Induction of Pluripotent Stem Cells
from Mouse Embryonic and Adult
Fibroblast Cultures by Defined Factors
Kazutoshi Takahashi1
and Shinya Yamanaka1,2,
*
1
Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
2
CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
*Contact: yamanaka@frontier.kyoto-u.ac.jp
DOI 10.1016/j.cell.2006.07.024
SUMMARY
Differentiated cells can be reprogrammed to an
embryonic-like state by transfer of nuclear contents
into oocytes or by fusion with embryonic
stem (ES) cells. Little is known about factors
that induce this reprogramming. Here, we demonstrate
induction of pluripotent stem cells
from mouse embryonic or adult fibroblasts by
introducing four factors, Oct3/4, Sox2, c-Myc,
and Klf4, under ES cell culture conditions.
Unexpectedly, Nanog was dispensable. These
cells, which we designated iPS (induced pluripotent
stem) cells, exhibit the morphology and
growth properties of ES cells and express ES
cell marker genes. Subcutaneous transplantation
of iPS cells into nude mice resulted in
tumors containing a variety of tissues from all
three germ layers. Following injection into blastocysts,
iPS cells contributed to mouse embryonic
development. These data demonstrate
that pluripotent stem cells can be directly generated
from fibroblast cultures by the addition
of only a few defined factors.
INTRODUCTION
Embryonic stem (ES) cells, which are derived from the inner
cell mass of mammalian blastocysts, have the ability
to grow indefinitely while maintaining pluripotency and
the ability to differentiate into cells of all three germ layers
(Evans and Kaufman, 1981; Martin, 1981). Human ES cells
might be used to treat a host of diseases, such as Parkinson’s
disease, spinal cord injury, and diabetes (Thomson
et al., 1998). However, there are ethical difficulties regarding
the use of human embryos, as well as the problem of
tissue rejection following transplantation in patients. One
way to circumvent these issues is the generation of pluripotent
cells directly from the patients’ own cells.
Somatic cells can be reprogrammed by transferring
their nuclear contents into oocytes (Wilmut et al., 1997)
or by fusion with ES cells (Cowan et al., 2005; Tada
et al., 2001), indicating that unfertilized eggs and ES cells
contain factors that can confer totipotency or pluripotency
to somatic cells. We hypothesized that the factors that
play important roles in the maintenance of ES cell identity
also play pivotal roles in the induction of pluripotency in
somatic cells.
Several transcription factors, including Oct3/4 (Nichols
et al., 1998; Niwa et al., 2000), Sox2 (Avilion et al., 2003),
and Nanog (Chambers et al., 2003; Mitsui et al., 2003),
function in the maintenance of pluripotency in both early
embryos and ES cells. Several genes that are frequently
upregulated in tumors, such as Stat3 (Matsuda et al.,
1999; Niwa et al., 1998), E-Ras (Takahashi et al., 2003),
c-myc (Cartwright et al., 2005), Klf4 (Li et al., 2005), and
b-catenin (Kielman et al., 2002; Sato et al., 2004), have
been shown to contribute to the long-term maintenance
of the ES cell phenotype and the rapid proliferation of
ES cells in culture. In addition, we have identified several
other genes that are specifically expressed in ES cells
(Maruyama et al., 2005; Mitsui et al., 2003).
In this study, we examined whether these factors could
induce pluripotency in somatic cells. By combining four
selected factors, we were able to generate pluripotent
cells, which we call induced pluripotent stem (iPS) cells,
directly from mouse embryonic or adult fibroblast cul-
tures.
RESULTS
We selected 24 genes as candidates for factors that
induce pluripotency in somatic cells, based on our
hypothesis that such factors also play pivotal roles in the
maintenance of ES cell identity (see Table S1 in the
Supplemental Data available with this article online). For
b-catenin, c-Myc, and Stat3, we used active forms,
S33Y-b-catenin (Sadot et al., 2002), T58A-c-Myc (Chang
et al., 2000), and Stat3-C (Bromberg et al., 1999), respectively.
Because of the reported negative effect of Grb2
on pluripotency (Burdon et al., 1999; Cheng et al., 1998),
we included its dominant-negative mutant Grb2DSH2
(Miyamoto et al., 2004) as 1 of the 24 candidates.
Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 663
To evaluate these 24 candidate genes, we developed
an assay system in which the induction of the pluripotent
state could be detected as resistance to G418 (Figure 1A).
We inserted a bgeo cassette (a fusion of the b-galactosidase
and neomycin resistance genes) into the mouse
Fbx15 gene by homologous recombination (Tokuzawa
et al., 2003). Although specifically expressed in mouse
ES cells and early embryos, Fbx15 is dispensable for the
maintenance of pluripotency and mouse development.
ES cells homozygous for the bgeo knockin construct
(Fbx15bgeo/bgeo
) were resistant to extremely high concentrations
of G418 (up to 12 mg/ml), whereas somatic cells
derived from Fbx15bgeo/bgeo
mice were sensitive to a normal
concentration of G418 (0.3 mg/ml). We expected that
even partial activation of the Fbx15 locus would result in
resistance to normal concentrations of G418.
We introduced each of the 24 candidate genes into
mouse embryonic fibroblasts (MEFs) from Fbx15bgeo/bgeo
Figure 1. Generation of iPS Cells from MEF Cultures via 24 Factors
(A) Strategy to test candidate factors.
(B) G418-resistant colonies were observed 16 days after transduction with a combination of 24 factors. Cells were stained with crystal violet.
(C) Morphology of ES cells, iPS cells (iPS-MEF24, clone 1-9), and MEFs. Scale bars = 200 mm.
(D) Growth curves of ES cells, iPS cells (iPS-MEF24, clones 2-1–4), and MEFs. 33 105
cells werepassaged every 3 days into each wellof six-wellplates.
