Induction of Pluripotent Stem Cells
from Mouse Embryonic and Adult
Fibroblast Cultures by Defined Factors
Kazutoshi Takahashi1 and Shinya Yamanaka1,2,*
1Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
2CREST, 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 con-
tents into oocytes or by fusion with embryonic
stem (ES) cells. Little is known about factors
that induce this reprogramming. Here, we dem-
onstrate 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 plurip-
otent stem) cells, exhibit the morphology and
growth properties of ES cells and express ES
cell marker genes. Subcutaneous transplanta-
tion of iPS cells into nude mice resulted in
tumors containing a variety of tissues from all
three germ layers. Following injection into blas-
tocysts, iPS cells contributed to mouse embry-
onic development. These data demonstrate
that pluripotent stem cells can be directly gen-
erated from fibroblast cultures by the addition
of only a few defined factors.
INTRODUCTION
Embryonic stem (ES) cells, which are derived from the in-
ner 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 Parkin-
son’s disease, spinal cord injury, and diabetes (Thomson
et al., 1998). However, there are ethical difficulties regard-
ing 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 plu-
ripotent 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), respec-
tively. 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-galactosi-
dase 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 concen-
trations of G418 (up to 12 mg/ml), whereas somatic cells
derived from Fbx15bgeo/bgeo mice were sensitive to a nor-
mal 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) GrowthcurvesofEScells, iPScells (iPS-MEF24, clones2-1–4), andMEFs. 33105cellswerepassagedevery 3days into eachwell of six-well plates.
(E) RT-PCRanalysis of EScellmarker genes in iPScells (iPS-MEF24, clones1-5, 1-9, and1-18), EScells, andMEFs.Nat1wasusedasa loading control.
(F) Bisulfite genomic sequencing of the promoter regions ofOct3/4,Nanog, andFbx15 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.