ES-cell lines as
described previously30. Established iPSC and ES-cell lines were evaluated on the
basis of their morphologies, stem cell markers, neuploidy and pluripotency
(Supplementary Fig. 1 and Supplementary Table 1). Skin and bone marrow were
prepared from fullychimaeric adultmalemice developedfrom either iPSCsorES
cells. The C57BL/6 and C57BL/6-Tg(CAG-EGFP) mouse strains were used as
Full Methods and any associated references are available in the online version of
Received 22 May; accepted 22 November 2012.
Published online 9 January 2013.
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Supplementary Information is available in the online version of the paper.
Acknowledgements We thank S. Yamanaka for providing the 2A plasmid vectors;
K. Ito, N. Nakajima and K. Kawabata for technical advice on the transplantation
experiments; H. Yoshida, K. Nishikawa, A. Ishibashi, R. Watanabe, M. Nakamura and
T. Maeda for technical assistance. This work was partially funded by a research grant
from Precursory Research for Embryonic Science and Technology (PRESTO), Japan
Science and Technology Agency.
Author Contributions R.A. and M.A. designed the experiments, analysed the data and
wrote the manuscript. R.A. and Y.H. established the iPSC and ES-cell lines. M.U., S.A.,
M.N. and Y.H. performed teratoma formation assays and transplantation assays. M.
A.N. performed immunohistochemical analyses.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
and requests for materials should be addressed to M.A. (email@example.com).
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Mice. C57BL/6J Jms Slc mice were used for iPSC and ES cell generation, and
C57BL/6J Jms Slc, C57BL/6-Tg(CAG-EGFP)18and Balb/c mice were used for
transplantation assays (Japan SLC). Only males were used for cell-line establish-
ment and transplantation experiments. Animal experiments were performed in
accordance with Institutional Animal Care and Use Committee guidelines.
c-Myc plasmids (Addgene) as described previously14,15. Briefly, mouse embryonic
fibroblasts (MEFs) were isolated from embryonic day (E)13.5 embryos of male
after tranfection. We carried out PCR analysis to confirm the absence of plasmid
vector integration14. Established iPSC lines were evaluated on the basis of their
morphologies, stem cell markers and pluripotency. Neuploidy was also investi-
gated.Weestablished EScelllinesasdescribedpreviously30. Briefly, E3.5embryos
feeder MEFs. Established ES cell lines were evaluated on the basis of their
morphologies, stem cell markers, neuploidy and pluripotency (Supplementary
Fig. 1 and Supplementary Table 1).
injecting cells (33106cells per injection) subcutaneously into the flanks of the
mice. Four or five weeks after injection, the tumours were surgically dissected,
fixed and embedded in paraffin. Tumour sections were immunostained with
anti-CD3 antibody MCA-1477 (AbD serotec) as described previously31.
Statistics.Normally distributed data areexpressedas the means6s.e.m.,andthe
Immunocytochemistry of iPSC colonies. For immunocytochemical staining,
anti-Nanog (1:50, ReproCELL), anti-Oct3/4 h-134 (1:100, Santa Cruz Bio-
technology) and anti Sox-2 Y-17 (1:100, Santa Cruz Biotechnology) antibodies
Generation of chimaeric mice from iPSCs and ES cells. Chimaeric mice were
produced via the aggregation method, using 8-cell embryos or tetraploid embryo
Skin transplantation. Adult male chimaeric mice were used to prepare donor
tail as donor tissue. Because mouse tail tissue is resistant to ischaemia it can be
transplanted efficiently without any technical troubles, and sensitive assays using
tails have been developed for long-term (.1 month) investigations33,34. Briefly,
donor skin (535mm) was grafted onto the back of recipient mice, and then
covered with a bandage. The bandage was removed after 7 days, followed by
analysis each day.