(E) RT-PCR analysis of ES cell marker genes in iPS cells (iPS-MEF24, clones 1-5, 1-9, and 1-18), ES cells, and MEFs. Nat1 was used as aloading control.
(F) Bisulfite genomic sequencing of the promoter regions of Oct3/4, Nanog, and Fbx15 in iPS cells (iPS-MEF24, clones 1-5, 1-9, and 1-18), ES cells, and
MEFs. Open circles indicate unmethylated CpG dinucleotides, while closed circles indicate methylated CpGs.
664 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.
embryos by retroviral transduction (Morita et al., 2000).
Transduced cells were then cultured on STO feeder cells
in ES cell medium containing G418 (0.3 mg/ml). We did
not, however, obtain drug-resistant colonies with any single
factor, indicating that no single candidate gene was
sufficient to activate the Fbx15 locus (Figure 1B; see
also Table S2, which summarizes all of the transduction
experiments in this study).
In contrast, transduction of all 24 candidates together
generated 22 G418-resistant colonies (Figure 1B). Of the
12 clones for which we continued cultivating under selection,
5 clones exhibited morphology similar to ES cells,
including a round shape, large nucleoli, and scant cytoplasm
(Figure 1C). We repeated the experiments and observed
29 G418-resistant colonies, from which we picked
6 colonies. Four of these clones possessed ES cell-like
morphology and proliferation properties (Figure 1D). The
doubling time of these cells (19.4, 17.5, 18.7, and 18.6
hr) was equivalent to that of ES cells (17.0 hr). We designated
these cells iPS-MEF24 for ‘‘pluripotent stem cells induced
from MEFs by 24 factors.’’ Reverse transcription
PCR (RT-PCR) analysis revealed that the iPS-MEF24
clones expressed ES cell markers, including Oct3/4,
Nanog, E-Ras, Cripto, Dax1, and Zfp296 (Mitsui et al.,
2003) and Fgf4 (Yuan et al., 1995) (Figure 1E). Bisulfite
genomic sequencing demonstrated that the promoters
of Fbx15 and Nanog were demethylated in iPS cells
(Figure 1F). By contrast, the Oct3/4 promoter remained
methylated in these cells. These data indicate that some
combination of these 24 candidate factors induced the
expression of ES cell marker genes in MEF culture.
Next, to determine which of the 24 candidates were critical,
we examined the effect of withdrawal of individual
factors from the pool of transduced candidate genes on
the formation of G418-resistant colonies (Figure 2A). We
identified 10 factors (3, 4, 5, 11, 14, 15, 18, 20, 21, and
22) whose individual withdrawal from the bulk transduction
pool resulted in no colony formation 10 days after
Figure 2. Narrowing down the Candidate Factors
(A) Effect of the removal of individual factors from the pool of 24 transduced factors on the formation of G418-resistant colonies. Fbx15bgeo/bgeo
MEFs
were transduced with the indicated factors and selected with G418 for 10 days (white columns) or 16 days (black columns).
(B) Effect of the removal of individual factors from the selected 10 factors on the formation of G418-resistant colonies 16 days after transduction.
(C) Effect of the transduction of pools of four, three, and two factors on the formation of G418-resistant colonies 16 days after transduction.
(D) Morphologies of iPS-MEF4 (clone 7), iPS-MEF10 (clone 6), and iPS-MEF3 (clone 3). Scale bars = 200 mm.
Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 665
transduction and fewer colonies 16 days after transduction.
Combination of these 10 genes alone produced
more ES cell-like colonies than transduction of all 24
genes did (Figure 2B).
We next examined the formation of colonies after withdrawal
of individual factors from the 10-factor pool transduced
into MEFs (Figure 2B). G418-resistant colonies did
not form when either Oct3/4 (factor 14) or Klf4 (factor 20)
was removed. Removal of Sox2 (factor 15) resulted in
only a few G418-resistant colonies. When we removed
c-Myc (factor 22), G418-resistant colonies did emerge,
but these had a flatter, non-ES-cell-like morphology. Removal
of the remaining factors did not significantly affect
colony numbers. These results indicate that Oct3/4, Klf4,
Sox2, and c-Myc play important roles in the generation
of iPS cells from MEFs.
Combination of the four genes produced a number of
G418-resistant colonies similar to that observed with the
pool of 10 genes (Figure 2C). We continued cultivation of
12 clones for each transduction and were able to establish
4 iPS-MEF4 and 5 iPS-MEF10 clones. In addition, we
could generate iPS cells (iPS-MEF4wt) with wild-type
c-Myc instead of the T58A mutant (Table S2). These data
demonstrate that iPS cells can be induced from MEF
culture by the introduction of four transcription factors,
Oct3/4, Sox2, c-Myc, and Klf4.
No combination of two factors could induce the formation
of G418-resistant colonies (Figure 2C). Two combinations
of three factors—Oct3/4, Sox2, and c-Myc (minus
Klf4) or Klf4, Sox2, and c-Myc (minus Oct3/4)—generated
a single, small colony in each case, but these could not be
maintained in culture. With the combination of Oct3/4,
Klf4, and Sox2 (minus c-Myc), we observed the formation
of 36 G418-resistant colonies, which, however, exhibited
a flat, non-ES-cell-like morphology. With the combination
of Oct3/4, Klf4, and c-Myc (minus Sox2), we observed
the formation of 54 G418-resistant colonies, of which we
picked 6. Although all 6 clones could be maintained over
several passages, the morphology of these cells (iPSMEF3)
differed from that of iPS-MEF4 and iPS-MEF10
cells, with iPS-MEF3 colonies exhibiting rough surfaces
(Figure 2D). These data indicate that the combination of
Oct3/4, c-Myc, and Klf4 can activate the Fbx15 locus,
but the change induced by these three factors alone is different
from that seen in iPS-MEF4 or iPS-MEF10 cells.
We performed RT-PCR to examine whether ES cell
marker genes were expressed in iPS cells (Figure 3A).
We used primers that would amplify transcripts of the endogenous
gene but not transcripts of the transgene. iPSMEF10
and iPS-MEF4 clones expressed the majority of
marker genes, with the exception of Ecat1 (Mitsui et al.,
2003). The expression of several marker genes, including
Oct3/4, was higher in iPS-MEF4-7, iPS-MEF10-6, and
iPS-MEF10-7 clones than in the remaining clones. Sox2
was only expressed in iPS-MEF10-6. The iPS-MEF4wt
clone also expressed many of the ES cell marker genes
(Figure S1). Chromatin immunoprecipitation analyses
showed that the promoters of Oct3/4 and Nanog had
increased acetylation of histone H3 and decreased
dimethylation of lysine 9 of histone H3 (Figure 3B). CpG
dinucleotides in these promoters remained partially
methylated in iPS cells (Figure 3C). iPS-MEF4 and iPSMEF10
cells were positive for alkaline phosphatase and
SSEA-1 (Figure 3D) and showed high telomerase activity
(Figure S2). These results demonstrate that iPS-MEF4
and iPS-MEF10 cells are similar, but not identical, to ES
cells.
In iPS-MEF3 clones, Ecat1, Esg1, and Sox2 were not
activated (Figure 3A). Nanog was induced, but to a lesser
extent than in iPS-MEF4 and iPS-MEF10 clones. Oct3/4
was weakly activated in iPS-MEF3-3, -5, and -6 but was
not activated in the remaining clones. By contrast, E-Ras
and Fgf4 were activated more efficiently in iPS-MEF3
than in iPS-MEF10 or iPS-MEF4. These data confirm
that iPS-MEF3 cells are substantially different from iPSMEF10
and iPS-MEF4 cells.
We compared the global gene-expression profiles of
ES cells, iPS cells, and Fbx15bgeo/bgeo
MEFs using DNA
microarrays (Figure 4A). In addition, we examined
Fbx15bgeo/bgeo
MEFs in which the four factors had been
introduced without G418 selection, immortalized MEFs
expressing K-RasV12, and NIH 3T3 cells transformed
with H-RasV12. Pearson correlation analysis revealed
that iPS cells are clustered closely with ES cells but separately
from fibroblasts and their derivatives (Figure 4A).
The microarray analyses identified genes that were commonly
upregulated in ES cells and iPS cells, including
Myb, Kit, Gdf3, and Zic3 (group I, Figure 4B and Table
S3). Other genes were upregulated more efficiently in ES
cells, iPS-MEF4, and iPS-MEF10 than in iPS-MEF3
clones, including Dppa3, Dppa4, Dppa5, Nanog, Sox2,
Esrrb, and Rex1 (group II). Lower expression of these
genes may account for the lack of pluripotency in iPSMEF3
cells. In addition, we found genes that were upregulated
more prominently in ES cells than in iPS cells, including
Dnmt3a, Dnmt3b, Dnmt3l, Utf1, Tcl1, and the LIF
receptor gene (group III). These data confirm that iPS cells
are similar, but not identical, to ES cells.
We examined the pluripotency of iPS cells by teratoma
formation (Figure 5A; Table S6 and Figure S3). We obtained
tumors with 5 iPS-MEF10 clones, 3 iPS-MEF4
clones, 1 iPS-MEF4wt clone, and 6 iPS-MEF3 clones
after subcutaneous injection into nude mice. Histological
examination revealed that 2 iPS-MEF10 clones (3 and 6),
2 iPS-MEF4 clones (2 and 7), and the iPS-MEF4wt-4 clone
differentiated into all three germ layers, including neural
tissues, cartilage, and columnar epithelium. iPS-MEF10-
6 could give rise to all three germ layers even after 30 passages
(Table S6 and Figure S3). We confirmed differentiation
into neural and muscle tissues by immunostaining
(Figure 5B) and RT-PCR (Figure S4). By contrast, these
teratomas did not express the trophoblast marker Cdx2
(Figure S4). iPS-MEF10-1 tumors differentiated into ectoderm
and endoderm, but not mesoderm, and no signs of
differentiation were observed in tumors derived from the
remaining iPS-MEF10 (7 and 10) or from iPS-MEF4-10.
666 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.
These data demonstrate that the majority of, but not all,
iPS-MEF10 and iPS-MEF4 clones exhibit pluripotency.
In contrast, all tumors derived from iPS-MEF3 clones
were composed entirely of undifferentiated cells (Table
S6 and Figure S3). Thus, although the three factors
(Oct3/4, c-Myc, and Klf4) could induce the expression of
some ES cell marker genes, they were not able to induce
pluripotency.
iPS-MEF10, iPS-MEF4, and iPS-MEF3 cells formed embryoid
bodies in noncoated plastic dishes (Figure 5C).
When grown in tissue culture dishes, the embryoid bodies
from iPS-MEF10 and iPS-MEF4 cells attached to the dish
bottom and initiated differentiation. After 3 days, immunostaining
detected cells positive for a-smooth muscle actin
(mesoderm marker), a-fetoprotein (endoderm marker),
and bIII tubulin (ectoderm marker) (Figure 5D). By contrast,
embryoid bodies from iPS-MEF3 cells remained undifferentiated
even when cultured in gelatin-coated dishes (Figure
5C). These data confirmed pluripotency of iPS-MEF10
and iPS-MEF4 and nullipotency of iPS-MEF3 in vitro.
Figure 3. Gene-Expression Profiles of iPS Cells
(A) RT-PCR analysis of ES marker genes in iPS cells, ES cells, and MEFs. We used primer sets that amplified endogenous but not transgenic
transcripts.