Bone marrow transplantation. Donor bone marrows were harvested from the
either iPSCs or ES cells. We used 7–8-week-old C57BL/6 or C57BL/6-Tg(CAG-
cells. The animals were killed and the tibia and femurs were clipped into small
pieces with scissors after removing the muscle and connective tissues. Bone chips
were thoroughly rinsed with complete medium (DMEM supplemented with 5%
FCS, 2mM L-glutamine and antibiotics) in a 50-ml tube. The washed solution
containing bone marrow cells was then transferred into a 50-ml tube after filtra-
tion through a 40-mm nylon mesh filter, and centrifuged at 600g for 10min. The
mixed for 1min on ice. After addition of a further 1ml of 1.6% NaCl, the mixture
was centrifuged at 600g for 10min and the pellet was resuspended in complete
medium. Cells were counted and 107cells in 0.4ml of PBS were injected into the
lateral tail veins using a 26-gauge needle for all animals, except for recipient
without irradiation IR-2, for which 0.73107cells were injected.
from the venous plexus of eye socket of the recipient mice. A 5-ml aliquot of the
blood was diluted with PBS without haemolysis and used for the measurement of
GFP-positive cells using flow cytometry, performed with an Epics XL instrument
(Beckman Coulter). To detect B lymphocytes, T lymphocytes and granulo-
cytes derived from donor bone marrow, the remaining collected whole blood
was haemolysed using Versalyse (Beckman-Coulter) to stain the cell-surface
markers B220 (CD45R)-APC (clone; RA3-6B2, code no. 17-0452; eBioscience),
CD3e-PE (clone; 145-2C11, code no. 12-0031; e-Bioscience), Gr-1 (Ly-6G)-PE
(clone; RB6-8C5, code no. 12-5931; e-Bioscience) and Mac-1(CD11b)-PE (clone;
M1/70, code no. 101208; Biolegend). Four-colour flow cytometric analysis
was performed using FACSCalibur, and the data were analysed using CellQuest
software (BD Biosciences). In addition, using FACSaria (BD Bioscience), we
sorted B220-positive, CD3e-PE-positive and Gr-1/Mac-1-positive fractions
from the bone marrow preparations for semi-quantitative measurement of
Genotyping. Allele-specific PCR was performed to discriminate between donor
and recipient tissues in the bone marrow transplantation experiments. Sequences
of the primers used to characterize Nnt loci are listed in Supplementary Fig. 835.
Genomic DNAs were prepared using the DNeasy Blood & Tissue Kit (Qiagen).
PCR reactions were performed usingTitaniumTaqDNApolymerase(Clontech),
and semi-quantitative PCR was performed using SYBR Premix EX Taq (Takara
Bio) with the StepOnePlus Real-Time PCR System (Applied Biosystems).
Semi-quantitative expression analysis. Teratomas and tissues were homoge-
using the RNeasy Mini kit (Qiagen) in accordance with the manufacturer’s pro-
tocol. All RT–PCR analyses were performed using the QuantiTect SYBR Green
RT–PCR Kit (Qiagen). The data were normalized by comparison with Gapdh
gene expression. The primers used were 59-CATCACCGCCTTCCGTAT-39
and 59-CGTTGAAACTTGTGCCTG A-39 for Zg16, and 59-GCTGACACCAAG
AAAGCAAG-39 and 59-TTGGGACTTCTCCCACATTT-39 for Hormad1.
Immunogenicity test for cardiomyocytes. Embryoid bodies were prepared with
Iscove’s modified Dulbecco’s medium (Invitrogen) supplemented with 15% FBS,
1mM sodium pyruvate (Invitrogen), 0.1mM non-essential amino acids, 0.1mM
200ml per well) using 96-well low cell-adhesion plates (Lipidure Coat; NOF
Corp.)36, then transferred to 0.3% gelatin-coated 12-well dishes (BD Falcon) in
aMEM supplemented with 10% FBS, 0.1mM non-essential amino acids, 0.1mM
6-Tg(CAG-EGFP) recipient mice. Tissue sections were immunostained with
anti-CD3 antibody (MCA-1477, AbD serotec).
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