(B) The promoters of Oct3/4 and Nanog were analyzed by ChIP for dimethylation status of lysine 9 of histone H3 and acetylation status of histone H3 in
ES cells, MEFs, and iPS cells (MEF4-7 and MEF10-6). Data were quantified by real-time PCR. Shown are the averages and standard deviations of
relative values compared to ES cells (n = 3). *p < 0.05; **p < 0.01 compared to MEFs.
(C) The promoters of Oct3/4, Nanog, and Fbx15 were analyzed with bisulfite genomic sequencing for DNA methylation status in iPS-MEF4-7 and
iPS-MEF10-6. The DNA methylation status of these promoters in ES cells and MEFs is shown in Figure 1F.
(D) iPS-MEF4-7 and iPS-MEF10-6 clones were stained with a mouse monoclonal antibody against SSEA-1 (480, Santa Cruz) or with an alkaline
phosphatase kit (Sigma). Scale bars = 500 mm (SSEA1) and 1 mm (AP).
Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 667
Figure 4. Global Gene-Expression
Analyses by DNA Microarrays
(A) Pearson correlation analysis of 10,517
probes was performed to cluster ES cells,
iPS cells (MEF4- 7, MEF10-6, MEF3-2, and
MEF3-3), MEFs, MEFs expressing the four
factors, immortalized MEFs expressing
K-RasV12, and NIH 3T3 cells transformed
by H-RasV12. Red indicates increased
expression compared to median levels of
the eight samples, whereas green means
decreased expression.
(B) Genes upregulated in ES and/or iPS cells.
Genes in group I are genes upregulated in ES
cells and iPS cells. Genes in group II are
upregulated more in ES cells, iPS-MEF4-7,
and iPS-MEF10-6 than in iPS-MEF3 cells.
Genes in group III are upregulated more in
ES cells than in iPS cells. Lists of genes are
shown in Tables S3–S5.
668 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.
We next introduced the four selected factors into tail-tip
fibroblasts (TTFs) of four 7-week-old male Fbx15bgeo/bgeo
mice on a C57/BL6-129 hybrid background. We obtained
3 G418-resistant colonies, from each of which we could
establish iPS cells (iPS-TTF4). We also introduced the
four factors into TTFs from a 12-week-old female
Fbx15bgeo/bgeo
mouse, which also constitutively expressed
green fluorescent protein (GFP) from the CAG
promoter and had a C57/BL6-129-ICR hybrid background.
Of the 13 G418-resistant colonies obtained, we
isolated 6 clones from which we could establish iPS cells
(iPS-TTFgfp4, clones 1–6). In addition, we established
another iPS-TTFgfp4 (clone 7), in which the cDNA for
each of the four factors was flanked with two loxP sites
in the transgene. These cells were morphologically indistinguishable
from ES cells (Figure 6A). RT-PCR showed
that clones 3 and 7 of iPS-TTFgfp4 expressed the majority
of ES cell marker genes at high levels and the others at
lower levels (Figure 6B). In another attempt, we used either
the T58A mutant or the wild-type c-Myc for transduction
and established 5 iPS-TTFgfp4 clones (clones 8–12) and
3 iPS-TTFgfp4wt clones (clones 1–3) (Figure S5). RT-PCR
Figure 5. Pluripotency of iPS Cells
Derived from MEFs
(A) Various tissues present in teratomas derived
from iPS-MEF4-7 cells. Histology of other
teratomas is shown in Figure S3 and Table S6.
(B) Immunostaining confirming differentiation
into neural tissues and muscles in teratomas
derived from iPS-MEF4-7.
(C) In vitro embryoid body formation (upper
row) and differentiation (lower row). Scale
bars = 200 mm.
(D) Immunostaining confirming in vitro differentiation
into all three germ layers. Scale bars =
100 mm. Secondary antibodies were labeled
with Cy3 (red), except for a-fetoprotein in iPSMEF10-6,
with which Alexa 488 (green) was
used.
Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 669
showed that iPS-TTFgfp4wt cells also expressed most
of the ES cell marker genes (Figure S6).
We transplanted 2 iPS-TTF4 and 6 iPS-TTFgfp4 clones
into nude mice, all of which produced tumors containing
tissues of all three germ layers (Table S6 and Figure S3).
We then introduced 2 clones of iPS-TTFgfp4 cells (clones
3 and 7) into C57/BL6 blastocysts by microinjection. With
iPS-TTFgfp4-3, we obtained 18 embryos at E13.5, 2 of
which showed contribution of GFP-positive iPS cells
(Figure 6C). Histological analyses confirmed that iPS cells
contributed to all three germ layers (Figure 6D). We observed
GFP-positive cells in the gonad but could not determine
whether they were germ cells or somatic cells.
With iPS-TTFgfp4-7, we obtained 22 embryos at E7.5, 3
of which were positive for GFP. With the 2 clones, we
had 27 pups born, but none of them were chimeric mice.
In addition, iPS-TTFgfp4 cells could differentiate into all
three germ layers in vitro (Figure S7). These data demonstrate
that the four selected factors could induce pluripotent
cells from adult mouse fibroblast cultures.
Figure 6. Characterization of iPS Cells Derived from Adult Mouse Tail-Tip Fibroblasts
(A) Morphology of iPS-TTFgfp4-3 on STO feeder cells.
(B) RT-PCR analysis of ES marker gene expression in iPS-TTFgfp4 cells (clones 1–5 and 7). We used primer sets that amplified endogenous but not
transgenic transcripts.
(C) Contribution of iPS-TTFgfp4-7 and iPS-TTFgfp4-3 cells to mouse embryonic development. iPS cells were microinjected into C57/BL6 blastocysts.
Embryos were analyzed with a fluorescence microscope at E7.5 (upper panels, iPS-TTFgfp4-7) or E13.5 (lower panels, iPS-TTFgfp4-3). Scale bars =
200 mm (upper panels) and 2 mm (lower panels).
(D) The E13.5 chimeric embryo was sectioned and stained with anti-GFP antibody (brown). Cells were counterstained with eosin (blue).
670 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.
We further characterized the expression of the four factors
and others in iPS cells. Real-time PCR confirmed that
endogenous expression of Oct3/4 and Sox2 was lower in
iPS cells than in ES cells (Figure S8). However, the total
amount of the four factors from the endogenous genes
and the transgenes exceeded the normal expression
levels in ES cells. In contrast, Western blot analyses
showed that the total protein amounts of the four factors
in iPS cells were comparable to those in ES cells (Figure
7A; Figure S8). We could detect Nanog and E-Ras proteins
in iPS cells, but at lower levels than those in ES cells
(Figures 7A and 7B; Figure S8). The p53 levels in iPS cells
were lower than those in MEFs and equivalent to those in
ES cells (Figure 7A; Figure S9). The p21 levels in iPS cells
varied in each clone and were between those in ES cells
and MEFs (Figure S9). Upon differentiation in vitro, the total
mRNA expression levels of Oct3/4 and Sox2 decreased
but remained much higher than in ES cells. In contrast,
their protein levels decreased to comparable levels in
iPS cells and ES cells (Figure 7B).
Southern blot analyses showed that each iPS clone has
a unique transgene integration pattern (Figure 7C). Karyotyping
analyses of the iPS-TTFgfp4 (clones 1, 2, 3, 7, and
11) and iPS-TTFgfp4wt (clones 1–3) demonstrated that 2
iPS-TTFgfp4 clones and all of the iPS-TTFgfp4wt clones
showed a normal karyotype of 40XX (Figure 7D), while
the other 3 iPS-TTFgfp4 clones were 39XO, 40XO +10,
and 40Xi(X). Analyses of PCR-based simple sequence
length polymorphisms (SSLPs) demonstrated that iPSMEF
clones have a mixed background of C57/BL6 and
129 (Table S7), whereas iPS-TTFgfp clones have a mixed
background of ICR, C57/BL6, and 129 (Table S8). Finally,
we found that iPS cells could not remain undifferentiated
when cultured in the absence of feeder cells, even with
the presence of LIF (Figure 7E). These results, together
with the different gene-expression patterns, exclude the
possibility that iPS cells are merely contamination of preexisting
ES cells. Finally, subclones of iPS cells were positive
for alkaline phosphatase and could differentiate into
all three germ layers in vitro (Figure S10), confirming their
clonal nature.
DISCUSSION
Oct3/4, Sox2, and Nanog have been shown to function
as core transcription factors in maintaining pluripotency
(Boyer et al., 2005; Loh et al., 2006). Among the three,
we found that Oct3/4 and Sox2 are essential for the generation
of iPS cells. Surprisingly, Nanog is dispensable. In
addition, we identified c-Myc and Klf4 as essential factors.
These two tumor-related factors could not be replaced by
other oncogenes including E-Ras, Tcl1, b-catenin, and
Stat3 (Figures 2A and 2B).
The c-Myc protein has many downstream targets that
enhance proliferation and transformation (Adhikary and
Eilers, 2005), many of which may have roles in the generation
of iPS cells. Of note, c-Myc associates with histone
acetyltransferase (HAT) complexes, including TRRAP,
which is a core subunit of the TIP60 and GCN5 HAT complexes
(McMahon et al., 1998), CREB binding protein
(CBP), and p300 (Vervoorts et al., 2003). Within the mammalian
genome, there may be up to 25,000 c-Myc binding
sites (Cawley et al., 2004), many more than the predicted
number of Oct3/4 and Sox2 binding sites (Boyer et al.,
2005; Loh et al., 2006). c-Myc protein may induce global
histone acetylation (Fernandez et al., 2003), thus allowing
Oct3/4 and Sox2 to bind to their specific target loci.
Klf4 has been shown to repress p53 directly (Rowland
et al., 2005), and p53 protein has been shown to suppress
Nanog during ES cell differentiation (Lin et al., 2004). We
found that iPS cells showed levels of p53 protein lower
than those in MEFs (Figure 7A). Thus, Klf4 might contribute
to activation of Nanog and other ES cell-specific genes
through p53 repression. Alternatively, Klf4 might function
as an inhibitor of Myc-induced apoptosis through the repression
of p53 in our system (Zindy et al., 1998). On the
other hand, Klf4 activates p21CIP1
, thereby suppressing
cell proliferation (Zhang et al., 2000). This antiproliferation
function of Klf4 might be inhibited by c-Myc, which suppresses
the expression of p21CIP1
(Seoane et al., 2002).
The balance between c-Myc and Klf4 may be important
for the generation of iPS cells.
One question that remains concerns the origin of our
iPS cells. With our retroviral expression system, we estimated
that only a small portion of cells expressing the
four factors became iPS cells (Figure S11). The low frequency
suggests that rare tissue stem/progenitor cells
that coexisted in the fibroblast cultures might have given
rise to the iPS cells. Indeed, multipotent stem cells have
been isolated from skin (Dyce et al., 2004; Toma et al.,
2001, 2005). These studies showed that 0.067% of
mouse skin cells are stem cells. One explanation for the
low frequency of iPS cell derivation is that the four factors
transform tissue stem cells. However, we found that the
four factors induced iPS cells with comparably low efficiency
even from bone marrow stroma, which should be
more enriched in mesenchymal stem cells and other multipotent
cells (Tables S2 and S6). Furthermore, cells induced
by the three factors were nullipotent (Table S6
and Figure S3). DNA microarray analyses suggested that
iPS-MEF4 cells and iPS-MEF3 cells have the same origin
(Figure 4). These results do not favor multipotent tissue
stem cells as the origin of iPS cells.
There are several other possibilities for the low frequency
of iPS cell derivation. First, the levels of the four
factors required for generation of pluripotent cells may
have narrow ranges, and only a small portion of cells expressing
all four of the factors at the right levels can acquire
ES cell-like properties. Consistent with this idea, a mere
50% increase or decrease in Oct3/4 proteins induces
differentiation of ES cells (Niwa et al., 2000). iPS clones
overexpressed the four factors when RNA levels were analyzed,
but their protein levels were comparable to those in
ES cells (Figures 7A and 7B; Figure S8), suggesting that the
iPS clones possess a mechanism (or mechanisms) that
Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 671
Figure 7. Biochemical and Genetic Analyses of iPS Cells
(A) Western blot analyses of the four factors and other proteins in iPS cells (MEF4-7, MEF10-6, TTFgfp4-3, and TTFgfp4-7), ES cells, and MEFs.
(B) Changes in RNA (left) and protein (right) levels of Oct3/4, Sox2, and Nanog in iPS cells and ES cells that were undifferentiated on STO feeder cells
(U) or induced to differentiated in vitro through embryoid body formation (D). Shown are relative expression levels compared to undifferentiated ES
cells. Data of MEFs and TTFs are also shown. RNA levels were determined with real-time PCR using primers specific for endogenous transcripts
672 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.
tightly regulates the protein levels of the four factors. We
speculate that high amounts of the four factors are required
in the initial stage of iPS cell generation, but, once
they acquire ES cell-like status, too much of the factors
are detrimental for self-renewal. Only a small portion of
transduced cells show such appropriate transgene expression.
Second, generation of pluripotent cells may
require additional chromosomal alterations, which take
place spontaneously during culture or are induced by
some of the four factors. Although the iPS-TTFgfp4 clones
had largely normal karyotypes (Figure 7D), we cannot rule
out the existence of minor chromosomal alterations. Sitespecific
retroviral insertion may also play a role. Southern
blot analyses showed that each iPS clone has 20 retroviral
integrations (Figure 7C). Some of these may have caused
silencing or fusion with endogenous genes. Further studies
will be required to determine the origin of iPS cells.
Another unsolved question is whether the four factors
we identified play roles in reprogramming induced by fusion
with ES cells or nuclear transfer into oocytes. Since
the four factors are expressed in ES cells at high levels,
it is reasonable to speculate that they are involved in the
reprogramming machinery that exists in ES cells. Our result
is also consistent with the finding that the reprogramming
activity resides in the nucleus, but not in the cytoplasm,
of ES cells (Do and Scholer, 2004). However, iPS
cells were not identical to ES cells, as shown by the global
gene-expression patterns and DNA methylation status. It
is possible that we have missed additional important factors.
One such candidate is ECAT1, although its forced
expression in iPS cells did not consistently upregulate
ES cell marker genes (Figure S12).
More obscure are the roles of the four factors, especially
Klf4 and c-Myc, in the reprogramming observed in oocytes.
Both Klf4 and c-Myc are dispensable for preimplantation
mouse development (Baudino et al., 2002; Katz
et al., 2002). Furthermore, c-myc is not detected in oocytes
(Domashenko et al., 1997). In contrast, L-myc is expressed
maternally in oocytes. Klf17 and Klf7, but not Klf4,
are found in expressed sequence-tag libraries derived
from unfertilized mouse eggs. Klf4 and c-Myc might be
compensated by these related proteins. It is highly likely
that other factors are also required to induce complete
reprogramming and totipotency in oocytes.
It is likely that the four factors from the transgenes are
required for maintaining the iPS cells since the expression
of Oct3/4 and Sox2 from the endogenous genes remained
low (Figure 7B; Figure S8). We intended to prove this by
using transgenes flanked by two loxP sites and obtained
an iPS clone (TTF4gfp4-7). However, we noticed that
these cells contain multiple loxP sites on multiple chromosomes,
and, thus, the Cre-mediated recombination would
cause not only deletion of the transgenes but also interand
intrachromosomal rearrangements. Studies with
conditional expression systems, such as the tetracyclinemediated
system, are required to answer this question.
We showed that the iPS cells can differentiate in vitro
and in vivo even with the presence of the retroviral vectors
containing the four factors. We found that Oct3/4 and
Sox2 proteins decreased significantly during in vitro differentiation
(Figure 7B). Retroviral expression has been
shown to be suppressed in ES cells and further silenced
upon differentiation by epigenetic modifications, such as
DNA methylation (Yao et al., 2004). The same mechanisms
are likely to play roles in transgene repression in iPS cells
since they express Dnmt3a, 3b, and 3l, albeit at lower
levels than ES cells do (Table S5). In addition, we found
that iPS cells possess a mechanism (or mechanisms)
that lowers protein levels of the transgenes and Nanog
(Figure 7B; Figure S8). The same mechanism may be
enhanced during differentiation. However, silencing of
Oct3/4 in iPS-TTFgfp4-3 cells was not complete, which
may explain our inability to obtain live chimeric mice after
blastocyst microinjection of iPS cells.
An unexpected finding in this study was the efficient activation
of Fgf4 and Fbx15 by the combination of the three
factors devoid of Sox2 since these two genes have been
shown to be regulated synergistically by Oct3/4 and
Sox2 (Tokuzawa et al., 2003; Yuan et al., 1995). It is also
surprising that Nanog is dispensable for induction and
maintenance of iPS cells. More detailed analyses of iPS
cells will enhance our understanding of transcriptional
regulation in pluripotent stem cells.
Our findings may have wider applications, as we have
found that transgene reporters with other ES cell marker
genes, such as Nanog, can replace the Fbx15 knockin during
selection (K. Okita and S.Y., unpublished data). However,
we still do not know whether the four factors can generate
pluripotent cells from human somatic cells. Use of
c-Myc may not be suitable for clinical applications, and
the process may require specific culture environments.
Nevertheless, the finding is an important step in controlling
pluripotency, which may eventually allow the creation of
pluripotent cells directly from somatic cells of patients.
EXPERIMENTAL PROCEDURES
Mice
Fbx15bgeo/bgeo
mice were generated with 129SvJae-derived RF8 ES
cells as described previously (Tokuzawa et al., 2003) and were
(white columns) or those common for both endogenous and transgenic transcripts (white and black columns). RNA expression levels are shown on
logarithmic axes. Protein levels were determined by Western blot normalized with b-actin. Protein levels are shown as the averages and standard
deviations on linear axes (n = 4). *p < 0.05 compared to undifferentiated cells.
(C) Southern blot analyses showing the integration of transgenes. Genomic DNA isolated from iPS cells and ES cells was digested with EcoRI and
BamHI, separated on agarose gel, transferred to a nylon membrane, and hybridized with a Klf4 cDNA probe.
(D) Normal karyotype of iPS-TTFgfp4-2 clone.
(E) Morphology of ES cells and iPS cells cultured without feeder cells. One thousand cells were cultured on gelatin-coated six-well plates for 5 days,
with or without LIF. Scale bars = 200 mm.
Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 673
backcrossed to the C57/BL6 strain for at least five generations. These
mice were used for primary mouse embryonic fibroblast (MEF) and tailtip
fibroblast (TTF) preparations. To generate Fbx15bgeo/bgeo
mice with
constitutive expression of GFP, an Fbx15bgeo/bgeo
mouse (C57/BL6-
129 background) was mated with an ICR mouse with the GFP transgene
driven by the constitutive CAG promoter (Niwa et al., 1991).
The resulting Fbx15bgeo/+,GFP/+
mice were intercrossed to generate
Fbx15bgeo/bgeo,GFP/GFP
mice. Nude mice (BALB/Jcl-nu) were purchased
from CLEA.
Cell Culture
RF8 ES cells and iPS cells were maintained on feeder layers of mitomycin
C-treated STO cells as previously described (Meiner et al., 1996).
As a source of leukemia inhibitory factor (LIF), we used conditioned
medium (1:10,000 dilution) from Plat-E cell cultures that had been
transduced with a LIF-encoding vector. ES and iPS cells were passaged
every 3 days. Plat-E packaging cells (Morita et al., 2000), which
were also used to produce retroviruses, were maintained in DMEM
containing 10% FBS, 50 units/50 mg/ml penicillin/streptomycin,
1 mg/ml puromycin (Sigma), and 100 mg/ml of blasticidin S (Funakoshi).
For MEF isolation, uteri isolated from 13.5-day-pregnant mice were
washed with phosphate-buffered saline (PBS). The head and visceral
tissues were removed from isolated embryos. The remaining bodies
were washed in fresh PBS, minced using a pair of scissors, transferred
into a 0.1 mM trypsin/1 mM EDTA solution (3 ml per embryo), and
incubated at 37
C for 20 min. After incubation, an additional 3 ml per
embryo of 0.1 mM trypsin/1 mM EDTA solution was added, and the
mixture was incubated at 37
C for 20 min. After trypsinization, an equal
amount of medium (6 ml per embryo DMEM containing 10% FBS) was
added and pipetted up and down a few times to help with tissue dissociation.
After incubation of the tissue/medium mixture for 5 min at
room temperature, the supernatant was transferred into a new tube.
Cells were collected by centrifugation (200 3 g for 5 min at 4
C) and
resuspended in fresh medium. 1 3 106
cells (passage 1) were cultured
on 100 mm dishes at 37
C with 5% CO2. In this study, we used MEFs
within three passages to avoid replicative senescence.
To establish TTFs, the tails from adult mice were peeled, minced into
1 cm pieces, placed on culture dishes, and incubated in MF-start medium
(Toyobo) for 5 days. Cells that migrated out of the graft pieces
were transferred to new plates (passage 2) and maintained in DMEM
containing 10% FBS. We used TTFs at passage 3 for iPS cell induction.
Retroviral Infection
The day before transduction, Plat-E cells (Morita et al., 2000) were
seeded at 8 3 106
cells per 100 mm dish. On the next day, pMXs-based
retroviral vectors were introduced into Plat-E cells using Fugene 6
transfection reagent (Roche) according to the manufacturer’s recommendations.
Twenty-seven microliters of Fugene 6 transfection reagent
was diluted in 300 ml DMEM and incubated for 5 min at room temperature.
Nine micrograms of plasmid DNA was added to the mixture,
which was incubated for another 15 min at room temperature. After incubation,
the DNA/Fugene 6 mixture was added drop by drop onto
Plat-E cells. Cells were then incubated overnight at 37
C with 5% CO2.
Twenty-four hours after transduction, the medium was replaced.
MEFs or TTFs were seeded at 8 3 105
cells per 100 mm dish on mitomycin
C-treated STO feeders. After 24 hr, virus-containing supernatants
derived from these Plat-E cultures were filtered through a
0.45 mm cellulose acetate filter (Schleicher & Schuell) and supplemented
with 4 mg/ml polybrene (Nacalai Tesque). Target cells were incubated
in the virus/polybrene-containing supernatants for 4 hr to
overnight. After infection, the cells were replated in 10 ml fresh
medium. Three days after infection, we added G418 at a final concentration
of 0.3 mg/ml. Clones were selected for 2 to 3 weeks.
Plasmid Construction
To generate pMXs-gw, we introduced a Gateway cassette rfA (Invitrogen)
into the EcoRI/XhoI site of the pMXs plasmid. Primers used are
listed in Table S9. Mutations in b-catenin, c-myc, and Stat3 were introduced
by PCR-based site-directed mutagenesis. For forced expression,
we amplified the coding regions of candidate genes by RT-PCR,
cloned these sequences into pDONR201 or pENTR-D-TOPO (Invitrogen),
and recombined the resulting plasmids with pMXs-gw by LR
reaction (Invitrogen).
Teratoma Formation and Histological Analysis
ES cells or iPS cells were suspended at 1 3 107
cells/ml in DMEM containing
10% FBS. Nude mice were anesthetized with diethyl ether. We
injected 100 ml of the cell suspension (1 3 106
cells) subcutaneously
into the dorsal flank. Four weeks after the injection, tumors were surgically
dissected from the mice. Samples were weighed, fixed in PBS
containing 4% formaldehyde, and embedded in paraffin. Sections
were stained with hematoxylin and eosin.
Bisulfite Genomic Sequencing
Bisulfite treatment was performed using the CpGenome modification
kit (Chemicon) according to the manufacturer’s recommendations.
PCR primers are listed in Table S9. Amplified products were cloned
into pCR2.1-TOPO (Invitrogen). Ten randomly selected clones were sequenced
with the M13 forward and M13 reverse primers for each gene.
Determination of Karyotypes and SSLP by PCR
Karyotypes were determined with quinacrine-Hoechst staining at the
International Council for Laboratory Animal Science (ICLAS) Monitoring
Center (Japan). We obtained PCR primer sequences for SSLP
from the Mouse Genome Informatics website (The Jackson Laboratory,
http://www.informatics.jax.org). Allele sizes were approximated
on the basis of the known allele sizes in various inbred strains.
Western Blot Analyses
Western blot was performed as previously described (Takahashi et al.,
2003). The primary antibodies used were anti-Oct3/4 monoclonal
antibody (C-10, Santa Cruz), anti-Sox2 antiserum (Maruyama et al.,
2005), anti-Klf4 polyclonal antibody (H-180, Santa Cruz), anti-c-Myc
polyclonal antibody (A-14, Santa Cruz), anti-Nanog antiserum (Mitsui
et al., 2003), anti-E-Ras antiserum (Takahashi et al., 2003), anti-p53
polyclonal antibody (FL-393, Santa Cruz), and anti-b-actin monoclonal
antibody (A5441, Sigma).
RT-PCR for Marker Genes
We performed reverse transcription reactions using ReverTra Ace -a(Toyobo)
and the oligo dT20 primer. PCR was done with ExTaq
(Takara). Real-time PCR was performed with Platinum SYBR Green
qPCR SuperMix-UDG with ROX (Invitrogen) according to manufacturer’s
instructions. Signals were detected with an ABI7300 RealTime
PCR System (Applied Biosystems). Primer sequences are listed
in Table S9.
DNA Microarray
Total RNA from ES cells, iPS cells, or MEFs were labeled with Cy3.
Samples were hybridized to a Mouse Oligo Microarray (G4121B, Agilent)
according to the manufacturer’s protocol. Arrays were scanned
with a G2565BA Microarray Scanner System (Agilent). Data were analyzed
using GeneSpring GX software (Agilent).
In Vitro Differentiation of iPS Cells
Cells were harvested by trypsinization and transferred to bacterial culture
dishes in the ES medium without G418 or LIF. After 3 days, aggregated
cells were plated onto gelatin-coated tissue culture dishes and
incubated for another 3 days. The cells were stained with antia-smooth
muscle actin monoclonal antibody (N1584, Dako), anti-afetoprotein
polyclonal antibody (N1501, Dako) or anti-bIII tubulin
monoclonal antibody (CBL412, Abcam) along with 40
-6-diamidino-
2-phenylindole (Sigma). Total RNA derived from plated embryoid
bodies on day 6 was used for RT-PCR analysis.
674 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.
Chromatin Immunoprecipitation Assay
We performed chromatin immunoprecipitation (ChIP) as previously
described (Maruyama et al., 2005). Antibodies used in this experiment
were anti-dimethyl K9 H3 rabbit polyclonal antibody (ab7312-100,
Abcam) and anti-acetyl H3 rabbit polyclonal antibody (06-599,
Upstate). PCR primers are listed in Table S9.
Statistical Analyses
Data are shown as averages and standard deviations. We used
Student’s t test for protein-level analyses and one-factor ANOVA
with Scheffe’s post hoc test for ChIP analyses. All statistical analyses
were done with Excel 2003 (Microsoft) with the Statcel2 add-on (OMS).
Supplemental Data
Supplemental Data include 12 figures and 9 tables and can be found
with this article online at http://www.cell.com/cgi/content/full/126/4/
663/DC1/.
ACKNOWLEDGMENTS
We are grateful to Tomoko Ichisaka for preparation of mice and Mitsuyo
Maeda and Yoshinobu Toda for histological analyses. We thank
Megumi Kumazaki, Mirei Murakami, Masayoshi Maruyama, and Noriko
Tsubooka for technical assistance; Masato Nakagawa, Keisuke
Okita, and Koji Shimozaki for scientific comments; and Yumi Ohuchi
for administrative assistance. We also thank Dr. Robert Farese, Jr.
for RF8 ES cells and Dr. Toshio Kitamura for the Plat-E cells and
pMX retroviral vectors. This work was supported in part by research
grants from the Ministry of Education, Culture, Sports, Science and
Technology of Japan to S.Y. This work is also supported in part by
the Takeda Science Foundation, the Osaka Cancer Research Foundation,
the Inamori Foundation, the Mitsubishi Pharma Research Foundation,
and the Sankyo Foundation of Life Science and by a Grantin-Aid
from the Japan Medical Association to S.Y. K.T. was supported
by a fellowship from the Japan Society for the Promotion of Science.
Received: April 24, 2006
Revised: June 18, 2006
Accepted: July 20, 2006
Published online: August 10, 2006
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Accession Numbers
Microarray data are available in GEO (Gene Expression Omnibus,
http://www.ncbi.nlm.nih.gov/projects/geo/index.cgi) with the accession
number GSE5259.
676 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.